Plasma Proinflammatory Cytokines Research Articles

30 Prenatally Stressed Brahman Bull Calves had Increased Pro-Inflammatory Cytokines and Transportation-Induced Mitochondrial Impairments

Abstract Prenatal transportation stress has been reported to alter behavior and adrenal glucocorticoid secretion in calves; however, the impact on mitochondrial and inflammatory responses to an industry-relevant stressor have not been explored. Sixteen Brahman bull calves were used to test the hypothesis that calves previously stressed by transportation in utero (PNS; n = 8; mean ± SD, 654 ± 71 kg, 11.7 ± 0.4 months) would have impaired mitochondrial capacity and greater plasma concentrations of two pro-inflammatory cytokines [interleukin (IL)-6 and tumor necrosis factor (TNF)-a] following a 3-hr transportation stressor compared with calves that experienced no prenatal stress (CON; n = 8; 645 ± 90 kg, 11.8 ± 0.5 months). Longissimus thoracis muscle samples were collected from calves, pre-transportation, and 6- and 24-hour post-transportation and were analyzed for citrate synthase activity (CS) as a marker of mitochondrial volume density and for mitochondrial oxidative phosphorylation (P) and electron transport (E) capacities via high resolution respirometry. Plasma was collected pre-transportation and post-transportation at hour 0, 1, 6, 24, and 48 for analysis of cytokine concentrations using custom MesoScale Discovery U-Plex kits. Data were analyzed using PROC MIXED with repeated measure (time) in SAS v9.4 with time, treatment, and time × treatment as fixed effects. Integrative (relative to tissue wet weight) E with complex II only (ECII), intrinsic (relative to CS) ECII, the contribution of P with complex I (PCI) to total E (flux control ratio, FCRPCI), and FCR ECII decreased from pre- to 6 hour post-transport (P ≤ 0.04) and remained suppressed at 24 hour post-transport (P ≤ 0.02) in all animals regardless of prenatal treatment. Integrative PCI was also decreased 24-hour post-transportation compared with pre-transport (P = 0.02). Intrinsic ECII tended to be greater in CON than PNS bulls overall (P = 0.07). Intrinsic Leak, integrative Leak, and FCR Leak tended to be impacted by the interaction of treatment and time (P ≤ 0.10) whereby all 3 measures were greater in PNS than CON bulls at 6-hour post-transport (P ≤ 0.02) but were similar between treatments at 24-hour post-transport. Both plasma IL-6 and TNF-a were greater in PNS than CON bulls overall (P ≤ 0.008) but neither cytokine was affected by postnatal transport. Bull calves subjected to prenatal transportation stress had sustained increased plasma pro-inflammatory cytokine concentrations and an increase in mitochondrial leak with a decrease in complex II supported electron transfer following a single postnatal transportation stressor. Importantly, the postnatal stressor utilized in this study did not affect plasma cytokines. A more stressful postnatal stimulus may provide a clearer assessment of potential impacts of prenatal stress on the progeny’s physiological responses to stress.

Comparison of oxidative stress parameters, thiol-disulfide homeostasis, and pro-inflammatory cytokines levels in patients with bipolar disorder and their first-degree relatives

BackgroundIn this study, we aimed to compare the oxidative stress parameters, thiol-disulfide homeostasis, and plasma pro-inflammatory cytokines levels of patients with bipolar disorder (BD), BD patients' first-degree relatives (FDRs), and the healthy controls (HCs). MethodsThirty-five patients with BD, 35 FDRs of BD, and 35 healthy controls (HCs) were included. The individuals' ages varied from 28 to 58, and the groups were well-matched in terms of age and gender. The total thiol (TT), native thiol (NT), disulfide (DIS), total oxidant status (TOS), total antioxidant status (TAS), IL-1β, IL-6, and TNF-α concentrations were measured from serum samples. The oxidative stress index (OSI) was calculated using mathematical formulas. ResultsTOS was significantly higher in both patients and FDRs than HCs (p < 0.01 for all pairwise comparisons). OSI, DIS, oxidized thiol, and the ratio of thiol oxidation-reduction levels were significantly higher in both patients with BD and FDRs than HCs (p < 0.01 for all pairwise comparisons). TAS, TT, NT, and reduced thiol levels were significantly lower in both patients with BD and FDRs than HCs (p < 0.01 for all pairwise comparisons). IL-1β, IL-6, and TNF-α were significantly higher in both patients and FDRs than HCs (p < 0.01 for all pairwise comparisons). LimitationsSmall sample size. ConclusionsEarly diagnosis is important for treating of bipolar disorder. TT, NT, DIS, TOS, TAS, OSI, IL1-β, IL-6, and TNF-α can be used as potential biomarkers in the early diagnosis and intervention of BD. Furthermore, oxidative/antioxidative markers and plasma pro-inflammatory cytokine parameters may guide the determination of the disease's activity and response to treatment.

