Decreased expression of Glucagon-like peptide-1 receptor and Sodium-glucose co-transporter 2 in patients with proliferative diabetic retinopathy.

Purpose: To investigate the expression of Glucagon-like peptide-1 receptor (GLP-1R), sodium-glucose co-transporter (SGLT) 1, SGLT2, Glucose transporter type 1 (GLUT1) and GLUT2 in patients with diabetic retinopathy (DR). Methods: We obtained peripheral blood mononuclear cells (PBMCs) and vitreous samples from 26 proliferative DR (PDR) patients, 25 non-proliferative DR (NPDR) patients, 25 non-DR (NDR) patients, and 26 nondiabetic patients with idiopathic epiretinal membranes (ERMs, control). The protein level and mRNA expression level of GLP-1R were quantified by immunoblot and qRT-PCR and the levels of SGLT1, SGLT2, GLUT1, and GLUT2 expression were determined by PCR. Their association with clinical parameters and PBMCs/vitreous cytokine was analyzed. Furthermore, immunofluorescence staining of GLP-1R and SGLT2 was carried out on samples of fibrovascular membranes (FVMs) retrieved from 26 patients with PDR and 26 patients with ERMs. Results: The transcriptional levels of GLP-1R and SGLT2 in PBMCs were significantly more decreased in PDR patients than in patients without DR and controls, which was simultaneously associated with an increased level of expression of tumor necrosis factor (TNF)-α and interferon (IFN)-γ. The expression levels of GLUT1 and GLUT2 were tightly correlated with their SGLT partners, respectively. Further, Immunofluorescence staining showed no positive staining of GLP-1R and SGLT2 was detected in the FVMs from PDR. Conclusions: GLP-1R and SGLT2 were significantly decreased in PDR patients which was associated with an increased level of expression of TNF-α and IFN-γ. These findings implicate that defective GLP-1R and SGLT2 signaling may potentially correlate with immune response cytokines in patients with PDR.

Protective role of ellagic acid and taurine against fluoxetine induced hepatotoxic effects on biochemical and oxidative stress parameters, histopathological changes, and gene expressions of IL-1ß, NF-κB, and TNF-α in male Wistar rats.

PURPOSES: Hepatic bioactivation of fluoxetine (FXN) could increase free radicals' generation provoking hepatotoxicity. Therefore, the protective effects of ellagic acid (EA) and taurine (TAU) treatments against fluoxetine-induced liver damage in rats were examined. MATERIALS AND METHODS: Sixty four male Wistar rats were randomly assigned to 8 groups (n = 8). Group (1) Control, group (2) FXN, group (3) FXN + EA, group (4) FXN + TAU, group (5) FXN + EA + TAU, group (6) EA, group (7) TAU, and group (8) EA + TAU. Then, the serum and tissue parameters of the oxidative stress were examined. KEY FINDINGS: FXN significantly raised serum MDA, protein carbonyl, lipid profile, ALT, AST, ALP, total bilirubin, serum IL-1ß; and gene expressions of IL-1ß, NF-κB, and TNF-α. Moreover, it significantly decreased HDL-C, ferric reducing antioxidant power (FRAP), catalase activity, vitamin C, and SOD activity in the liver compared to group 1. When compared to group 2, EA and TAU treatment dramatically increased antioxidant capacity and lowered hepatotoxic biochemical markers and cellular inflammation. Results also showed a protective effect of treatment against oxidative damage caused by hepatocytes' cytoarchitecture. SIGNIFICANCE: Our study concluded the beneficial effects of EA and TAU on FXN-induced hepatotoxicity. These effects were derived from free radical scavenging properties and the anti-inflammatory effects related to IL-1ß, NF-κB, and TNF-α gene expression inhibition.

Activation and increased production of interleukin-17 and tumour necrosis factor-α of mucosal-associated invariant T cells in patients with periodontitis.

