MCP-1 Levels Research Articles

Functional characterization of the immunomodulatory properties of human urine-derived stem cells.

BackgroundUrine-derived stem cells (USCs) have been widely researched as a novel cell source for stem cell therapy, but their immunomodulatory characteristics remain to be investigated. This study aimed to characterize the immunomodulatory properties of human USCs.MethodsHuman USCs were isolated from fresh voiding urine samples from healthy male donors and expanded. Their cell surface markers were characterized by flow cytometry analysis and the telomerase activities for several USCs clones were determined. The immunosuppressive potential of USCs was evaluated by the performing the mixed lymphocyte reaction (MLR) [co-culture with peripheral blood mononuclear cells (PBMNCs)] and natural killer cells (NK) cytotoxicity assay. USCs cytokines release profile was determined by using human cytokine proteome array.ResultsUSCs exhibited high cell surface expression of embryonic/mesenchymal stem cells (MSCs) markers CD29, CD44, CD54, CD73, CD90, CD146, and CD166, while lacked expression of hematopoietic stem cell markers CD11, CD14, CD19, CD31, CD34, CD45, B cell marker CD79, and co-stimulatory factors CD80 and CD86, thus, exhibiting the phenotype of MSCs. MLR indicated that USCs significantly inhibited the proliferation of PBMNCs, as compared to that of the human smooth muscle cells (SMCs). In cell cytotoxicity assays, NK cells displayed less cytotoxicity against USCs than against bone marrow mesenchymal stem cells (BMSCs) and SMCs. Furthermore, upon PBMNCs stimulation, USCs secreted higher levels of immunomodulatory cytokines, including IL-6, IL-8, MCP-1, RANTES, GROα, and GM-CSF, compared to those of BMSCs, especially when directly contact mix-culture with PBMNCs.ConclusionsUSCs secreted immunoregulatory cytokines and possessed immunomodulatory properties, comparable to those of BMSCs.

Abstract 40: SARS-CoV-2/ACE2 Induces Vascular Inflammatory Responses In Human Microvascular Endothelial Cells Independently Of Viral Replication

SARS-CoV-2, the virus responsible for COVID19, binds to ACE2, via its spike protein S1 subunit, leading to viral infection and respiratory disease. COVID-19 is associated with cardiovascular disease and systemic inflammation. Since ACE2 is expressed in vascular cells we questioned whether SARS-CoV-2 induces vascular inflammation and whether this is related to viral infection. Human microvascular endothelial cells (EC) were exposed to recombinant S1p (rS1p) 0.66 μg/mL for 10 min, 5h and 24h. Gene expression was assessed by RT-PCR and levels of IL6 and MCP1, as well as ACE2 activity, were assessed by ELISA. Expression of ICAM1 and PAI1 was assessed by immunoblotting. ACE2 activity was blocked by MLN4760 (ACE2 inhibitor) and siRNA. Viral infection was assessed by exposing Vero E6 (kidney epithelial cells; pos ctl) and EC to 10 5 pfu of SARS-CoV-2 where virus titre was measured by plaque assay. Co-IP coupled mass spectrometry protein identification and label free proteomics were used to investigate ACE2-mediated signalling. rS1p increased IL6 mRNA (14.2±2.1 vs. C:0.61±0.03 2^-ddCT) and levels (1221.2±18.3 vs. C:22.77±3.2 pg/mL); MCP1 mRNA (5.55±0.62 vs. C:0.65±0.04 2^-ddCT) and levels (1110±13.33 vs. C:876.9±33.4 pg/mL); ICAM1 (17.7±3.1 vs. C:3.9±0.4 AU) and PAI1 (5.6±0.7 vs. C: 2.9±0.2), p<0.05. MLN4760, but not rS1p, decreased ACE2 activity (367.4±18 vs. C: 1011±268 RFU, p<0.05) and blocked rS1p effects on ICAM1 and PAI1. ACE2 siRNA blocked rS1p-induced IL6 release, ICAM1, and PAI1 responses as well as rS1p-induced NFκB activation. Proteomics analysis of the global effect of rS1, identified biological process enrichment of proteins from virus transcription and NFκB signalling. ACE2 Co-IP identified 216 interacting proteins (filtered with ≥1 unique peptide, 1% FDR), linked to cytokine production and inflammation. EC were not susceptible to SARS-CoV-2 infection, while the virus replicated well in Vero E6. In conclusion, we demonstrate that rS1p induces an inflammatory response through ACE2 in endothelial cells. These effects seem to be independent of viral infection. Our findings suggest that vascular inflammation in COVID-19 involves activation of ACE2-mediated pro-inflammatory signalling that may be unrelated to viral replication.