Effects of PTSD-Dependent Neurogenic Hypertension and Inflammation on Thoracic Aortic Wall Homeostasis

Objective: Post-traumatic stress disorder (PTSD) in patients is associated with hypertension, increased heart rate, and elevated plasma proinflammatory cytokines, which also are risk factors for the development of cardiovascular conditions, such as thoracic aortic aneurysm (TAA). Although the association between stress and aortic pathology has been suggested, the exact mechanism linking the two is unknown. Our hypothesis is that PTSD-induced changes in mechanical and inflammatory signaling alter thoracic aortic wall homeostasis, which can predispose to aortic disease. Methods: Adult male and female C57BL/6 mice (n=7) underwent PTSD induction consisting of inescapable foot shock (five x 1 sec, 1 mA foot shocks over 6 min paired with a 20 sec 80 db tone (conditioned stimulus)) followed by single prolonged stress events. Mice were subjected to behavioral testing at 4-week intervals over a 16-week paradigm to determine their PTSD-like phenotype. The behavioral tests were chosen to assess for each of the human criteria of PTSD based on the DSM-5. A composite t-score was generated for each mouse derived from their mean individual z-score for each parameter, of each behavioral test. Mice with a composite t-score above 1.96 were considered to have a PTSD-like phenotype. Blood pressure was measured (CODA, tail-cuff) at baseline and at 4-week intervals. At terminal study, thoracic aortic diameter was measured by digital microscopy. Plasma was collected to measure circulating cytokines and aortic tissue was harvested for biochemistry and histology. Aortic structure was examined for changes in elastic lamellae, collagen deposition, and cellular content. Control mice (n=8) underwent the same procedures except for PTSD induction. Results: Our protocol generated a PTSD-like phenotype in 71% (5 of 7) of the mice. PTSD mice had significantly higher systolic blood pressures following a conditioned stimulus (PTSD (n=5), 153 ± 8mmHg; control (n=8), 129 ± 7mmHg, p&lt;0.05). PTSD mice also had increased plasma proinflammatory cytokines (IL1-α, IL-3, IL-5, IL-7, IL-9, all p&lt;0.05), enhanced extracellular matrix remodeling (ECM), and larger aortic diameters than control mice (PTSD (n=5), 712.9 ± 97.6μm; control (n=8), 584.2 ± 102.7μm, p &lt;0.05). Conclusion: Our PTSD-like mouse model recapitulated the human phenotype showing elevated systolic blood pressure and increased circulating proinflammatory cytokines. Increase thoracic aortic diameter and changes in the ECM suggest that PTSD can alter structural homeostasis in the aortic wall, predisposing the thoracic aorta to development of further pathology. Future studies will examine PTSD effects on TAA progression in a mouse model. NIH-NCATS TL1 TR00145-08 &amp; UL1TR001450-08 (HH) and VA Merit Award I01-BX000904-08A1 (JAJ) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cd13 controls myeloid cell-cell fusion

Abstract Macrophages have the ability to undergo phenotypic and functional changes in response to microenvironmental cues. One such change is the formation of multinucleated giant cells (MGCs) via intercellular fusion triggered by certain bacterial or parasitic infections or implantation of medical devices that induces deleterious MGC formation in response to the foreign body. Despite advances in biomaterials, implants ultimately corrode to produce particles that initiate a foreign body response characterized by macrophage fusion into pro-inflammatory, Foreign Body Giant Cells that directly define the pathology of loosening of medical implants. We have shown that the transmembrane protein CD13 is a multifunctional molecule that regulates diverse processes such as cell-cell adhesion, receptor endocytosis and recycling and organization of the actin cytoskeleton, all of which are critical in cell-cell fusion. We demonstrate that subcutaneous foreign implants in CD13 KOmice produce significantly more MGCs with increased levels of plasma pro-inflammatory cytokines. In vitro induction of fusion in CD13-deficient myeloid progenitors generated from bone marrow resulted in hyperfusion to generate MGCs that were considerably larger in size and contained many more nuclei than those from wild type progenitors, suggesting that CD13 regulates MGC formation at the stage of fusion. While expression of the key fusion proteins are typically downregulated in mature cells post-fusion, expression of these proteins is sustained at high levels in multinucleated cells lacking CD13. Thus, CD13 acts as a brake to prevent aberrant myeloid cell-cell fusion and may be a novel therapeutic target in pathological conditions mediated by abnormal cell fusion. Supported by grants from NIH (1R21 AI15 )

Activated CD90/Thy-1 fibroblasts co-express the Δ133p53β isoform and are associated with highly inflamed rheumatoid arthritis

BackgroundThe p53 isoform Δ133p53β is known to be associated with cancers driven by inflammation. Many of the features associated with the development of inflammation in rheumatoid arthritis (RA) parallel those evident in cancer progression. However, the role of this isoform in RA has not yet been explored. The aim of this study was to determine whether Δ133p53β is driving aggressive disease in RA.MethodsUsing RA patient synovia, we carried out RT-qPCR and RNAScope-ISH to determine both protein and mRNA levels of Δ133p53 and p53. We also used IHC to determine the location and type of cells with elevated levels of Δ133p53β. Plasma cytokines were also measured using a BioPlex cytokine panel and data analysed by the Milliplex Analyst software.ResultsElevated levels of pro-inflammatory plasma cytokines were associated with synovia from RA patients displaying extensive tissue inflammation, increased immune cell infiltration and the highest levels of Δ133TP53 and TP53β mRNA. Located in perivascular regions of synovial sub-lining and surrounding ectopic lymphoid structures (ELS) were a subset of cells with high levels of CD90, a marker of ‘activated fibroblasts’ together with elevated levels of Δ133p53β.ConclusionsInduction of Δ133p53β in CD90+ synovial fibroblasts leads to an increase in cytokine and chemokine expression and the recruitment of proinflammatory cells into the synovial joint, creating a persistently inflamed environment. Our results show that dysregulated expression of Δ133p53β could represent one of the early triggers in the immunopathogenesis of RA and actively perpetuates chronic synovial inflammation. Therefore, Δ133p53β could be used as a biomarker to identify RA patients more likely to develop aggressive disease who might benefit from targeted therapy to cytokines such as IL-6.