AIM: Mucosal-associated invariant T (MAIT) cells are known to be resident in oral mucosal tissue, but their roles in periodontitis are unknown. This study aimed to examine the level and function of MAIT cells in periodontitis patients. MATERIALS AND METHODS: Frequency, activation, and function of MAIT cells from 28 periodontitis patients and 28 healthy controls (HCs) were measured by flow cytometry. RESULTS: Circulating MAIT cells were numerically reduced in periodontitis patients. Moreover, they exhibited higher expression of CD69 and annexin V, together with more increased production of interleukin (IL)-17 and tumour necrosis factor (TNF)-α, in periodontitis patients than in HCs. Interestingly, periodontitis patients had higher frequencies of MAIT cells in gingival tissue than in peripheral blood. In addition, circulating MAIT cells had elevated expression of tissue-homing chemokine receptors such as CCR6 and CXCR6, and the corresponding chemokines (i.e., CCL20 and CXCL16) were more strongly expressed in inflamed gingiva than in healthy gingiva. CONCLUSIONS: This study demonstrates that circulating MAIT cells are numerically deficient with an activated profile toward the production of IL-17 and TNF-α in periodontitis patients. Furthermore, circulating MAIT cells have the potential to migrate to inflamed gingival tissues.

Characterization and Validation of In Vitro and In Vivo Models to Investigate TNF-α-Induced Inflammation in Retinal Diseases.

Purpose: Inflammation is implicated in the etiology of diverse retinopathies including uveitis, age-related macular degeneration or diabetic retinopathy. Tumor necrosis factor alpha (TNF-α) is a well-known proinflammatory cytokine that is described as a biomarker for inflammation in diverse retinopathies and therefore emerged as an interesting target to treat inflammation in the eye by neutralizing anti-TNF-α antibodies. Methods: Recently, we have demonstrated that Adeno-associated virus (AAV)-mediated expression of human TNF-α in the murine eye induces retinal inflammation including vasculitis and fibrosis, thereby mimicking human disease-relevant pathologies. In a proof-of-mechanism study, we now tested whether AAV-TNF-α induced pathologies can be reversed by neutralizing TNF-α antibody treatment. Results: Strikingly, a single intravitreal injection of the TNF-α antibody golimumab reduced AAV-TNF-α-induced retinal inflammation and retinal thickening. Furthermore, AAV-TNF-α-mediated impaired retinal function was partially rescued by golimumab as revealed by electroretinography recordings. Finally, to study TNF-α-induced vasculitis in human in vitro cell culture assays, we established a monocyte-to-endothelium adhesion co-culture system. Indeed, also in vitro TNF-α induced monocyte adhesion to human retinal endothelial cells, which was prevented by golimumab. Conclusions: Overall, our study describes valuable in vitro and in vivo approaches to study the function of TNF-α in retinal inflammation and demonstrated a preclinical proof-of-mechanism treatment with golimumab. Translational Relevance: The AAV-based model expressing human TNF-α allows us to investigate TNF-α-driven pathologies supporting research in mechanisms of retinal inflammation.

Exercise in the treatment of ankylosing spondylitis.

Seronegative spondyloarthritis is a progressive inflammatory disease with a wide and varied range of symptoms. Ankylosing spondylitis (AS) is a chronic inflammatory rheumatic disease characterized by pain and structural changes. Ankylosing spondylitis is characterized by functional disorders such as reduced mobility and axial deformity, leading to a decrease in patient quality of life. The purpose of this article is to provide basic information on therapeutic options for exercise in the treatment of ankylosing spondylitis. Therapeutic exercise is the most important non-pharmacological intervention in patients. In addition to positive metabolic effects, exercise also plays an important role in improving spinal mobility and muscle strength. Maintaining proper postural posture appears to play a key role in patients with ankylosing spondylitis. Muscle contraction stimulates and promotes the secretion of cytokines called myokines. The best known is interleukin-6 (IL-6). This myokine also acts indirectly in the expression of tumor necrosis factor alpha (TNF-α). Exercise improves quality of life, physical condition, and symptoms associated with ankylosing spondylitis.

Photochemically-Mediated Inflammation and Cross-Presentation of Mycobacterium bovis BCG Proteins Stimulates Strong CD4 and CD8 T-Cell Responses in Mice.