Regulatory role of Apelin-13-mediated PI3K/AKT signaling pathway in the glucose and lipid metabolism of mouse with gestational diabetes mellitus

ObjectiveTo investigate the regulatory mechanism of Apelin-13-mediated PI3K/AKT signaling pathway in the glucose and lipid metabolism of gestational diabetes mellitus (GDM) mouse. MethodsGDM mice models were established and treated with Apelin-13 and/or PI3K/AKT inhibitor LY294002. Then, the indicators of glucose and lipid metabolism and the levels of inflammatory factors were detected. Besides, the levels of indicators of oxidative stress in the placenta of mice were measured. Western blotting was also carried out to determine the expression of PI3K/AKT pathway-related proteins in all groups. ResultsIn comparison with the Control group, mice in the GDM group presented with the continuous increase in the level of FBG as the time went on, while FINS level decreased evidently. Besides, the fetus alive ratio in the GDM group was much lower with significant increased weight of fetal mouse and weight of placenta; the mice had significant decreased levels of IL-6, IL-1β, TNF-α and MCP-1, and in the placenta, the levels of SOD, GPx, GSH and CAT were also reduced evidently, with significant downregulation of p-PI3K/PI3K and p-AKT/AKT. However, indicators above in the GDM mice treated with Apelin-13 had significant improvement as compared to those in the GDM group, and the improvement was reversed by LY294002 treatment. ConclusionApelin-13, possibly by activating the PI3K/AKT pathway, could improve the glucose and lipid metabolism, reduce the damage caused by oxidative stress and inflammatory reaction, and protect the pancreas islet, thereby improving the pregnancy outcome of GDM mice.

Abstract P127: Myeloid-specific PKM2 Deletion Reduces Atherosclerosis By Limiting Inflammation

Introduction: The underlying cause of coronary artery disease (CAD) is atherosclerosis, which is a pathological response to chronic inflammation and hyperlipidemia. The onset of atherogenesis is characterized by infiltration of myeloid cells, including monocytes followed by fatty streak formation and progressive accumulation of smooth muscle cells (SMCs). These microenvironmental changes dictate the balance between inflammatory and anti-inflammatory macrophages. Pyruvate kinase M2 (PKM2), a glycolytic enzyme, is highly expressed in activated proinflammatory macrophages. The mechanistic role of PKM2 in atherosclerosis remains unknown. Hypothesis: We hypothesize that PKM2 promotes macrophage migration in response to MCP-1 and mediates atherosclerosis by increasing inflammation. Methods and Results: PKM2 was upregulated in macrophages of Ldlr -/- mice fed a high-fat "Western" diet compared with a control chow diet. We generated the novel myeloid cell-specific PKM2 fl/fl LysMCre +/- on a Ldlr-deficient background (PKM2 fl/fl LysMCre +/- Ldlr -/- ) and evaluated atherosclerosis after 14 weeks high-fat “Western” diet feeding. Controls were littermate PKM2 fl/fl LysMCre -/- Ldlr -/- mice. Myeloid cell-specific deletion of PKM2 led to a significant reduction in lesions in the whole aorta and aortic sinus despite high cholesterol and triglyceride levels. (P<0.05, n=10-12 mice/group). Furthermore, we found decreased macrophage content in the lesions of myeloid cell-specific PKM2 -/- mice compared with control mice that was associated with decreased plasma levels of pro-inflammatory cytokines, including MCP-1, and reduced transmigration of macrophages in response to MCP-1. Macrophages isolated from myeloid-specific PKM2 -/- mice fed a high-fat "Western" diet exhibited reduced expression of pro-inflammatory genes, including MCP-1, IL-1β, IL-12, and increased expression of the anti-inflammatory genes Arg1 and IL-10. Inhibiting PKM2 nuclear translocation in bone marrow-derived macrophages led to a significant reduction in MCP-1 and IL-1β levels and reduced transmigration of macrophages. Conclusion: Genetic deletion of PKM2 in myeloid cells reduces atherosclerosis by limiting inflammation.

Cytokine Profiles Associated With Worse Prognosis in a Hospitalized Peruvian COVID-19 Cohort.