340 The Effects of PTSD-Dependent Neurogenic Hypertension and Inflammation on Thoracic Aortic Aneurysm Progression

OBJECTIVES/GOALS: Nearly all thoracic aortic aneurysm patients suffer from hypertension leading to elevated wall tension and abnormal extracellular matrix remodeling. PTSD patients have higher blood pressure both at rest and in response to stimuli. Although stress is associated with cardiovascular disease, the exact mechanism linking the two is still unknown. METHODS/STUDY POPULATION: Adult C57BL/6 mice underwent a PTSD induction protocol consisting of inescapable foot shock followed by single prolonged stress. The mice were assessed incrementally for their PTSD-like phenotype using specific behavioral tests chosen to assess for each of the human criteria of PTSD according to the DSM-V. Tail cuff blood pressure measurements were taken serially throughout the 16-week protocol. At terminal study, thoracic aortic diameter measurements were obtained through digital microscopy and plasma was harvested for cytokine analysis. Thoracic aortic aneurysms (TAA) were induced through periadventitial application of a calcium chloride solution on the descending thoracic aorta in BPH/2J and BPN/3J adult mice. The thoracic aortic diameter was measured at terminal study through digital microscopy. RESULTS/ANTICIPATED RESULTS: Using our PTSD-like mouse model we have demonstrated that PTSD-like mice have significantly higher systolic blood pressure following a reminder of the traumatic event than control mice recapitulating the human phenotype. They also had increased plasma proinflammatory cytokines and larger thoracic aortic diameters than control mice. Although the increased thoracic aortic diameter is not an aneurysm, it suggests ECM remodeling is occurring predisposing the aorta to aneurysm formation. Finally, we have shown that in neurogenic hypertensive mice, TAA formation was accelerated by 12 weeks with roughly 70% dilation at 4 weeks post-TAA induction surgery as compared to roughly a 20% dilation in control mice. DISCUSSION/SIGNIFICANCE: Altogether, these studies reinforce the link between stress and TAA development, and our mouse model will allow for the underlying mechanism to be elucidated. Better understanding of the mechanism linking PTSD and TAA will allow for the creation of novel therapeutics to treat PTSD symptoms while also delaying TAA progression.

Prospective study on Maresin-1 and cytokine levels in medication-naïve adolescents with first-episode major depressive disorder.

Inflammation and immune activation may play a role in the pathological mechanism of Major Depressive Disorder (MDD). Evidence from cross-sectional and longitudinal studies of adolescents and adults has shown that MDD is associated with increased plasma pro-inflammatory cytokines (e.g., IL-1β, IL-6). It has been reported that Specialized Pro-resolving Mediators (SPMs) mediate inflammation resolution, and Maresin-1 can activate the process of inflammation and promote inflammation resolution by promoting macrophage phagocytosis. However, no clinical studies have been conducted to evaluate the relationship between the levels of Maresin-1 and cytokine and the severity of MDD symptomatology in adolescents. 40 untreated adolescent patients with primary and moderate to severe MDD and 30 healthy participants as the healthy control (HC) group aged between 13 and 18 years old were enrolled. They received clinical and Hamilton Depression Rating Scale (HDRS-17) evaluation and then, blood samples were collected. Patients in the MDD group were re-evaluated for HDRS-17, and blood samples were taken after a six to eight-week fluoxetine treatment. The adolescent patients with MDD had lower serum levels of Maresin-1 and higher serum levels of interleukin 6 (IL-6) compared with the HC group. Fluoxetine treatment alleviated depressive symptoms in MDD adolescent patients, which was reflected by higher serum levels of Maresin-1 and IL-4 and lower HDRS-17 scores, serum levels of IL-6, and IL-1β. Moreover, the serum level of Maresin-1 was negatively correlated with the depression severity scores on the HDRS-17. Adolescent patients with primary MDD had lower levels of Maresin-1 and higher levels of IL-6 compared with the HC group, implying that the peripheral level of pro-inflammatory cytokines may be elevated in MDD, resulting in the insufficiency of inflammation resolution. The Maresin-1 and IL-4 levels increased after anti-depressant treatment, whereas IL-6 and IL-1β levels decreased significantly. Moreover, Maresin-1 level negatively correlated with depression severity, suggesting that reduced levels of Maresin-1 promoted the progression of MDD.

Repeated injection of multivitamins and multiminerals during the transition period enhances immune response by suppressing inflammation and oxidative stress in cows and their calves.