Conventional vaccines are very efficient in the prevention of bacterial infections caused by extracellular pathogens due to effective stimulation of pathogen-specific antibodies. In contrast, considering that intracellular surveillance by antibodies is not possible, they are typically less effective in preventing or treating infections caused by intracellular pathogens such as Mycobacterium tuberculosis. The objective of the current study was to use so-called photochemical internalization (PCI) to deliver a live bacterial vaccine to the cytosol of antigen-presenting cells (APCs) for the purpose of stimulating major histocompatibility complex (MHC) I-restricted CD8 T-cell responses. For this purpose, Mycobacterium bovis BCG (BCG) was combined with the photosensitiser tetraphenyl chlorine disulfonate (TPCS2a) and injected intradermally into mice. TPCS2a was then activated by illumination of the injection site with light of defined energy. Antigen-specific CD4 and CD8 T-cell responses were monitored in blood, spleen, and lymph nodes at different time points thereafter using flow cytometry, ELISA and ELISPOT. Finally, APCs were infected and PCI-treated in vitro for analysis of their activation of T cells in vitro or in vivo after autologous vaccination of mice. Combination of BCG with PCI induced stronger BCG-specific CD4 and CD8 T-cell responses than treatment with BCG only or with BCG and TPCS2a without light. The overall T-cell responses were multifunctional as characterized by the production of IFN-γ, TNF-α, IL-2 and IL-17. Importantly, PCI induced cross-presentation of BCG proteins for stimulation of antigen-specific CD8 T-cells that were particularly producing IFN-γ and TNF-α. PCI further facilitated antigen presentation by causing up-regulation of MHC and co-stimulatory proteins on the surface of APCs as well as their production of TNF-α and IL-1ß in vivo. Furthermore, PCI-based vaccination also caused local inflammation at the site of vaccination, showing strong infiltration of immune cells, which could contribute to the stimulation of antigen-specific immune responses. This study is the first to demonstrate that a live microbial vaccine can be combined with a photochemical compound and light for cross presentation of antigens to CD8 T cells. Moreover, the results revealed that PCI treatment strongly improved the immunogenicity of M. bovis BCG.

Precise Thermal Regulation of Engineered Bacteria Secretion for Breast Cancer Treatment In Vivo.

For the biomedical application of engineered bacteria, strictly regulating the function of engineered bacteria has always been the goal pursued. However, the existing regulation methods do not meet the needs of the in vivo application of engineered bacteria. Therefore, the exploration of the precise regulation of engineered bacteria is necessary. Herein, heat-sensitive engineered bacteria that can respond to thermal stimuli within 30 min were constructed, and the precise control of functions was verified in the intestines of various model organisms (including C. elegans, bees, and mice). Subsequently, heat-sensitive engineered bacteria were shown to colonize the mouse tumor microenvironment. Finally, thermal stimulation was proven to control engineered bacteria to produce the therapeutic protein tumor necrosis factor α (TNF-α) in the tumor. After three heat stimulation treatments, the growth of the tumor was significantly inhibited, suggesting that heat can be used as a strategy to precisely control engineered bacteria in vivo.

RI75, a curcumin analogue, inhibits tumor necrosis factor-α and interleukin-6 production and exhibits antiallodynic and antiedematogenic activities in mice.