Cytokines, chemokines and growth factors present different expression profiles related to the prognosis of COVID-19. We analyzed clinical parameters and assessed the expression of these biomarkers in patients with different disease severity in a hospitalized Peruvian cohort to determine those associated with worse prognosis. We measured anti-spike IgG antibodies by ELISA and 30 cytokines by quantitative suspension array technology in 123 sera samples. We analyzed differences between patients with moderate, severe and fatal COVID-19 by logistic regression at baseline and in longitudinal samples. Significant differences were found among the clinical parameters: hemoglobin, neutrophils, lymphocytes and C-reactive protein (CRP), creatinine and D-dimer levels. Higher anti-spike IgG antibody concentrations were associated to fatal patient outcomes. At hospitalization, IL-10, IL-6, MIP-1α, GM-CSF, MCP-1, IL-15, IL-5, IL1RA, TNFα and IL-8 levels were already increased in fatal patients´ group. Meanwhile, multivariable analysis revealed that increased GM-CSF, MCP-1, IL-15, and IL-8 values were associated with fatal outcomes. Moreover, longitudinal analysis identified IL-6 and MCP-1 as the main risk factors related to mortality in hospitalized COVID-19 patients. In this Peruvian cohort we identified and validated biomarkers related to COVID-19 outcomes. Further studies are needed to identify novel criteria for stratification of SARS-CoV-2 infected patients at hospital entry. BackgroundIn the most severe forms of SARS-CoV-2 infection, large numbers of innate and adaptive immune cells become activated and begin to produce pro-inflammatory cytokines, establishing an exacerbated feedback loop of inflammation.MethodsA total of 55 patients with laboratory-confirmed COVID-19 admitted to the Hospital Nacional Guillermo Almenara Irigoyen in Lima, Peru were enrolled during August-October 2020. Of these, 21 had moderate disease, 24 severe diseases and 10 died. We measured 30 cytokines and chemokines by quantitative suspension array technology and anti-spike IgG antibodies using a commercial ELISA. We evaluated these parameters in peripheral blood every 2-5 days until patient discharge or death. Patient information and clinical parameters related were obtained from the respective clinical histories.ResultsThe frequency of obesity differed among the 3 groups, being most frequent in patients who died. There were also significant differences in clinical parameters: hemoglobin, segmented neutrophils, lymphocytes,C-reactive protein, creatinine and D-dimer levels. Greater anti-spike IgG antibody concentrations were associated to fatal outcomes. In univariate analyses, higher baseline concentrations of IL-6, MIP-1α, GM-CSF, MCP-1, IL-15, IL-5, IL1RA, TNFα, IL-8 and IL-12p70 correlated with severity, while multivariable analysis showed that increased concentrations in 4 biomarkers (GM-CSF, MCP-1, IL-15, IL-8) were associated with fatal outcomes. Longitudinal analysis showed IL-6 (hazard ratio [HR] 6.81, 95% confidence interval [CI] 1.6-28.7) and MCP-1 (HR 4.61, 95%CI 1.1-19.1) to be related to mortality in hospitalized COVID-19 patients.ConclusionsCytokine, chemokine and growth factor profiles were identified and validated related to severity and outcomes of COVID-19. Our findings may be useful to identify novel criteria for COVID-19 patient stratification at hospital entry.

1-deoxynojirimycin alleviates liver fibrosis induced by type 2 diabetes in mice

To investigate the effect of 1-deoxynojirimycin (DNJ) for improving diabetic liver fibrosis and explore the underlying mechanism. Mouse models of type 2 diabetes were established in 10 Kunming mice by high-fat diet feeding for 8 weeks and intraperitoneal injection of STZ, with 5 mice receiving intraperitoneal injection of citrate buffer solution with normal feeding as the control group. The mouse models were randomized into two groups (n=5) for further highfat feeding (model group) and additional treatment with 10% DNJ in drinking water (200 mg · kg-1 per day; DNJ group) for 8 weeks. The mice were monitored for changes in body weight (BW), blood glucose, serum total cholesterol (TC), triglyceride (TG) and superoxide dismutase (SOD) levels. The pathological changes in the liver tissue were observed using HE and Sirius Red staining, and the solubility of collagens in the liver tissues was determined. The expression levels of MCP-1, TNF-α, IL-1β and TGF-β1 mRNA were detected with real-time PCR, and the protein expressions of α-SMA and collagen2 (ColA2) were determined with Western blotting. In the in vitro experiment, mouse fibroblasts L929 cells were pretreated with DNJ (10 μg/ mL) or PBS for 30 min followed by culture in high-glucose medium for 24 h, and the level of ROS production was measured using dihydroethidium (DHE) staining. In the mouse model of type 2 diabetes, DNJ treatment significantly lowered serum level of glucose, TC, and TG (P < 0.05) and increased serum SOD activity (P < 0.05). DNJ obviously attenuated liver fibrosis in the diabetic mice, as shown by alleviated cross-linking of collagens and reduced contents of pepsin-solubilized collagen (PSC) and total collagen (P < 0.05). DNJ treatment also significantly reduced the overexpression of the proinflammatory cytokines and fibrosis-related cytokines induced by diabetes (P < 0.05). In L929 cells exposed to high glucose, pretreatment with DNJ significantly lowered the intensity of red fluorescence in DHE staining. DNJ can attenuate type 2 diabetes-induced liver fibrosis in mice through its hypoglycemic, anti-inflammatory and anti-oxidative effects.

