Pro-inflammatory Markers Research Articles

Longitudinal soluble marker profiles reveal strong association between cytokine storms resulting from macrophage activation and disease severity in COVID-19 disease

SARS-CoV2 infection results in a range of disease severities, but the underlying differential pathogenesis is still not completely understood. At presentation it remains difficult to estimate and predict severity, in particular, identify individuals at greatest risk of progression towards the most severe disease-states. Here we used advanced models with circulating serum analytes as variables in combination with daily assessment of disease severity using the SCODA-score, not only at single time points but also during the course of disease, to correlate analyte levels and disease severity. We identified a remarkably strong pro-inflammatory cytokine/chemokine profile with high levels for sCD163, CCL20, HGF, CHintinase3like1 and Pentraxin3 in serum which correlated with COVID-19 disease severity and overall outcome. Although precise analyte levels differed, resulting biomarker profiles were highly similar at early and late disease stages, and even during convalescence similar biomarkers were elevated and further included CXCL3, CXCL6 and Osteopontin. Taken together, strong pro-inflammatory marker profiles were identified in patients with COVID-19 disease which correlated with overall outcome and disease severity.

Intergenerational Impact of Parental Zinc Deficiency on Metabolic and Redox Outcomes in Drosophila melanogaster.

Zinc deficiency is a common nutritional disorder with detrimental health consequences. Whether parental zinc deficiency induces intergenerational effects remains largely unknown. We investigated the effects of a combined maternal and paternal zinc deficiency on offspring's metabolic outcomes and gene expression changes in Drosophila melanogaster. The parent flies were raised on zinc-deficient diets throughout development, and their progeny were assessed. Offspring from zinc-deprived parents exhibited a significant (p < 0.05) increase in body weight and whole-body zinc levels. They also displayed disrupted glucose metabolism, altered lipid homeostasis, and diminished activity of antioxidant enzymes. Gene expression analysis revealed significant (p < 0.05) alterations in zinc transport genes, with increases in mRNA levels of dZIP1 and dZnT1 for female and male offspring, respectively. Both sexes exhibited reduced dZnT35C mRNA levels and significant (p < 0.05) increases in the mRNA levels of DILP2 and proinflammatory markers, Eiger and UPD2. Overall, female offspring showed higher sensitivity to parental zinc deficiency. Our findings underscore zinc's crucial role in maintaining health and the gender-specific responses to zinc deficiency. There is the need for further exploration of the underlying mechanisms behind these intergenerational effects.

Developmental aspects of metabolic syndrome in children under treatment with gastrointestinal lipase inhibitors