Curcumin and its analogues exhibited anti-inflammatory activity in different experimental models. Recently, we synthesized (2E,3E)-3-buten-2-one-4-(4-hydroxy-3-methoxyphenyl)-2-(4-(4-methoxyphenyl)-2-thiazolyl)hydrazone (RI75), a curcumin analogue with a thiazolyl hydrazone moiety. In the present study, we investigated the effects induced by RI75 in different models of inflammation and pain in mice, as well as some underlying mechanisms. Pre-treatment with RI75 (40 mg/kg, intraperitoneal; i.p.) or curcumin (40 mg/kg, i.p.) reduced the mechanical allodynia and paw edema induced by intraplantar (i.pl) injection of carrageenan. RI75 antiallodynic activity was reduced by pre-treatment with naltrexone (5 and 10 mg/kg, i.p.) and cyproheptadine (10 mg/kg, i.p.), but not glibenclamide (20 and 40 mg/kg, i.p.). In a model of neuropathic pain, a single i.p. administration of RI75 (40 mg/kg) or curcumin (40 mg/kg) attenuated the ongoing mechanical allodynia induced by repeated administrations of paclitaxel. Pre-treatment with RI75 (40 mg/kg, i.p.) or curcumin (40 mg/kg, i.p.) also reduced tumor necrosis factor-α and interleukin-6 production and myeloperoxidase activity induced by carrageenan. The results of the present study demonstrate that RI75, a synthetic curcumin analogue, exhibits antiallodynic and antiedematogenic activities. Activation of opioidergic and serotonergic mechanisms and reduced production of inflammatory mediators and neutrophil recruitment may underlie RI75 activities.

Kupffer cells play a crucial role in monocrotaline-induced liver injury by producing TNF-α.

Monocrotaline (MCT), an unsaturated pyrrolizidine alkaloid (PA) in plants, is mainly toxic to the lung and liver of mammals. As a commonly used tool for liver injury model, the mechanism of MCT hepatoxicity has still not been fully clarified. Kupffer cells (KCs) are the liver-resident macrophages and have various responses to different toxicants and liver damage. However, the role of KCs in MCT-induced liver injury remains controversial. In current work, we investigated the effects of KCs on MCT-induced liver injury, especially on MCT-induced hepatocyte death. KCs were depleted in Balb/c mice by liposome-entrapped clodronate (Lip/Clo) (0.2 mL/mouse, i.p.) or GdCl3 (0.7 mg/kg, i.p.) before MCT administration (300 mg/kg, i.p.), we found that the Lip/Clo group showed higher efficiency in KCs depletion and stronger hepatoprotective effects against MCT. We also found TNF-α was remarkably decreased after KCs depletion, the experiment of administering anti-TNF-α antibody (20 µg/mouse, i.p.) to MCT-treated animals generated the similar results. To further elaborate the function of KCs, we compared the ALT levels released from co-culturing murine hepatic parenchymal cells (HPCs) and RAW264.7 cells with that from HPCs alone. After the treatment of MCT, the released ALT levels in co-culture system were shown to be dependent on the number of RAW264.7 cells, while the anti-TNF-α antibody could suppress it. Finally, we discovered RIPK3/MLKL pathway might be activated by TNF-α released from KCs, and subsequently induced hepatocyte necrosis. Noteworthy, the known mechanisms including ER stress and NF-κB pathways were also found to be involved in the activation of KCs. In conclusion, our study reveals a further mechanism to MCT-induced hepatoxicity mediated directly by KCs via producing TNF-α.

Sensitization of primary cultures from rat dorsal root ganglia with lipopolysaccharide (LPS) requires a robust inflammatory response.

OBJECTIVE: We investigated whether it is possible to induce a state of "LPS-sensitization" in neurons of primary cultures from rat dorsal root ganglia by pre-treatment with ultra-low doses of LPS. METHODS: DRG primary cultures were pre-treated with low to ultra-low doses of LPS (0.001-0.1 µg/ml) for 18 h, followed by a short-term stimulation with a higher LPS-dose (10 µg/ml for 2 h). TNF-α in the supernatants was measured as a sensitive read out. Using the fura-2 340/380 nm ratio imaging technique, we further investigated the capsaicin-evoked Ca2+-signals in neurons from DRG, which were pre-treated with a wide range of LPS-doses. RESULTS: Release of TNF-α evoked by stimulation with 10 µg/ml LPS into the supernatant was not significantly modified by pre-exposure to low to ultra-low LPS-doses. Capsaicin-evoked Ca2+-signals were significantly enhanced by pre-treatment with LPS doses being above a certain threshold. CONCLUSION: Ultra-low doses of LPS, which per se do not evoke a detectable inflammatory response, are not sufficient to sensitize neurons (Ca2+-responses) and glial elements (TNF-α-responses) of the primary afferent somatosensory system.