Nicotine self-administration with menthol and audiovisual cue facilitates differential packaging of CYP2A6 and cytokines/chemokines in rat plasma extracellular vesicles

In this study, we investigated whether intravenously self-administered nicotine with menthol and audiovisual cue modulates nicotine-metabolizing CYP2A6, oxidative stress modulators, and cytokines/chemokines in plasma extracellular vesicles (EVs) in rats. We assigned rats to self-administered nicotine with: (a) audiovisual cue (AV), (b) menthol, and (c) menthol and AV cue. We found increased levels of CD9 in plasma EVs after self-administered nicotine with menthol and AV cue. Moreover, expression of CYP2A6 in plasma EVs was significantly increased after self-administered nicotine in response to menthol and AV cue. However, despite an upward trend on SOD1 and catalase, increase was not found to be statistically significant, while total antioxidant capacity was found to be significantly increased in plasma and plasma EVs obtained after self-administered nicotine with menthol and AV cue. Among cytokine and chemokine profiling, we found a significant increase in the levels of MCP-1 after self-administered nicotine with menthol and AV cue and complete packaging of IL-1β in EVs. Taken together, the study provides evidence that nicotine in response to menthol and AV cues can package altered levels of CYP2A6, and cytokines/chemokines in plasma EVs that may contribute to cell–cell communication, nicotine metabolism, and inflammation upon cigarette smoking.

Protective effects of intravitreal administration of mesenchymal stem cell-derived exosomes in an experimental model of optic nerve injury

Traumatic optic neuropathy results in the loss of retinal ganglion cells (RGCs), leading to unavoidable visual impairment. However, there is no effective therapy by far. Accumulated studies support the perception that mesenchymal stem cells (MSCs) secrete exosomes that serve as a protective paracrine factor. The study aimed to explore and evaluate the potential therapeutic effects of intravitreal transplantation of MSC-derived exosomes (MSC-exos) in an experimental model of optic nerve crush (ONC). Exosomes were isolated from rat MSCs and characterized by transmission electron microscope and western blotting. At the onset of ONC, a single intravitreal injection of exosomes or PBS was administered to the rats. At day 30, hematoxylin and eosin staining, immunohistochemistry, and βIII-tubulin staining were performed to evaluate the survival of RGCs. Moreover, TUNEL assay was used to examine the apoptosis of RGCs. Inflammation-relevant factors were identified via quantitative polymerase chain reaction. The expression levels of cell apoptosis-related molecules and key members of the PI3K/AKT signaling pathway were determined via western blot analysis. We found that MSC-exos exhibited typical characteristic morphologies (cup-shaped) and sizes (peak size of 93 nm). Furthermore, they exhibited substantial expression of the exosome markers CD63 and TSG101, but lacked the expression of the cellular marker GM130. Treatment with intravitreal MSC-exos notably promoted the survival of RGCs in ONC rats. The level of pro-inflammatory cytokines, including TNF-α, IL-1β, IL-6, IL-8, and MCP-1, were reduced, whereas those of the anti-inflammatory factor IL-10 were increased. Moreover, the apoptosis induced by ONC was decreased by the administration of MSC-exos via upregulation of the Bcl-2/Bax ratio and downregulation of caspase-3 activity. Furthermore, MSC-exos significantly stimulated AKT phosphorylation, whereas LY294002 restored the apoptosis-preventing effects of MSC-exos. The results of our results demonstrated that intravitreal administration of MSC-exos ameliorates ONC-induced injury in a rat model. These findings might aid in the development of effective exosome-based therapeutic strategies for the treatment of optic nerve degeneration.

The Effects of Maternal Smoking on Pregnancy and Offspring: Possible Role for EGF?

Cigarette smoke exposure during pregnancy and lactation is associated with adverse pregnancy outcomes. Here, we investigated the effects of maternal smoke exposure on pregnancy and offspring immunity and explored whether, epidermal growth factor (EGF), an important growth-promoting factor in human colostrum and milk, might be a possible missing link in maternal smoke exposure and changes in infants’ immune responses. Pregnant BALB/c mice were exposed to either cigarette smoke or air during gestation and lactation, and effects on pulmonary inflammation in dams and immune responses in offspring were examined. Maternal smoke exposure increased airway hyperresponsiveness and accumulation of inflammatory cells in the lungs of pregnant dams compared to non-pregnant dams. The E-cadherin protein expression was reduced in mammary glands of cigarette smoke-exposed pregnant dams. EGF levels were higher in mammary glands and serum of smoke-exposed pregnant dams compared to air-exposed pregnant dams. Offspring from cigarette smoke-exposed dams exhibited elevated levels of IL-17A, MCP-1, IL-22, and IL-13 in anti-CD3 stimulated spleen cell culture supernatants. EGF levels were also increased in serum of offspring from smoke-exposed dams. A positive correlation was observed between serum EGF levels and neutrophil numbers in bronchoalveolar lavage fluid of the dams. Interestingly, IL-17A, MCP-1, IL-22, IL13, and IFN-γ levels in anti-CD3 stimulated spleen cell culture supernatants of male pups also showed a positive correlation with EGF serum levels. In summary, our results reveal that maternal smoke exposure predisposes dams to exacerbated airway inflammation and offspring to exacerbated immune responses and both phenomena are associated with elevated EGF concentrations.