Alongside the increasing trends in obesity rates, there is a documented rise in the prevalence of metabolic syndrome (MetS) in children, dictating that each component of MetS should be identified as early as possible;and the treatment goal consists of reducing obesity, managing metabolic complications, insulin resistance, and also addressing hypertension (HTN).Aim of the study. To determine risk factors, the role of certain proinflammatory markers (TNF α, hs-CRP), and adipokines (leptin, adiponectin) in the onset of MetS in children and their evolution under treatment with gastrointestinal lipase inhibitors (GLI). Material and methods. The presented results were obtained as part of a project within the State Program 2020-2023 titled “Evolutionary Aspects of Metabolic Syndrome in Children Under Treatment with Gastrointestinal Lipase Inhibitors”, project number 20.80009.8007.33. The study included 57 children with MS (IDF, 2007). Depending on the pharmacological therapy associated with non-pharmacological treatment, the children in the research were divided into 3 groups: Group I- 23 children who received orlistat (orlip) - GLI, Group II - 15 children who received Angiotensin Converting Enzyme Inhibitors (ACEIs), and Group III - 19 children who received both GLI and ACEIs. The control group consisted of 50 normotensive and normal-weight children of similar age (gender ratio: 1:1). Risk factors, body mass index (BMI), waist circumference (WC), blood pressure values, total cholesterol (TC), LDL-C, HDL-C, triglycerides (TG), basal glycemia (BG), serum insulin, TNFα, hs-CRP, leptin, and adiponectin were analyzed initially and at the 3-month interval.The study protocol was approved by the Medical Ethics Committee of the Institute of Cardiology, Republic of Moldova (minutes no.2 dated February 20, 2020).Results: The majority of children in the study had a history of hereditary aggravated HTN and obesity, were sedentary or moderately sedentary, and had poor dietary habits. Among the components of MetS (IDF, 2007) associated with abdominal obesity, the following predominated: hypertension in 38 (66.7%) children, hypertriglyceridemia - 32 (56.1%%) children, increased basal blood glucose (BG) - 17(29.8%) children and hypo-HDL-C - 11 (19.3%) children. BMI at the 3-month interval from the initiation of medication decreased most significantly in group I (-4.025±1.267 kg/ m2), followed by group II (-3.33±0.972 kg/m2), and III (-3.046±0.840 kg/m2). WC decreased most notably in group III (-10.63±3.732 cm) compared to group II (-9.167±1.783 cm) and group I (-4.700±2.352 cm). Similar trends were observed for blood pressure values: group III (-18.75±1.897 mm Hg) vs group II (-12.167±1.359 mm Hg) vs group I (-3.900±4.613 mm Hg). LDL-C decreased essentially in group II (-0.46±0.211 mmol/l), while TG decreased in group III (-0.77±0.325 mmol/l) and II (-0.53±0.112 mmol/l). HDL-C increased by +0.60±0.274 mmol/l in group I, by +0.15±0.066 mmol/l in group II, and by +0.12±0.031 mmol/l in group III. Serum insulin decreased predominantly in group III (-9.36±4.53 µU/ mL). Similar trends were observed for BG (group III -1.24±0.322 mmol/l). TNF α decreased most significantly in group III (-2.65±0.911 pg/ml) compared to group I (-1.24±0.508 pg/ml) and group II (-1.61±1.018 pg/ml). hs-CRP decreased mainly in group I (-0.69±0.785 mg/l) and group II (-0.74±0.593 mg/l) compared to group III (-0.42±0.617 mg/l). Leptin values decreased significantly in all study groups at the 3-month interval from the initiation of medication (group I by-8.80±3.64 ng/ml, group II by -10.92±4.084 ng/ml, group III by -9.75±6.172 ng/ml). Adiponectin values increased by+1.83±1.087 µg/L in group III of the study.Conclusion: In combination with a calorie-restricted diet, regular physical exercise, and behavioral modifications, treatment with ILG and ACEIs in children with MetS in the study contributed to a decrease in the degree of obesity, blood pressure values, and normalization of the lipid and glucose profile, respectively. Additionally, it highlighted both the importance of prevention, early detection, and management of obesity, as well as the causal link between obesity, hypertension, and chronic inflammation.

Impact of Piezo and other Osteotomy Models on Soft Tissue, Blood Oxidative Stress, and Proinflammatory Markers

Background: Rhinoplasty is a common surgical procedure used in nose esthetics and pathologies. Shaping the nasal bones is a crucial step in achieving successful rhinoplasty surgery. However, complications such as excessive bleeding, edema, mucosal damage, and periosteal damage may occur during osteotomy for nose shaping. Aim: To investigate the damage to soft tissue and the effects on oxidative stress and proinflammatory cytokines in the blood caused by osteotomy performed on rabbits, using different osteotomy methods. Methods: Thirty-two albino New Zealand rabbits were divided into four groups. Group A was the sham group (n = 8), Group B the piezoelectric device group (n = 8), Group C the manual saw group (n = 8), and Group D the classical osteotomy group (n = 8). About 3 ml of blood was drawn to compare preoperative and postoperative interleukin-1ß (IL-1ß), thiobarbituric acid-reactive substances (TBARS), tumor necrosis factor-alpha (TNF-alpha), nitric oxide (NO), interleukin-10 (IL-10), and glutathione (GSH) levels. A 1 mm3 piece of soft tissue from the nasal bone of each animal in the study groups was sent for histopathological examination. The Chi-square test was used to analyze the incidence of postoperative necrosis, inflammation, and edema in the groups. Results: Histopathologically, edema was significantly higher in Group C and Group D compared to Group B. Inflammation was increased in all groups. The necrosis was significantly higher in Group B compared to Group C and Group D. Except for two parameters, no significant changes were found in the biochemical markers for all groups. Conclusions: The piezoelectric device was found to be a better option for reducing edema and inflammation, while manual saws and classical osteotomy may lead to more tissue damage.

Harnessing IL-10 induced anti-inflammatory response in maturing macrophages in presence of electrospun dexamethasone-loaded PLLA scaffold.