Fluvastatin enhances IL-33-mediated mast cell IL-6 and TNF production.

Statins are HMG-CoA reductase inhibitors prescribed for lowering cholesterol. They can also inhibit inflammatory responses by suppressing isoprenylation of small G proteins. Consistent with this, we previously found that fluvastatin suppresses IgE-mediated mast cell function. However, some studies have found that statins induced pro-inflammatory cytokines in macrophages and NK cells. In contrast to IgE signaling, we show that fluvastatin augments IL-33-induced TNF and IL-6 production by mast cells. This effect required the key mast cell growth factor, stem cell factor (SCF). Treatment of IL-33-activated mast cells with mevalonic acid or isoprenoids reduced fluvastatin effects, suggesting fluvastatin acts at least partly by reducing isoprenoid production. Fluvastatin also enhanced IL-33-induced NF-κB transcriptional activity and promoted neutrophilic peritonitis in vivo, a response requiring mast cell activation. Other statins tested did not enhance IL-33 responsiveness. Therefore, this work supports observations of unexpected pro-inflammatory effects of some statins and suggests mechanisms by which this may occur. Because statins are candidates for repurposing in inflammatory disorders, our work emphasizes the importance of understanding the pleiotropic and possible unexpected effects of these drugs.

A New Hybrid Protein Is a Novel Regulator for Experimental Colitis in Rats.

We newly developed a hybrid protein, tentatively named rMIKO-1, using gene technology. We herein investigated the effects of rMIKO-1 on activated macrophages and discussed its potential as a suppressor of experimental colitis. Fluorescent microscopy was used to observe the dynamic mobility of rMIKO-1 in macrophages. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, fluorescent immunochemical staining, flow cytometry, enzyme-linked immunosorbent assays, a polymerase chain reaction/quantitative polymerase chain reaction, and hematoxylin and eosin staining were conducted to assess the potential activity of rMIKO-1. A large amount of bleeding was observed in rats treated with 5% dextran sulfate sodium (DSS) alone on day 8 after treatment initiation, but not in those treated with 5% DSS plus rMIKO-1. In the in vitro assay, rMIKO-1 rapidly bound to macrophages, immediately entered cells by an unknown mechanism, and then migrated inside the nucleus. This result suggests that rMIKO-1 plays important immunological roles in the nucleus. Despite the activation of macrophages by lipopolysaccharide, the mRNA expression of pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin-6, and interleukin-1ß, was significantly suppressed in macrophages preliminarily treated with rMIKO-1 for 1 h. Complexes of rMIKO-1 with nuclear factor-kappa B (NF-κB)/p65 and ß-actin formed in activated macrophages, which attenuated experimental colitis in rats. These results strongly suggest that rMIKO-1 negatively regulates excessively activated macrophages through the NF-κB/p65 signaling pathway. Therefore, rMIKO-1 is a novel suppressor of experimental colitis in rats through the negative regulation of activated macrophages.

SARS-CoV-2 Nsp5 Activates NF-κB Pathway by Upregulating SUMOylation of MAVS.

The COVID-19 is an infectious disease caused by SARS-CoV-2 infection. A large number of clinical studies found high-level expression of pro-inflammatory cytokines in patients infected with SARS-CoV-2, which fuels the rapid development of the disease. However, the specific molecular mechanism is still unclear. In this study, we found that SARS-CoV-2 Nsp5 can induce the expression of cytokines IL-1ß, IL-6, TNF-α, and IL-2 in Calu-3 and THP1 cells. Further research found that Nsp5 enhances cytokine expression through activating the NF-κB signaling pathway. Subsequently, we investigated the upstream effectors of the NF-κB signal pathway on Nsp5 overexpression and discovered that Nsp5 increases the protein level of MAVS. Moreover, Nsp5 can promote the SUMOylation of MAVS to increase its stability and lead to increasing levels of MAVS protein, finally triggering activation of NF-κB signaling. The knockdown of MAVS and the inhibitor of SUMOylation treatment can attenuate Nsp5-mediated NF-κB activation and cytokine induction. We identified a novel role of SARS-CoV-2 Nsp5 to enhance cytokine production by activating the NF-κB signaling pathway.