Periparturient dairy cows undergo major physiological and metabolic changes as well as immunosuppression, associated with decrease in plasma concentrations of various minerals and vitamins. The present study was conducted to investigate effects of repeated injections of vitamins and minerals on oxidative stress, innate and adaptive immune response in periparturient dairy cows and their offspring. Experiment was carried out on 24 peripartum Karan-Fries cows, randomly divided into four groups (n=6): control, Multi-mineral (MM), Multi-vitamin (MV) and Multi-minerals and Multi-vitamin (MMMV). Five ml of MM (Zinc 40 mg/ml, Manganese 10 mg/ml, Copper 15 mg/ml, Selenium 5 mg/ml) and five ml of MV (Vitamin E 5 mg/ml, Vitamin A 1000 IU/ml, B-Complex 5 mg/ml, and Vitamin D3 500 IU/ml) were injected intramuscularly (IM) to the MM and MV groups. MMMV group cows were injected with both. In all treatment groups, injections and blood sampling were carried out on 30th, 15th, 7th days before and after expected date of parturition and at calving. In calves, blood was collected at calving and on 1, 2, 3, 4, 7, 8, 15, 30 and 45 days post-calving. Colostrum/milk were collected at calving and at days 2, 4, and 8 post-calving. A lower percentage of total neutrophils and immature neutrophils, higher percentage of lymphocytes together with increased phagocytic activity of neutrophils and proliferative capacity of lymphocytes found in blood of MMMV cows/calves. Lower relative mRNA expression of TLRs and CXCRs and higher mRNA expression of GR-α, CD62L, CD11b, CD25 and CD44 found in blood neutrophils of MMMV groups. Total antioxidant capacity was higher, activity of antioxidant enzymes (SOD and CAT), TBARS levels were lower in the blood plasma of treated cows/calves. In both cows/calves, plasma pro-inflammatory cytokines (IL-1α, IL-1β, IL-6, IL-8, IL-17A, IFN-γ and TNF-α) increased, whereas anti-inflammatory cytokines (IL-4 and IL-10) decreased in MMMV groups. Total immunoglobulins increased in colostrum/milk of MMMV injected cows and plasma of their calves. Results indicate that repeated injections of multivitamins and multiminerals to peripartum dairy cows could be a major strategy to improve immune response and decrease in inflammation and oxidative stress in transition dairy cows and their calves.

Associations Between Inflammatory Mediators and Bone Outcomes in Postmenopausal Women: A Cross-Sectional Analysis of Baseline Data from the Prune Study.

Hypoestrogenism triggers increased production of inflammatory mediators, which contribute to bone loss during postmenopausal osteoporosis. This study aimed to investigate the association between circulating inflammatory markers and bone outcomes in postmenopausal women. We conducted a cross-sectional, secondary analysis of baseline data from participants who completed a 12-month randomized controlled trial, The Prune Study (NCT02822378), which included healthy postmenopausal women (n=183, 55-75 years old) with bone mineral density (BMD) T-score between 0.0 and -3.0 at any site. BMD was measured using dual-energy X-ray absorptiometry, and bone geometry and strength were measured using peripheral quantitative computed tomography. Blood was collected at baseline to measure (1) serum biomarkers of bone turnover, including procollagen type 1 N-terminal propeptide (P1NP) and C-terminal telopeptide and (2) inflammatory markers, including serum high-sensitivity C-reactive protein (hs-CRP) and plasma pro-inflammatory cytokines, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-8, and monocyte chemoattractant protein (MCP)-1, using enzyme-linked immunosorbent assay. The associations between bone and inflammatory outcomes at baseline were analyzed using correlation and regression analyses. Serum hs-CRP negatively correlated with P1NP (r=-0.197, p=0.042). Plasma IL-1β, IL-6, IL-8, and TNF-α negatively correlated with trabecular bone score at the lumbar spine (all p<0.05). In normal-weight women, plasma IL-1β, IL-6, and IL-8 negatively correlated (p<0.05) with trabecular and cortical bone area, content, and density at various sites in the tibia and radius. Serum hs-CRP positively predicted lumbar spine BMD (β=0.078, p=0.028). Plasma IL-6 negatively predicted BMD at the total body (β=-0.131, p=0.027) and lumbar spine (β=-0.151, p=0.036), whereas plasma TNF-α negatively predicted total hip BMD (β=-0.114, p=0.028). At baseline, inflammatory markers were inversely associated with various estimates of bone density, geometry, and strength in postmenopausal women. These findings suggest that inflammatory markers may be an important mediator for postmenopausal bone loss.

Severe primary graft dysfunction of the heart transplant is associated with increased plasma and intragraft proinflammatory cytokine expression

Heart transplant results have constantly improved but primary left ventricle graft dysfunction (LV-PGD) remains a devastating complication early after transplantation. Donor and recipient systemic inflammatory response may be involved in immune activation of the transplant, and LV-PGD development. Here, we investigated donor and recipient plasma and intragraft cytokine profiles preoperatively and during LV-PGD and searched for predictive markers for LV-PGD. Donor and recipient plasma samples (n=74) and myocardial biopsies of heart transplants (n=64) were analyzed. Plasma and intragraft cytokine levels were determined by multiplexed and next-generation sequencing platforms, respectively. The development of LV-PGD during the first 24 hours, and graft function and mortality up to 1 year after transplantation, were examined. Severe LV-PGD, but not mild or moderate LV-PGD, was significantly associated with early mortality, plasma high-sensitivity troponin elevation, and an increase in intragraft and plasma proinflammatory cytokines during reperfusion. Preoperative donor and recipient plasma cytokine levels failed to predict LV-PGD. Cytokine network analysis identified interleukins -6, -8, -10, and -18 as key players during reperfusion. Prolonged cold and total ischemia time, and increased need for red blood cell transfusions during operation were identified as clinical risk factors for severe LV-PGD. Severe LV-PGD was associated with a poor clinical outcome. Donor and recipient plasma cytokine profile failed to predict LV-PGD, but severe LV-PGD was associated with an increase in post-reperfusion intragraft and recipient plasma proinflammatory cytokines. Identified key cytokines may be potential therapeutic targets to improve early and long-term outcomes after heart transplantation.