The ultimate goal of tissue engineering is to repair and regenerate damaged tissue or organ. Achieving this goal requires blood vessel networks to supply oxygen and nutrients to new forming tissues. Macrophages are part of the immune system whose behavior plays a significant role in angiogenesis and blood vessel formation. On the other hand, macrophages are versatile cells that change their behavior in response to environmental stimuli. Given that implantation of a biomaterial is followed by inflammation; therefore, we reasoned that this inflammatory condition in tissue spaces modulates the final phenotype of macrophages. Also, we hypothesized that anti-inflammatory glucocorticoid dexamethasone improves modulating macrophages behavior. To check these concepts, we investigated the macrophages that had matured in an inflammatory media. Furthermore, we examined macrophages' behavior after maturation on a dexamethasone-containing scaffold and analyzed how the behavioral change of maturing macrophages stimulates other macrophages in the same environment. In this study, the expression of pro-inflammatory markers TNFa and NFκB1 along with pro-healing markers IL-10 and CD163 were investigated to study the behavior of macrophages. Our results showed that macrophages that were matured in the inflammatory media in vitro increase expression of IL-10, which in turn decreased the expression of pro-inflammatory markers TNFa and NFκB in maturing macrophages. Also, macrophages that were matured on dexamethasone-containing scaffolds decreased the expression of IL-10, TNFa, and NFκB and increase the expression of CD163 compared to the control group. Moreover, the modulation of anti-inflammatory response in maturing macrophages on dexamethasone-containing scaffold resulted in increased expression of TNFa and CD163 by other macrophages in the same media. The results obtained in this study, proposing strategies to improve healing through controlling the behavior of maturing macrophages and present a promising perspective for inflammation control using tissue engineering scaffolds.

Development of an injectable oxidized dextran/gelatin hydrogel capable of promoting the healing of alkali burn-associated corneal wounds

The cornea serves as an essential shield that protects the underlying eye from external conditions, yet it remains highly vulnerable to injuries that could lead to blindness and scarring if not promptly and effectively treated. Excessive inflammatory response constitute the primary cause of pathological corneal injury. This study aimed to develop effective approaches for enabling the functional repair of corneal injuries by combining nanoparticles loaded with anti-inflammatory agents and an injectable oxidized dextran/gelatin/borax hydrogel. The injectability and self-healing properties of developed hydrogels based on borate ester bonds and dynamic Schiff base bonds were excellent, improving the retention of administered drugs on the ocular surface. In vitro cellular assays and in vivo animal studies collectively substantiated the proficiency of probucol nanoparticle-loaded hydrogels to readily suppress proinflammatory marker expression and to induce the upregulation of anti-inflammatory mediators, thereby supporting rapid repair of rat corneal tissue following alkali burn-induced injury. As such, probucol nanoparticle-loaded hydrogels represent a prospective avenue to developing long-acting and efficacious therapies for ophthalmic diseases.

Decreased atherosclerosis and abdominal aortic aneurysm in mice deficient in polymeric immunoglobulin receptor

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): La Caixa Banking Foundation and Comunidad de Madrid Background Polymeric immunoglobulin receptor (PIGR) is a transmembrane protein involved in the transcytosis of polymeric immunoglobulins that is widely expressed in mucosal epithelial cells. Recent data from our group described increased PIGR levels in plasma and tissue of atherosclerotic subjects. However, the role for PIGR in the mechanisms involved in vascular remodeling has not been addressed. Purpose To analyse the role for PIGR in experimental mouse models of atherosclerosis and abdominal aortic aneurysm (AAA). Methods We analyzed PIGR expression, secretion and distribution in early atherosclerotic plaques and AAA tissues compared to healthy aortic samples, as well as in macrophages derived from the human monocytic THP-1 cell line. Next, we studied the effect of global PIGR deficiency in experimental atherosclerosis (Ldlr-/- Pigr-/- mice fed with atherogenic diet for 10 weeks). In addition, the contribution of hematopoietic PIGR was assessed in both experimental atherosclerosis and AAA (Ldlr-/- mice fed with atherogenic diet and 1 µg/Kg/min angiotensin II infusion for 28 days) by bone marrow transplantation experiments. Finally, bone marrow-derived macrophages (BMDMs) from Pigr-/- and control mice were isolated to assess their inflammatory expression profile and modulation of polarization in response to oxidized LDL (ox-LDL), as well as their ability to promote foam cell formation. Results PIGR expression was increased in the intima of early human atherosclerotic lesions and showed colocalization with CD68+ expression. PIGR expression was also increased during the in vitro differentiation of THP-1 monocytes to macrophages with phorbol myristate acetate. Both global and hematopoietic-specific PIGR deficient mice displayed reduced plaque size in the aortic sinuses as well as a strong decrease in macrophage infiltration (CD68+ cells), while no changes in smooth muscle cells (α-SMA+ cells) and collagen content were observed. Regarding AAA, increased PIGR levels were found in human AAA tissues, associated with infiltrating CD68+ cells. PIGR deficiency in hematopoietic cells also showed significantly reduced AAA dilatation and reduced macrophage infiltration. In vitro experiments with BMDMs obtained from Pigr-/- mice and stimulated with ox-LDL showed reduced proinflammatory gene expression (TNF-a and IL-6) and M1 markers (iNOS and CD11c) as well as diminished foam cell formation. Conclusions PIGR deficiency in hematopoietic cells decreases atherosclerosis and AAA progression by modulating macrophage inflammatory responses, suggesting a novel proinflammatory role for PIGR in vascular pathologies. Future experiments will test the potential therapeutic effect of modulating PIGR to dampen the inflammatory profile in vascular diseases.Results in human and mouse studies