Comparative effects of dexmedetomidine and propofol on brain and lung damage in experimental acute ischemic stroke.

Acute ischemic stroke is associated with pulmonary complications, and often dexmedetomidine and propofol are used to decrease cerebral metabolic rate. However, it is unknown the immunomodulatory actions of dexmedetomidine and propofol on brain and lungs during acute ischemic stroke. The effects of dexmedetomidine and propofol were compared on perilesional brain tissue and lung damage after acute ischemic stroke in rats. Further, the mean amount of both sedatives was directly evaluated on alveolar macrophages and lung endothelial cells primarily extracted 24-h after acute ischemic stroke. In twenty-five Wistar rats, ischemic stroke was induced and after 24-h treated with sodium thiopental (STROKE), dexmedetomidine and propofol. Dexmedetomidine, compared to STROKE, reduced diffuse alveolar damage score [median(interquartile range); 12(7.8-15.3) vs. 19.5(18-24), p = 0.007)], bronchoconstriction index [2.28(2.08-2.36) vs. 2.64(2.53-2.77), p = 0.006], and TNF-α expression (p = 0.0003), while propofol increased VCAM-1 expression compared to STROKE (p = 0.0004). In perilesional brain tissue, dexmedetomidine, compared to STROKE, decreased TNF-α (p = 0.010), while propofol increased VCAM-1 compared to STROKE (p = 0.024). In alveolar macrophages and endothelial cells, dexmedetomidine decreased IL-6 and IL-1ß compared to STROKE (p = 0.002, and p = 0.040, respectively), and reduced IL-1ß compared to propofol (p = 0.014). Dexmedetomidine, but not propofol, induced brain and lung protection in experimental acute ischemic stroke.

Citrullinated myelin induces microglial TNFα and inhibits endogenous repair in the cuprizone model of demyelination.

BACKGROUND: Microglia are the primary phagocytes of the central nervous system and are responsible for removing damaged myelin following demyelination. Previous investigations exploring the consequences of myelin phagocytosis on microglial activation overlooked the biochemical modifications present on myelin debris. Such modifications, including citrullination, are increased within the inflammatory environment of multiple sclerosis lesions. METHODS: Mouse cortical myelin isolated by ultracentrifugation was citrullinated ex vivo by incubation with the calcium-dependent peptidyl arginine deiminase PAD2. Demyelination was induced by 6 weeks of cuprizone (0.3%) treatment and spontaneous repair was initiated by reversion to normal chow. Citrullinated or unmodified myelin was injected into the primary motor cortex above the cingulum bundle at the time of reversion to normal chow and the consequent impact on remyelination was assessed by measuring the surface area of myelin basic protein-positive fibers in the cortex 3 weeks later. Microglial responses to myelin were characterized by measuring cytokine release, assessing flow cytometric markers of microglial activation, and RNAseq profiling of transcriptional changes. RESULTS: Citrullinated myelin induced a unique microglial response marked by increased tumor necrosis factor α (TNFα) production both in vitro and in vivo. This response was not induced by unmodified myelin. Injection of citrullinated myelin but not unmodified myelin into the cortex of cuprizone-demyelinated mice significantly inhibited spontaneous remyelination. Antibody-mediated neutralization of TNFα blocked this effect and restored remyelination to normal levels. CONCLUSIONS: These findings highlight the role of post-translation modifications such as citrullination in the determination of microglial activation in response to myelin during demyelination. The inhibition of endogenous repair induced by citrullinated myelin and the reversal of this effect by neutralization of TNFα may have implications for therapeutic approaches to patients with inflammatory demyelinating disorders.

Mitochondrial aspartate regulates TNF biogenesis and autoimmune tissue inflammation.