Adult Human Multipotent Neural Cells Could Be Distinguished from Other Cell Types by Proangiogenic Paracrine Effects via MCP-1 and GRO.

Adult human multipotent neural cells (ahMNCs) are unique cells derived from adult human temporal lobes. They show multipotent differentiation potentials into neurons and astrocytes. In addition, they possess proangiogenic capacities. The objective of this study was to characterize ahMNCs in terms of expression of cell type-specific markers, in vitro differentiation potentials, and paracrine factors compared with several other cell types including fetal neural stem cells (fNSCs) to provide detailed molecular and functional features of ahMNCs. Interestingly, the expression of cell type-specific markers of ahMNCs could not be differentiated from those of pericytes, mesenchymal stem cells (MSCs), or fNSCs. In contrast, differentiation potentials of ahMNCs and fNSCs into neural cells were higher than those of other cell types. Compared with MSCs, ahMNCs showed lower differentiation capacities into osteogenic and adipogenic cells. Moreover, ahMNCs uniquely expressed higher levels of MCP-1 and GRO family paracrine factors than fNSCs and MSCs. These high levels of MCP-1 and GRO family mediated in vivo proangiogenic effects of ahMNCs. These results indicate that ahMNCs have their own distinct characteristics that could distinguish ahMNCs from other cell types. Characteristics of ahMNCs could be utilized further in the preclinical and clinical development of ahMNCs for regenerative medicine. They could also be used as experimental references for other cell types including fNSCs.

Multitarget molecule, PTUPB, to treat diabetic nephropathy in rats.

Diabetic nephropathy is a common complications related to high morbidity and mortality in type 2 diabetes. We investigated the action of the dual modulator, PTUPB, a soluble epoxide hydrolase and cyclooxygenase-2 inhibitor against diabetic nephropathy. Sixteen-week-old type 2 diabetic and proteinuric obese ZSF1 rats were treated with vehicle, PTUPB or enalapril for 8weeks. Measurements were made of epoxyeicosatrienoic acids, thromboxane B2 (TBX2 ) and prostaglandin E2 (PGE2 ) in the kidney of these and lean ZSF1 rats along with their blood pressure. Obese ZSF1 rats were diabetic with fivefold higher fasting blood glucose levels and markedly higher HbA1c levels compared with lean ZSF1 rats. PTUPB nor enalapril reduced fasting blood glucose or HbA1c but alleviated the development of diabetic nephropathy. In PTUPB-treated obese ZSF1 rats, glomerular nephrin expression was preserved. Enalapril also alleviated diabetic nephropathy. Diabetic renal injury in obese ZSF1 rats was accompanied by renal inflammation with six to sevenfold higher urinary MCP-1 (CCR2) level and renal infiltration of CD-68 positive cells. PTUPB and enalapril significantly reduced urinary MCP-1 levels and renal mRNA expression of cytokines. Both PTUPB and enalapril lowered blood pressure. PTUPB but not enalapril decreased hyperlipidaemia and liver injury in obese ZSF1 rats. Overall, the dual modulator PTUPB does not treat hyperglycaemia but can effectively alleviate hypertension, diabetic nephropathy, hyperlipidaemia and liver injury in type 2 diabetic rats. Our data further demonstrate that the renal actions of PTUPB are comparable with a current standard diabetic nephropathy treatment.

Taohong Siwu Decoction Regulates Cell Necrosis and Neuroinflammation in the Rat Middle Cerebral Artery Occlusion Model.