6-gingerol ameliorates weight gain and insulin resistance in metabolic syndrome rats by regulating adipocytokines

Metabolic syndrome (MetS) can lead to increase of insulin resistance (IR) and visceral adipose tissue production of adipocytokines. 6-gingerol is known to have antioxidant and anti-inflammatory activities. Aim of this study is to investigate the effects of 6-gingerol on high-fat high-fructose (HFHF) diet-induced weight gain and IR in rats through modulation of adipocytokines. To induce MetS, male Sprague-Dawley rats were fed with a HFHF diet for 16 weeks and at Week 8, single-dose low-dose streptozotocin (22 mg/kg) were intraperitoneally injected. After 8 weeks of HFHF diet feeding, the rats were treated orally with 6-gingerol (50, 100, and 200 mg/kg/day) once daily for 8 weeks. At the end of the study, all animals were terminated, serum, liver, and visceral adipose tissues were harvested for biochemical analysis including the measurements of total cholesterol, triglycerides, HDL-cholesterol, fasting plasma glucose, insulin, leptin, adiponectin, proinflammatory cytokines (TNF-α and IL-6) and liver and adipose tissue histopathology. Biochemical parameters namely serum total cholesterol (243.7 ± 127.6 vs 72.6 ± 3 mg/dL), triglycerides (469.2 ± 164.9 vs 49.3 ± 6.3 mg/dL), fasting plasma glucose (334 ± 49.5 vs 121 ± 8.5 mg/dL), HOMA-IR (0.70 ± 0.24 vs 0.32 ± 0.06), and leptin (6.19 ± 1.24 vs 3.45 ± 0.33 ng/mL) were significantly enhanced, whereas HDL-cholesterol (26.2 ± 5.2 vs 27.9 ± 1.1 mg/dL) and adiponectin level (14.4 ± 5.5 vs 52.8 ± 10.7 ng/mL) were lowered in MetS vs normal control. Moreover, MetS were marked a significant increase in body weight and proinflammatory cytokines. Treatment with 6-gingerol dose-dependently restored all of those alterations towards normal values as well as the accumulation of lipid in liver and adipose tissues. These findings demonstrate that 6-gingerol, in a dose-dependent mode, showed capability of improving weight gain and IR in MetS rats through modulation of adipocytokines.

Metformin Mitigates Memory Impairment of Diabetic Mice through Modulation of Plasma Pro-inflammatory Cytokines and Aβ1-42 Levels

Abstract Experimental and clinical studies reported that type 2 diabetes mellitus (T2DM) is associated with cognitive dysfunction and promotes the onset of dementia. Metformin is an antihyperglycemic drug used for the treatment of T2DM. A growing number of evidence revealed neuroprotective, antioxidant, and anti-inflammation effects exerted by metformin. The present study aimed to investigate the effect of metformin on cognitive function, systemic proinflammatory cytokines and amyloid-beta 1-42 (Aβ1-42) which is a pathological hallmark of Alzheimer’s disease (AD) in diabetic mice. C57BL/6N mice were divided into the following experimental groups: normal control group (NC); diabetes mellitus group (DM) induced by a high-fat diet combined with streptozotocin (STZ) injection; diabetes mellitus treated with metformin 100 mg/kg (DM+Met). Cognitive performance was evaluated by the novel object recognition test (NORT). Systemic proinflammatory cytokines and Aβ1-42 were assessed by the enzyme-linked immunosorbant assay (ELISA) test. We found that diabetic mice exhibited cognitive impairment in NORT whereas the treatment with metformin restored the cognitive function of diabetic mice. Moreover, diabetic mice presented an increase in plasma IL-6 and TNF-α levels while Aβ1-42 was decreased when compared to NC mice. Nevertheless, the administration of metformin allowed the levels of plasma IL-6, TNF-α, and Aβ1-42 to normalize in diabetic mice. Taken together, our findings suggest that metformin improves the cognitive function of diabetic mice possibly via the modulation of plasma pro-inflammatory cytokines and Aβ1-42 levels. Metformin may potentially be used as a therapeutic agent for patients with T2DM who show cognitive deficits. Keywords: Diabetes mellitus, Cognitive impairments, Pro-inflammatory cytokines, Amyloid-beta, Metformin

Dual Inhibition of NF-Kb and JAK/STAT Pathways Reverts Myelofibrosis-like Phenotype in an In Vivo Murine Model