Misdirected immunity gives rise to the autoimmune tissue inflammation of rheumatoid arthritis, in which excess production of the cytokine tumor necrosis factor (TNF) is a central pathogenic event. Mechanisms underlying the breakdown of self-tolerance are unclear, but T cells in the arthritic joint have a distinctive metabolic signature of ATPlo acetyl-CoAhi proinflammatory effector cells. Here we show that a deficiency in the production of mitochondrial aspartate is an important abnormality in these autoimmune T cells. Shortage of mitochondrial aspartate disrupted the regeneration of the metabolic cofactor nicotinamide adenine dinucleotide, causing ADP deribosylation of the endoplasmic reticulum (ER) sensor GRP78/BiP. As a result, ribosome-rich ER membranes expanded, promoting co-translational translocation and enhanced biogenesis of transmembrane TNF. ERrich T cells were the predominant TNF producers in the arthritic joint. Transfer of intact mitochondria into T cells, as well as supplementation of exogenous aspartate, rescued the mitochondria-instructed expansion of ER membranes and suppressed TNF release and rheumatoid tissue inflammation.

Multifunctional, TNF-α and IFN-γ-Secreting CD4 and CD8 T Cells and CD8High T Cells Are Associated With the Cure of Human Visceral Leishmaniasis.

Visceral leishmaniasis (VL) is a chronic and often fatal disease caused by protozoans of the genus Leishmania that affects millions of people worldwide. Patients with symptomatic VL have an impaired anti-Leishmania-specific CD4+ T-cell response, which is reversed after clinical cure. In contrast, the quality of the CD4+ and CD8+ T-cell responses involved in resistance and/or cure of VL relies on the capability of these cells to activate polyfunctional and memory responses, which are associated with the simultaneous production of three cytokines: IFN-γ, IL-2, and TNF-α. Models for the development of CD4 and CD8 T-cell quality in memory and protection to leishmaniasis have been described previously. We aimed to assess the functionality of the T cells involved in the recovery of the immune suppression throughout the VL treatment. Therefore, we cultured peripheral blood mononuclear cells (PBMCs) from VL patients and healthy controls in vitro with soluble Leishmania antigen (SLA). Cell surface markers and intracellular cytokine production were determined on days 7, 14, 21, 30, 60, 90, and 180 after the beginning of chemotherapy. We observed that the frequencies of CD4+TNF-α+IFN-γ+ and the multifunctional CD4+IL-2+TNF-α+IFN-γ+, together with CD4+TNF-α+ and CD4+IFN-γ+ T cells, increased throughout and at the end of the treatment, respectively. In addition, enhanced frequencies of CD8+IL-2+TNF-α+IFN-γ+ and CD8+TNF-α+IFN-γ T cells were also relevant in the healing process. Noteworthy, the frequencies of the CD4+ and CD8 central-memory T cells, which produce IL-2, TNF-α, and IFN-γ and ensure the memory response against parasite reinfection, are significantly enhanced in cured patients. In addition, the subset of the non-functional CD8Low population is predominant in VL untreated patients and decreases along the chemotherapy treatment. In contrast, a CD8High subset increased towards the cure. Furthermore, the cure due to treatment with meglumine antimoniate or with liposomal amphotericin B was associated with the recovery of the T-cell immune responses. We described the evolution and participation of functional T cells during the treatment of patients with VL. Our results disclosed that the clinical improvement of patients is significantly associated with the participation of the CD4+ and CD8+ cytokine-secreting T cells.

Traumatic brain injury and hemorrhage in a juvenile rat model of polytrauma leads to immunosuppression and splenic alterations.