Cell necrosis and neuroinflammation play an important role in brain injury induced by ischemic stroke. Previous studies reported that Taohong Siwu decoction (THSWD)can reduce heart muscle cell necrosis and has anti-inflammatory properties. In this study, we investigated the effects of THSWD on cell necrosis and neuroinflammation in a rat model of middle cerebral artery occlusion (MCAO). Thirty-six male Sprague-Dawley (SD) rats were randomly divided into three groups with 12 rats in each group. They were the sham operation group, MCAO model group, and MCAO + THSWD group. We used ELISA to determine the levels of TNF-α, Mcp-1, and IL-1β inflammatory factors in rat serum, qRT‐PCR to detect the expression of TNF‐α, Mcp‐1 and IL‐1β mRNA in rat brain, and immunohistochemistry to detect the number of microglia and neutrophils in rat brain. qRT-PCR and Western blot were used to detect the mRNA and protein expression levels of IBA-1 and MPO inflammatory factors and the TNF-α/RIP1/RIP3/MLKL pathway in the rat brain and protein expression levels. Compared with the sham operation group, the expression of MCP-1, IL-1β, IBA-1, and MPO inflammatory factors and the TNF-α/RIP1/RIP3/MLKL pathway were significantly upregulated in the MCAO group. Compared with the MCAO group, the expressions of MCP-1, IL-1β, IBA-1, and MPO inflammatory factors and the TNF-α/RIP1/RIP3/MLKL pathway were significantly downregulated in the MCAO + THSWD group. THSWD can reduce the expression levels of MCP-1, IL-1β, IBA-1, and MPO inflammatory factors as well as the TNF-α/RIP1/RIP3/MLKL pathway. Meanwhile, it can reduce the necrosis and inflammation of brain cells after cerebral ischemia, so as to protect the brain tissue of rats.

Long-term endogenous acetylcholine deficiency potentiates pulmonary inflammation in a murine model of elastase-induced emphysema

Acetylcholine (ACh), the neurotransmitter of the cholinergic system, regulates inflammation in several diseases including pulmonary diseases. ACh is also involved in a non-neuronal mechanism that modulates the innate immune response. Because inflammation and release of pro-inflammatory cytokines are involved in pulmonary emphysema, we hypothesized that vesicular acetylcholine transport protein (VAChT) deficiency, which leads to reduction in ACh release, can modulate lung inflammation in an experimental model of emphysema. Mice with genetical reduced expression of VAChT (VAChT KDHOM 70%) and wild-type mice (WT) received nasal instillation of 50 uL of porcine pancreatic elastase (PPE) or saline on day 0. Twenty-eight days after, animals were evaluated. Elastase instilled VAChT KDHOM mice presented an increase in macrophages, lymphocytes, and neutrophils in bronchoalveolar lavage fluid and MAC2-positive macrophages in lung tissue and peribronchovascular area that was comparable to that observed in WT mice. Conversely, elastase instilled VAChT KDHOM mice showed significantly larger number of NF-κB-positive cells and isoprostane staining in the peribronchovascular area when compared to elastase-instilled WT-mice. Moreover, elastase-instilled VAChT-deficient mice showed increased MCP-1 levels in the lungs. Other cytokines, extracellular matrix remodeling, alveolar enlargement, and lung function were not worse in elastase-instilled VAChT deficiency than in elastase-instilled WT-controls. These data suggest that decreased VAChT expression may contribute to the pathogenesis of emphysema, at least in part, through NF-κB activation, MCP-1, and oxidative stress pathways. This study highlights novel pathways involved in lung inflammation that may contribute to the development of chronic obstrutive lung disease (COPD) in cholinergic deficient individuals such as Alzheimer’s disease patients.

Presentation of cytokine profile in relation to oxidative stress parameters in patients with severe COVID-19: an observational pilot study.

Introduction: COVID-19 can be worsened by hyper-production of cytokines accompanied by increased level of oxidative stress. The aim of this study was to investigate the correlation between a set of cytokines and the markers of the oxidative stress. Methods: The levels of cytokines IL-2, IL-4, IL-6, IL8, IL-10, VEGF, IFN-γ, TNF-α, IL-1α, MCP-1 and EGF were determined by using High Sensitivity Evidence Investigator™ Biochip Array technology. The oxidative stress parameters (d-ROM, PAT, OS index) were measured in serum on FRAS5 analytical photometric system. Results: IL-6, IL-8, IL-10, VEGF, MCP-1 and EGF were significantly higher (p<0.05) in the patients with severe COVID-19 with increased levels of IL-2, IFN-g, TNF-a and IL-1α. The d-ROM, OS index, and PAT were significantly higher (p<0.05) in severe COVID-19 patients. IL-6 demonstrated the strongest correlation with all of the markers of the oxidative stress, d-ROM (r=0.9725, p=0.0001), PAT (r=0.5000, p=0.0001) and OS index (r=0.9593, p=0.012). Similar behavior was evidenced between IFN-g and d-ROM (r=0.4006, p=0.0001), PAT (r=0.6030, p=0.0001) and OS index (r=0.4298, p=0.012). Conclusion: The oxidative stress markers show good correlation with the tested cytokines which can be measured at the beginning of the disease in a primary care setting to predict the course of COVID-19.