Introduction: In JAK-STAT-mediated inflammatory cytokine production in myeloproliferative neoplasms (MPN), both malignant and non-malignant clones contribute. The NF-κB pathway is a mediator of inflammation-induced carcinogenesis. We have previously demonstrated the role of miR-146a, a NF-κB repressor through IRAK1/TRAF6, in the progression of MPN. Indeed, the rs2431697 TT genotype, associated with lower miR-146a levels, contributes to early progression to secondary myelofibrosis (MF); and miR-146a-/- (KO) mice, with MF-like phenotype, show an increase in p-Stat3 with aging (Ferrer-Marin F. et al. Leukemia 2020). Here, we postulate that in this inflammation-driven MF murine model, inhibition of both NF-κB and JAK/STAT pathways can synergistically dampen the inflammatory state and reverse the MF phenotype. Methods: 11-month-old miR-146a-/- micewere 1-month-oral ad libitum treated with either ruxolitinib (JAK1/2 inhibitor) at 75 mg/kg/day, BMS-345541 (BMS) (NF-κB pathway inhibitor at IKKα/β level) at 100 mg/kg/day, both inhibitors at the same concentrations, or pacritinib (JAK2 and NF-κB pathway inhibitor at IRAK1 level) at 150 mg/kg/day. Untreated KO and wild-type (WT) mice were used as controls (n=7-15 mice/group). We evaluated: cell counts in peripheral blood; proinflammatory plasma cytokines by Luminex; size, weight and myeloid (CD11b) and lymphoid (CD19) populations by flow cytometry in spleen; histology of spleen and bone marrow (BM) by hematoxylin-eosin and reticulin staining. SET2 cells were used as an in vitro human model of JAK2V617F clonal hematopoiesis for immunoblotting quantification of pTyrSTAT3 (JAK/STAT pathway) and IRAK1 and nuclear p-p65 (NF-κB pathway). Results: As expected, compared to WT mice, KO mice exhibited monocytopenia, anemia and thrombocytopenia (Table 1), splenomegaly and severe reticulin fibrosis with osteosclerosis in BM (Fig.1). Moreover, in line with their inflammatory phenotype, plasma IL6, IL1β and TNFα levels were elevated in KO (vs WT) mice (Table 1). Besides splenomegaly, KO mice had spleen architecture loss, decreased white pulp and increased megakaryocytes (Fig.1), as a sign of extramedullary hematopoiesis. Consistent with histology, at a cellular level, KO mice showed significantly increased myeloid (CD11b+) and decreased lymphoid (CD19+) populations in the spleen. NF-κB pathway inhibitors (BMS and pacritinib) improved the thrombocytopenia, but only pacritinib improved monocyte counts without worsening anemia; by contrast, ruxolitinib did not improve monocytes or platelet counts, and the combination of ruxolitinib/BMS worsened anemia (Table 1). Regarding the proinflammatory cytokines, IL6 levels were significantly decreased with ruxolitinib; IL1β levels decreased whenever NF-κB pathway was inhibited, although it only showed significance with BMS; and TNFα levels significantly decreased with all treatments, with only a tendency for ruxolitinib/BMS (Table 1). Although all treatments decreased splenomegaly and partially recovered spleen architecture, only pacritinib and BMS (NF-κB inhibitors) diminished splenic megakaryocytic counts (Fig.1). NF-κB inhibitors [pacritinib and BMS (+/- ruxolitinib)] decreased the number of mice with severe reticulin fibrosis or moderate/severe osteosclerosis in the BM, whereas ruxolitinib alone did not have this effect. Unlike pacritinib, however, the combination of ruxolitinib/BMS induced BM aplasia (Fig.1). Finally, in SET2 cells, monotherapy with ruxolitinib or BMS inhibited JAK-STAT or NF-κB signaling pathways, respectively, but only pacritinib and the combination were able to inhibit both pathways. Conclusions: miR-146a-/- mice constitute a useful model of MPN without driver mutations to evaluate the role of inflammation in these neoplasms. The dual inhibition of NF-κB and JAK/STAT with pacritinib reduced inflammatory cytokines, reverted monocytopenia and thrombocytopenia, and improved BM fibrosis without worsening anemia. While some of these benefits could be recapitulated in vivo by combining ruxolitinib with an NF-κB inhibitor, this combination resulted in worsening anemia and BM aplasia. These results highlight the therapeutic possibilities of dual JAK/NF-κB inhibitors, such as pacritinib, in patients with MF. Funding: PI18/00316; PI20/00136; PI21/00347; 20644/JLI/18; FPU18/03189. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Synthesis and antidepressant-like effects of new 5-epi-incensole and 5-epi- incensole acetate in chronic unpredictable mild stress model of depression; behavioural and biochemical correlates

In the current investigation, 5-epi-incensole (3) and 5-epi-incensole acetate (5) were synthesized from the most potent anti-depressant constituents incensole (1) and incensole acetate (2) of Boswellia papyrifera Hochst. The resulting compounds were evaluated for their ability to ameliorate depressive symptoms in forced swim test (FST) and tail suspension test (TST) in chronic unpredictable mild stress (CUMS) induced depression paradigm. The results demonstrated that compounds 3 and 5 at the doses of 1 and 3 mg/kg administered for 28 days, significantly reduced the immobility time in FST and TST and were devoid of any effect on locomotor activity in the open field test (OFT). Both compounds 3 and 5 also reversed CUMS-induced reduction in the weight of animals and aversion in sucrose preference. The tested compounds also inhibited Monoamine oxidase-A (MAO) enzyme and increased the levels of brain noradrenaline (NA) and 5-Hydroxytryptamine (5-HT), decreased plasma corticosterone and pro-inflammatory cytokines including TNF-α, IL-6 in hippocampal homogenates. Compounds 3 and 5 also significantly reduced the increased lipid peroxidation and nitrite levels; decreased glutathione levels, and catalase activities in mice undergoing CUMS protocol. The binding mode of compounds 3 and 5 was predicted at the monoamine oxidase substrate binding site by molecular docking having docking scores of > −6 kcal/mol. Taken together these data revealed that compounds 3 and 5 exerted antidepressant-like effects in chronic unpredictable mild stress-induced depression paradigm and are likely mediated via modulating the central oxidative stress, MAO-A activity with a consequent increase in brain NA and 5-HT levels in inflammatory pathways.

Positive Association of Plasma Trimethylamine-N-Oxide and Atherosclerosis in Patient with Acute Coronary Syndrome.