Exploring the role of air pollution on the development of atherosclerosis disorder in human endothelial cells

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): QU - NSPP Introduction Atherosclerosis, the main cause of cardiovascular and cerebrovascular diseases, is a global health threat. Rising cardiovascular disease prevalence, unexplained by traditional risk factors, prompts exploration of the impact of occupational exposure to environmental toxins as a risk factor. benzo[a]pyrene (BaP) is a polyaromatic compound that mediate its toxicity through activation of the Aryl hydrocarbon receptor (AhR). The molecular mechanisms of involvement of environmental toxins and the role of AhR/CYP1A pathway in atherosclerosis development remain not fully investigated. Purpose The current study explores the AhR pathway activation in endothelial cells upon exposure to environmental toxins in order to elucidate the molecular mechanisms of atherosclerotic cardiovascular diseases and plaque formation through investigating pro-inflammatory (CYP1A1, CYP1B1, TNF-a) and pro-apoptotic markers (BCL-2, Caspase-3). Methods EA. hy926 cell line underwent treatment with increasing concentrations of BaP (0-20 microM), as well as 10nm of TCDD, to confirm the results. DSMO was used as a control. Total RNA and protein were isolated to investigate mRNA expression and protein expression of AhR, CYP1A1, CYP1B1, as well as caspase-3, Bcl-2, and TNF-α using RT-PCR and Western Blot analysis. Statistical analysis was done using one-way ANOVA. Results The results showed that BaP induce significant dose-dependent upregulation of AhR and target genes (CYP1A1, CYP1B1). At 20uM, expression of AhR (3-fold), CYP1A1 (2.5-fold) and CYP1B1 (2-fold) was significantly high as compared to their respective controls and low doses. As for apoptotic markers, Bcl-2 was significantly downregulated at the highest concentrations 10uM and 20uM (0.7- and 0.6-fold, respectively). Caspase-3 showed no significant change but exhibited upregulation across all doses. TCDD 10nM significantly upregulated AhR (2.0-fold), CYP1A1 (1.5-fold), CYP1B1 (1.7-fold). Bcl-2 downregulated (0.6-fold), TNF-α significantly upregulated (1.8-fold) at 10nM. Caspase-3 increased 2-folds but was not statistically significant. Conclusion This study illustrated that AhR inducers, such as BaP, can accelerate the pathological progress of atherosclerosis by evoking oxidative stress and inflammation response molecules in human endothelial cells, demonstrated by the reduced expression of anti-apoptotic markers and the increased expression of inflammatory markers.

The extracellular matrix proteoglycan serglycin is associated with human atherosclerotic plaque inflammation