Interaction of miR-146a-5p with oxidative stress and inflammation in complications of type 2 diabetes mellitus in male rats: Anti-oxidant and anti-inflammatory protection strategies in type 2 diabetic retinopathy.

Objective(s):This study aimed to evaluate the role of miR-146a-5p in the pathogenesis of diabetic retinopathy and its interaction with oxidative stress and inflammation in the ocular tissue of rats with type 2 diabetes mellitus (T2DM). Materials and Methods:Twenty adult male Sprague Dawley rats (220 ±20 g) were randomly assigned to control and diabetic groups. A high-fat diet was used for three months to induce T2DM which was confirmed by the HOMA-IR index. After that, the levels of glucose and insulin in serum, HOMA-IR as an indicator of insulin resistance, the ocular level of oxidative markers, TNF-α, IL-1β, MIPs, and MCP-1 along with ocular gene expression of NF-κB, Nrf2, and miR-146a-5p were evaluated.Results:The level of lipid peroxidation along with metabolic and inflammatory factors significantly increased and the antioxidant enzyme activity significantly decreased in diabetic rats (P<0.05). The ocular expression of NF-κB and TNF-α increased and Nrf2, HO-1, and miR-146a-5p expression decreased in diabetic rats (P<0.05). In addition, a negative correlation between miR-146a-5p expression with NF-κB and HOMA-IR and a positive correlation between miR-146a-5p with Nrf2 were observed.Conclusion:It can be concluded that miR-146a-5p may regulate Nrf2 and NF-κB expression and inflammation and oxidative stress in the ocular tissue of diabetic rats.

Localized and Systemic Inflammatory Mediators in a Murine Acute Mastitis Model.

IntroductionMilk depression is the major driver of economic loss due to mastitis in dairy animals. The aim of this study was to identify potential mediators of milk depression by investigating the local and systemic changes in gene expression or cytokine production during endotoxin challenge of the mammary gland in a mouse model.MethodsThe left and right sides of the 4th pair of mouse mammary glands were alternatively injected with either lipopolysaccharide (LPS, Escherichia coli 055: B5, 50 μL of 0.4 mg/mL) or sterile PBS through the teat meatus 3 days postpartum (n = 9). The 4th glands were individually collected 12 h after LPS injection and analyzed to identify gene expression changes by RNA sequencing and real-time PCR, and the plasma was collected before and after LPS challenge and analyzed to determine the levels of 32 cytokines.ResultsTranscriptome analysis showed that in addition to strong pro-inflammatory responses, which included granulocyte and monocyte migration and cytokine production and signaling, the LPS-treated glands exhibited strong ubiquitin-mediated and immune-mediated proteasome activation and an increase in nitric oxide-mediated oxidative stress. Furthermore, LPS induced a down-regulation in vesicle membrane, vesicle-mediated trafficking, and metabolic processes of amino acids and other organic molecules in the mammary gland. Of the 32 cytokines analyzed, the levels of 24 (mainly IL-6, G-CSF, MCP-1, RANTES, MIG, MIP-1b, KC, MIP-2, IP-10, and TNFα) were increased or tended to increase in the blood after LPS treatment, and only the levels of IL-9 were decreased. In the mammary gland after LPS challenge, the levels of IL-5, IL-6, IP-10, LIF, MCP-1, MIP-2, and TNFα were significantly increased, and the levels of INFΥ, IL-2, IL-4, IL-10, and IL-12 (p40) were decreased.DiscussionThese observations provide potential markers and targets for further studies on the prevention and treatment of gram-negative bacteria-induced mastitis.

Protective effects of p-coumaric acid against high-fat diet-induced metabolic dysregulation in mice

p-Coumaric acid (PC), a naturally occurring phytochemical, possesses antioxidant and anti-inflammatory properties; however, the mechanisms underlying its protective effects against obesity-related metabolic dysfunction are largely unknown. Here, we treated C57BL/6J mice to a high-fat diet (HFD) with or without PC (10 mg/kg body weight/day) for 16 weeks to determine whether PC ameliorates HFD-induced obesity, insulin resistance, inflammation, and non-alcoholic fatty liver disease (NAFLD). We found no significant differences in food intake and body weight between the groups. However, PC-treated mice showed significantly lower white adipose tissue (WAT) weight, adipocyte size, and plasma leptin level, which were associated with decreased lipogenic enzyme activity and mRNA expression of their genes in the epididymal WAT. Moreover, hepatic lipogenic enzymes activities and expression of their genes and proteins were decreased with concomitant increases in hepatic fatty acid oxidation and mRNA expression of its gene; fecal lipid excretion was significantly increased, resulting in decreased liver weight, hepatic lipid levels, lipid droplet accumulation, and plasma aspartate aminotransferase and lipid levels. Additionally, PC-treated mice showed lower fasting blood glucose, plasma resistin, and MCP-1 levels, HOMA-IR, and mRNA expression of inflammatory genes in the epididymal WAT and liver. Our findings reveal potential mechanisms underlying the action of PC against HFD-induced adiposity, NAFLD, and other metabolic disturbances.