Atherosclerosis is the major cause of acute coronary syndrome (ACS) which is a significant contributor to both morbidity and mortality in the world. The microbiome-derived metabolite trimethylamine-N-oxide (TMAO) has aroused great interest and controversy as a risk factor of atherosclerosis. Therefore, in this study, we aimed at investigating whether plasma TMAO can be a risk factor of atherosclerosis in coronary artery of patients with ACS and how this relates to lipids and proinflammatory cytokines in plasma. We enrolled consecutive patients with ACS who underwent percutaneous coronary intervention (PCI). Gensini scoring was used to evaluate angiographic atherosclerosis in the coronary artery of the patients. 13 patients were divided into low (Gensini score < 25), 33 into intermediate (Gensini score 25-50), and 81 into severe atherosclerosis (Gensini score ≥50). Plasma TMAO, vasculitis factors, and cardiovascular biomarkers were measured by clinical biochemistry, intima-media thickness (IMT) of carotid artery was determined by the Color Doppler ultrasound, and the atherosclerotic lesion in coronary artery was assessed in PCI. Plasma TMAO concentrations were positively associated with Gensini score (OR = 0.629, p < 0.001) and Gensini subgroup (R = 0.604, p < 0.001). Plasma TMAO concentrations in patients with severe coronary atherosclerosis were higher than those of patients with moderate coronary atherosclerosis, and the plasma TMAO concentrations of patients with moderate coronary atherosclerosis were higher than those of patients with mild coronary atherosclerosis, the difference was statistically significant [4.73 (3.13, 4.62) versus 1.13 (0.63, 3.34) versus 0.79 (0.20, 1.29), p < 0.001], respectively. Furthermore, ROC analysis showed that plasma TMAO could identify the severity of atherosclerosis (p < 0.001). The AUC of TMAO for severe atherosclerosis was 0.852 (95%CI = 0.779 - 0.925). The sensitivity and specificity of TMAO for identifying severe atherosclerosis are 96.3% and 63.0% when the cut-off value of TMAO was set at 1.2715 pg/ml. Furthermore, logistic regression analysis showed plasma TMAO concentrations were positively associated with severity of atherosclerosis in coronary artery (OR = 1.934, 95%CI = 1.522 - 2.459, p < 0.001). For all that, negatively association was observed between TMAO and age (OR = -0.224, p < 0.05), B-type natriuretic peptide (BNP) (OR = -0.175, p < 0.05), and interleukin-8 (IL-8) (OR = -0.324, p < 0.001), while positive association was observed between TMAO and nitric oxide (NO) (OR = 0.234, p < 0.01). However, there is no obvious association was observed between Gensini score and cardiovascular biomarkers, vasculitis factors, and carotid IMT, respectively. Our cross-sectional observation suggested that plasma TMAO concentrations positively associated with coronary atherosclerosis in ACS patients and serve as a risk factor for severe atherosclerosis. Plasma TMAO also correlated with age, BNP, IL-8, and NO. However, no obvious association was found between atherosclerosis with vasculitis factors and cardiovascular biomarkers in this study, and there was no conclusive evidence showing TMAO enhance atherosclerosis via regulation of inflammation or lipid.

Synergism between Extracts of Garcinia mangostana Pericarp and Curcuma in Ameliorating Altered Brain Neurotransmitters, Systemic Inflammation, and Leptin Levels in High-Fat Diet-Induced Obesity in Male Wistar Albino Rats.

This study aims to explore the effects of Garcinia mangostana (mangosteen) and Curcuma&amp;nbsp;longa independently and synergistically in modulating induced inflammation and impaired brain neurotransmitters commonly observed in high-fat diet-induced obesity in rodent models. Male albino Wistar rats were divided into four experimental groups. Group I, control, obese, fed on a high-fat diet (HFD), and Group II-IV, fed on HFD then given mangosteen extract (400 mg/kg/day) and/or Curcuma (80 mg/kg/day), or a mixture of both for 6 weeks. Plasma pro-inflammatory cytokines, leptin, and brain serotonin, dopamine, and glutamate were measured in the five studied groups. G. mangostana and Curcuma longa extracts demonstrate antioxidant and DPPH radical scavenging activities. Both induced a significant reduction in the weight gained, concomitant with a non-significant decrease in the BMI (from 0.86 to 0.81 g/cm2). Curcuma either alone or in combination with MPE was more effective. Both extracts demonstrated anti-inflammatory effects and induced a significant reduction in levels of both IL-6 and IL-12. The lowest leptin level was achieved in the synergistically treated group, compared to independent treatments. Brain dopamine was the most affected variable, with significantly lower levels recorded in the Curcuma and synergistically treated groups than in the control group. Glutamate and serotonin levels were not affected significantly. The present study demonstrated that mangosteen pericarp extract (MPE) and Curcuma were independently and in combination effective in treating obesity-induced inflammation and demonstrating neuroprotective properties.

Rifaximin Modifies Gut Microbiota and Attenuates Inflammation in Parkinson's Disease: Preclinical and Clinical Studies.

Patients with Parkinson's disease (PD) exhibit distinct gut microbiota, which may promote gut-derived inflammation. Rifaximin is a nonabsorbable antibiotic that can modify gut microbiota. The present study investigated the effect of rifaximin on gut microbiota and inflammation status in PD. The study examined the effect of long-term rifaximin treatment on in vivo transgenic PD mice (MitoPark) and short-term rifaximin treatment on patients with PD. Rifaximin treatment caused a significant change in gut microbiota in the transgenic PD mice; in particular, it reduced the relative abundance of Prevotellaceae UCG-001 and increased the relative abundance of Bacteroides, Muribaculum, and Lachnospiraceae UCG-001. Rifaximin treatment attenuated serum interleukin-1β, interleukin-6 and tumor necrosis factor-α, claudin-5 and occludin, which indicated the reduction of systemic inflammation and the protection of the blood-brain barrier integrity. The rifaximin-treated MitoPark mice exhibited better motor and memory performance than did the control mice, with lower microglial activation and increased neuronal survival in the hippocampus. In the patients with PD, 7-day rifaximin treatment caused an increase in the relative abundance of Flavonifractor 6 months after treatment, and the change in plasma proinflammatory cytokine levels was negatively associated with the baseline plasma interleukin-1α level. In conclusion, the present study demonstrated that rifaximin exerted a neuroprotective effect on the transgenic PD mice by modulating gut microbiota. We observed that patients with higher baseline inflammation possibly benefited from rifaximin treatment. With consideration for the tolerability and safety of rifaximin, randomized controlled trials should investigate the disease-modification effect of long-term treatment on select patients with PD.