BACKGROUND: Traumatic brain injury (TBI) is a common cause of morbidity and mortality. We have previously shown that TBI with a concurrent extra-cranial injury reliably leads to post-injury suppression of the innate immune system, but the impact of this injury on the adaptive immune system is unknown. We present data showing that combined injury reduced immune response as assayed in both blood and spleen samples and that these changes parallel apoptosis in the spleen. To assess the clinical relevance of these changes, we examined lungs for spontaneous bacterial colonization. METHODS: For these studies, prepubescent (28 day old) rats were injured using a controlled cortical impact model and then 25% blood volume removal by arteriotomy, and injured animals were compared with sham injured animals. Blood and spleen samples at post-injury day 1 were incubated with or without immunostimulant and examined for IFN-γ production using an Eli-Spot assay. Spleen samples were also examined for apoptosis using Annexin V staining, and lungs were harvested and plated on blood agar to examine for spontaneous bacterial colonization. RESULTS: Stimulations of whole blood and spleen samples with phorbol 12-myristate 13-acetate/ionomycin (PMA/I) at post-injury day 1 were associated with significant decreases in IFN-γ-positive cells/million in injured animals. Stimulation of whole blood with either PMA/I or pokeweed mitogen led to reduced tumor necrosis factor alpha production. Spleen from injured animals showed a marked increase in apoptosis. Lung samples showed a 300% increase in colonies per plate in injured animals. CONCLUSIONS: These data suggest that the combined injury can lead to adaptive immunosuppression, and our findings further suggest a potential role for the spleen in altering leukocyte function following injury.

Selective Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Inhibition by the SCH772984 Compound Attenuates In Vitro and In Vivo Inflammatory Responses and Prolongs Survival in Murine Sepsis Models.

Sepsis is the leading cause of death in intensive care units worldwide. Current treatments of sepsis are largely supportive and clinical trials using specific pharmacotherapy for sepsis have failed to improve outcomes. Here, we used the lipopolysaccharide (LPS)-stimulated mouse RAW264.7 cell line and AlphaLisa assay for TNFa as a readout to perform a supervised drug repurposing screen for sepsis treatment with compounds targeting epigenetic enzymes, including kinases. We identified the SCH772984 compound, an extracellular signal-regulated kinase (ERK) 1/2 inhibitor, as an effective blocker of TNFa production in vitro. RNA-Seq of the SCH772984-treated RAW264.7 cells at 1, 4, and 24 h time points of LPS challenge followed by functional annotation of differentially expressed genes highlighted the suppression of cellular pathways related to the immune system. SCH772984 treatment improved survival in the LPS-induced lethal endotoxemia and cecal ligation and puncture (CLP) mouse models of sepsis, and reduced plasma levels of Ccl2/Mcp1. Functional analyses of RNA-seq datasets for kidney, lung, liver, and heart tissues from SCH772984-treated animals collected at 6 h and 12 h post-CLP revealed a significant downregulation of pathways related to the immune response and platelets activation but upregulation of the extracellular matrix organization and retinoic acid signaling pathways. Thus, this study defined transcriptome signatures of SCH772984 action in vitro and in vivo, an agent that has the potential to improve sepsis outcome.

Immunomodulatory Properties of Blackberry Anthocyanins in THP-1 Derived Macrophages.

An anthocyanin-rich diet is considered to protect against chronic inflammatory processes although the bioavailability of anthocyanins is regarded as rather low. Moreover, the immunomodulatory role of anthocyanins is not fully understood yet. In the present study, fractions of blackberry (Rubus fruticosus) juice were investigated in plasma-relevant concentrations with respect to their immunomodulatory properties in lipopolysaccharide (LPS)-challenged THP-1-derived macrophages. The complex blackberry extract acted ineffective as well as potential degradation products. Cyanidin-3O-glucoside (Cy3glc), the main constituent of blackberry anthocyanins, diminished TNF-α levels at a concentration of 0.02 µg/mL, indicating protective effects as measured with quantitative RT-PCR and multiplex cytokine assays. LPS-boosted activity of transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) of differentiated THP-1 reporter gene cells was marginally inhibited by Cy3glc. LPS-induced microRNA-155 was further increased, supporting the evidence of protection. Of note, fractions obtained from blackberry juice, in particular cyanidin-3O-(6″-dioxalylglucoside), were displaying potential pro-inflammatory properties as these elevated IL-6 and TNF-α levels. In conclusion, highly purified anthocyanin fractions of blackberry juice display both anti- and pro-inflammatory properties at plasma-relevant concentrations depending on their structure and substitution pattern.