A Potent Leukocyte Transmigration Blocker: GT-73 Showed a Protective Effect against LPS-Induced ARDS in Mice.

We recently developed a molecule (GT-73) that blocked leukocyte transendothelial migration from blood to the peripheral tissues, supposedly by affecting the platelet endothelial cell adhesion molecule (PECAM-1) function. GT-73 was tested in an LPS-induced acute respiratory distress syndrome (ARDS) mouse model. The rationale for this is based on the finding that the mortality of COVID-19 patients is partly caused by ARDS induced by a massive migration of leukocytes to the lungs. In addition, the role of tert-butyl and methyl ester moieties in the biological effect of GT-73 was investigated. A human leukocyte, transendothelial migration assay was applied to validate the blocking effect of GT-73 derivatives. Finally, a mouse model of LPS-induced ARDS was used to evaluate the histological and biochemical effects of GT-73. The obtained results showed that GT-73 has a unique structure that is responsible for its biological activity; two of its chemical moieties (tert-butyl and a methyl ester) are critical for this effect. GT-73 is a prodrug, and its lipophilic tail covalently binds to PECAM-1 via Lys536. GT-73 significantly decreased the number of infiltrating leukocytes in the lungs and reduced the inflammation level. Finally, GT-73 reduced the levels of IL-1β, IL-6, and MCP-1 in bronchoalveolar lavage fluid (BALF). In summary, we concluded that GT-73, a blocker of white blood cell transendothelial migration, has a favorable profile as a drug candidate for the treatment of ARDS in COVID-19 patients.

Cytokines, brain proteins, and growth factors in acute stroke patients: A pilot study.

Background: Immunomodulation and cell signaling involve several cytokines, proteins, and other mediators released in response to the trauma, inflammation, or other insults to the central nervous system. This pilot study is part of the registry designed to evaluate the temporal trends among these molecules after an acute ischemic stroke (AIS) in patients.Methods: Twelve AIS patients were enrolled within 24 hours of the symptoms onset. Two sets of plasma samples were collected: First at admission and second at 24 hours after admission. Cytokines/chemokines and other inflammatory molecules were measured using multiplex assay kit.Results: An increased trend in IL-6 (22 vs. 34 pg/ml), IL-8/CXCL8 (87 vs. 98 pg/ml), MMP-9 (16225 vs. 18450 pg/ml), and GMF-β (999 vs. 3739 pg/ml) levels was observed overtime after an AIS. Patients ≤60 years had lower levels of plasma MCP-1/CCL2 (50–647 vs. 150–1159 pg/ml), IL-6 (9–25 vs. 20–68 pg/ml), and IL-8 (30– 143 vs. 72–630 pg/ml), when compared with patients >60 years old.Conclusion: Cytokines/chemokines and other inflammatory mediators play an important role in the pathogenesis of stroke in addition to mediating poststroke inflammation. Further research is needed to evaluate and characterize the cumulative trends of these mediators for the clinical prognosis or as surrogate biomarkers.

The 3-Year Effect of the Mediterranean Diet Intervention on Inflammatory Biomarkers Related to Cardiovascular Disease.

The intervention with the Mediterranean diet (MD) pattern has evidenced short-term anti-inflammatory effects, but little is known about its long-term anti-inflammatory properties at molecular level. This study aims to investigate the 3-year effect of MD interventions compared to low-fat diet (LFD) on changes on inflammatory biomarkers related to atherosclerosis in a free-living population with a high-risk of cardiovascular disease (CD). Participants (n = 285) in the PREDIMED trial were randomly assigned into three intervention groups: MD with extra-virgin olive oil (EVOO) or MD-Nuts, and a LFD. Fourteen plasma inflammatory biomarkers were determined by Luminex assays. An additional pilot study of gene expression (GE) was determined by RT-PCR in 35 participants. After 3 years, both MDs showed a significant reduction in the plasma levels of IL-1β, IL-6, IL-8, TNF-α, IFN-γ, hs-CRP, MCP-1, MIP-1β, RANTES, and ENA78 (p < 0.05; all). The decreased levels of IL-1β, IL-6, IL-8, and TNF-α after MD significantly differed from those in the LFD (p < 0.05). No significant changes were observed at the gene level after MD interventions, however, the GE of CXCR2 and CXCR3 tended to increase in the control LFD group (p = 0.09). This study supports the implementation of MD as a healthy long-term dietary pattern in the prevention of CD in populations at high cardiovascular risk.