Abstract Funding Acknowledgements Type of funding sources: Other. Main funding source(s): This work was supported by grants from the Swedish Research Council, the Swedish Heart and Lung Foundation, Skåne University Hospital funds, Royal Physiographic Society in Lund and O.E. and Edla Johanssons Foundation. Background Atherosclerotic cardiovascular disease is caused by the formation of plaques in the arterial wall which contain lipids, cells, cell debris and a unique extracellular matrix (ECM) signature. These plaques may ultimately rupture or erode away causing thrombosis, thereby leading to myocardial infarction or stroke. Rupture-prone plaques generally bear a greater inflammatory activity, with ECM proteins suggested to influence the inflammatory activity. Among many ECM proteins, serglycin (SRGN) is highly expressed by inflammatory cells, and SRGN has been linked to modulation of the inflammatory response in disease such as cancer. Yet, if SRGN contributes to the inflammatory circuitry in human atherosclerotic plaques remains to be explored. Purpose We aimed to investigate if SRGN was associated with inflammation in atherosclerotic plaques. Methods Plaque protein levels of SRGN were measured by ELISA in human carotid plaques obtained from the Carotid Plaque Imaging Project (CPIP) biobank. Plaque RNA expression levels of SRGN and cell markers were assessed by plaque bulk RNA sequencing. SRGN expression and distribution were further examined in tissue plaque sections through histology and multispectral imaging. In vitro models were used to investigate the functional consequences of SRGN expression changes, using SRGN CRISPR knockout THP1 monocytic cells. Immunoprecipitation of SRGN and immunoblotting against SRGN, heparan and chondroitin sulfate chains were employed to structurally characterize plaque protein SRGN. Results SRGN protein levels were significantly higher in plaques from asymptomatic patients compared to symptomatic. Multispectral imaging showed that SRGN protein was observed in plaque areas rich in macrophages and lipids. Using immunoprecipitation and immunoblotting, we identified a unique low glycosylated plaque SRGN, with both heparan sulfate and chondroitin sulfate glycosaminoglycans decorating its protein core. This exclusive configuration suggests that the plaque cell origin of SRGN may be macrophages or mast cells. In support of this, plaque SRGN RNA expression correlated with RNA levels of pro-inflammatory macrophage cells markers like CD68, CD163, MRC1, ITGAX. Plaque SRGN protein levels correlated with plaque levels of oxidised lipoproteins, proatherogenic (CCL1, IL7, MCP-4 and TNFα) and antiatherogenic cytokines (IL33, IL16 and CXCL13), as well as with growth factors, such as TGF-β. On the one hand, in vitro suppression of SRGN expression in monocytes led to a reduction in the anti-inflammatory M2-polarized phenotype, while M1 pro-inflammatory macrophages were unskewed. Furthermore, our in vitro studies showed that upon SRGN knockdown M2 macrophages retained more oxidized lipoproteins. Conclusions Our results suggest that SRGN may be a crucial modulator of inflammation in human atherosclerotic plaques, potentially by affecting the bioavailability of effector molecules such as growth factors and cytokines.

Anti-interleukin-18 as therapy against atherosclerosis

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Established Investigator of the Dutch Heart Foundation Background Interleukin-18 (IL-18) is a pro-inflammatory cytokine and is secreted by macrophages after NLRP3-inflammasome activation. IL-18 is important for the induction of the Th1 response and overexpression of IL-18 aggravates atherosclerosis, suggesting that inhibition of IL-18 may act atheroprotective. In this study, we investigated the therapeutic potential of an IL-18 neutralizing antibody (anti-IL18) on atherosclerosis development. Methods and results Female ApoE-/- mice were fed a Western-type diet for 8 weeks and simultaneously received an antibody against IL-18 (100 µg/mouse; n=15) or a rat IgG2a control (100 µg/mouse; n=15) 3x per week intraperitoneally, after which the mice were sacrificed. First, we assessed macrophage phenotype in the peritoneal cavity. Expression of the proinflammatory markers CD80 (control: 46.3±1.4 *103; p&amp;lt;0.05 vs. anti-IL18: 40.4 ±1.7 *103) and iNOS (control: 15.5±1.8 *102 vs. anti-IL18: 8.0±1.9 *102; p&amp;lt;0.01) were reduced. In the aorta, MFIs of anti-inflammatory macrophage markers Arginase-1 (control: 389±99 vs. anti-IL18: 654±85; p=0.06) and CD206 (control: 55.2±2.4 *103 vs. anti-IL18: 62.8±3.7 *103; p=0.1) tended to be increased expression in the ascending aorta of anti-IL18 mice compared to controls. In addition, anti-IL18 treatment promoted the number of CD4 T cells (control: 20±1% vs. anti-IL18: 28±1%; p&amp;lt;0.0001) in the mediastinal lymph node and increased it’s regulatory phenotype (Tregs; Foxp3+) of CD4 (control: 23±1% vs. anti-IL18: 26±1%; p=0.07) and CD8 T cells (control: 0.4±0.02% vs. anti-IL18: 0.9±0.09%; p&amp;lt;0.0001). These effects however, did not translate into an effect on plaque size in the aortic root, as measured using an Oil-Red-O staining, which did not differ between the control and anti-IL18 treatment group (control: 47.1±2.4 *104 µm2 vs. anti-IL18: 43.4±2.3 *104 µm2). Conclusions Anti-IL18 treatment reduced the proinflammatory phenotype of peritoneal macrophages and promoted Tregs in the mediastinal lymph node. Further analyses on plaque stability and intraplaque macrophage subsets are currently being performed.