Can we attenuate ischaemia-reperfusion injury of allografts in a porcine left lung transplant models by adsorption of cytokines?

Primary graft dysfunction resulting from ischaemia-reperfusion injury remains a major obstacle after lung transplantation (LTx) and is associated with morbidity and mortality. Continuous release of inflammatory cytokines, due to the process of ischaemia and reperfusion, triggers a complex cascade of apoptosis and necrosis resulting in graft dysfunction. Previous studies demonstrated successful graft improvement by cytokine filtration during ex vivo lung perfusion. We hypothesize that plasma cytokine filtration with CytoSorb® during in vivo graft perfusion immediately after implantation may attenuate ischaemia-reperfusion injury after left LTx in a porcine model. Left porcine LTx was performed with allografts preserved for 24 h at 4°C. In the treatment group [T] (n = 7), a veno-venous shunt was created to insert the cytokine filter (CytoSorbents, Berlin, Germany). In the sham group [S] (n = 4), the shunt was created without the filter. Haemodynamic parameters, lung mechanics, blood gases and plasma cytokines were assessed during 6 h in vivo reperfusion. During 6 h of reperfusion, significant differences in plasma pro-inflammatory cytokine [interferon (IFN)-α, IFN-γ and interleukin (IL)-6] concentrations were observed between [T] and [S], but surprisingly with higher plasma levels in the [T] group. Plasma concentrations of other pro-inflammatory cytokines (IL-1β, IL-12p40, IL-4, IL-6, IL-8, IFN-α, IFN-γ and tumour necrosis factor-α) and anti-inflammatory cytokines (IL-10) did not find any evidence for a difference. Furthermore, our study failed to show meaningful difference in haemodynamics and blood gases. Also, no statistically significant differences were found between [T] and [S] in biopsies and wet-to-dry ratio at the end of the experiment. In our porcine left LTx model cytokine filtration did not achieve the intended effect. This is in contrast to previous studies with CytoSorb use during ex vivo lung perfusion as a surrogate LTx model. Our findings might highlight the fact that the theoretical benefit of inserting an additional cytokine adsorber to improve graft function in clinical practice should be critically evaluated with further studies.

Alterations of Plasma Pro-Inflammatory Cytokine Levels in Children with Refractory Epilepsies.

Paediatric epilepsy is a multifaceted neurological disorder with various aetiologies. Up to 30% of patients are considered drug-resistant. The background impact of interfering inflammatory and neuronal pathways has been closely linked to paediatric epilepsy. The characteristics of the inflamed state have been described not only in epilepsies, which are considered prototypes of an inflammatory pathophysiology, but also in patients with drug-resistant epilepsy, especially in epileptic encephalopathies. The imbalance of different cytokine levels was confirmed in several epileptic models. Chemokines are new targets for exploring neuroimmune communication in epileptogenesis, which control leukocyte migration and have a possible role in neuromodulation. Additionally, prostaglandin E2 (PGE2) is an important effector molecule for central neural inflammatory responses and may influence drug responsiveness. We measured the serum interictal quantitative levels of chemokines (CCL2, CCL4, CCL11) and PGE2 in correlation with the seizure frequency and severity in controlled and intractable childhood epilepsies. Our refractory seizure group demonstrated significantly increased concentrations of eotaxin (CCL11) compared to the controlled epilepsy group. The higher level of CCL11 was correlated with an increased seizure frequency, while the PGE2 levels were associated with the severity of seizure and epilepsy, supporting the findings that proinflammatory cytokines may contribute to epileptogenesis and possibly have a role in developing seizure resistance.

Immunologic effects of red blood cell and platelet transfusions in neonates.

Premature neonates are frequently transfused red blood cells (RBCs) or platelets to raise hemoglobin or platelet counts. However, these transfusions may have unintended effects on the immune system. This review will summarize the newest discoveries on the immunologic effects of RBC and platelet transfusions in neonates, and their potential impact on neonatal outcomes. Neonatal RBC transfusions are associated with increases in plasma pro-inflammatory cytokines, but recent findings suggest sex-specific differential responses. At least one cytokine (monocyte chemoattractant protein-1) rises in females receiving RBC transfusions, but not in males. These inflammatory responses correlate with poorer neurodevelopmental outcomes in heavily transfused female infants, while preterm male infants seem to be more sensitive to severe anemia. Platelet transfusions in preterm neonates are associated with increased neonatal mortality and morbidity. The underlying mechanisms are unknown, but likely related to the immune/inflammatory effects of transfused platelets. Adult platelets are different from neonatal platelets, with the potential to be more pro-inflammatory. Early preclinical data suggest that platelet transfusions alter the neonatal systemic inflammatory response and enhance immune cell migration. RBC and platelet transfusions alter neonatal immune and inflammatory responses. Their pro-inflammatory effects might worsen neonatal disease or affect neurodevelopmental outcomes.