Propionate and butyrate counteract renal damage and progression to chronic kidney disease.

Short-chain fatty acids (SCFAs), mainly acetate, propionate and butyrate, are produced by gut microbiota through fermentation of complex carbohydrates that cannot be digested by the human host. They affect gut health and can contribute at the distal level to the pathophysiology of several diseases, including renal pathologies. SCFA levels were measured in chronic kidney disease (CKD) patients (n=54) at different stages of the disease and associations with renal function and inflammation parameters were examined. The impact of propionate and butyrate in pathways triggered in tubular cells under inflammatory conditions was analysed using genome-wide expression assays. Finally, a pre-clinical mouse model of folic acid-induced transition from acute kidney injury to CKD was used to analyse the preventive and therapeutic potential of these microbial metabolites in the development of CKD. Faecal levels of propionate and butyrate in CKD patients gradually reduce as the disease progresses, and do so in close association with established clinical parameters for serum creatinine, blood urea nitrogen and the estimated glomerular filtration rate. Propionate and butyrate jointly downregulated the expression of 103 genes related to inflammatory processes and immune system activation triggered by TNF-α in tubular cells. In vivo, the administration of propionate and butyrate, either before or soon after injury, respectively prevented and slowed the progression of damage. This was indicated by a decrease in renal injury markers, the expression of pro-inflammatory and pro-fibrotic markers, and recovery of renal function over the long term. Propionate and butyrate levels are associated with a progressive loss of renal function in CKD patients. Early administration of these SCFAs prevents disease advancement in a pre-clinical model of acute renal damage, demonstrating their therapeutic potential independently of the gut microbiota.

Efficacy of Probiotic Strains Lactobacillus sakei Probio65 and Lactobacillus plantarum Probio-093 in Management of Obesity: An In Vitro and In Vivo Analysis.

The prevalence of obesity, characterized by an excessive accumulation of adipose tissue and adipocyte hypertrophy, presents a major public health challenge. This study investigates the therapeutic potential of two probiotic strains, Lactobacillus sakei Probio65 and Lactobacillus plantarum Probio-093, in the context of obesity. Utilizing 3T3-L1 cell-derived human adipocytes, we assessed Probio65's and Probio-093's capacity to mitigate triglyceride accumulation and influence adipocytokine production in vitro. Subsequently, an in vivo trial with male C57BL/6J mice examined the effects of both probiotic strains on adipose tissue characteristics, body weight, fat mass, and obesity-related gene expression. This study employed both live and ethanol-extracted bacterial cells. The results demonstrated significant reductions in the triglyceride deposition, body weight, and adipose tissue mass in the treated groups (p < 0.05). Furthermore, both strains modulated adipokine profiles by downregulating proinflammatory markers such as PAI-1, leptin, TNF-α, STAMP2, F4/80, resistin, and MCP-1, and upregulating the insulin-sensitive transporter GLUT4 and the anti-inflammatory adiponectin (p < 0.05). Our findings suggest that Lactobacillus sakei Probio65 and Lactobacillus plantarum Probio-093 are promising agents for microbiome-targeted anti-obesity therapies, offering the effective mitigation of obesity and improvement in adipocyte function in a murine model.

Electrochemical Degradation of Molecularly Imprinted Polymers for Future Applications of Inflammation Sensing in Cochlear Implants.

After cochlear implant (CI) insertion, there is a possibility of postoperative inflammation, which may involve proinflammatory markers such as interleukin-6. Detecting this inflammation promptly is crucial for administering anti-inflammatory drugs, if required. One potential method for detecting inflammation is using molecular imprinted polymers (MIPs). These MIPs, which can be deposited on the CI electrode, provide readout employing impedance measurements, a feature already available on the CI circuit. MIPs designed for this purpose should possess biocompatibility, conductivity, and degradability. The degradability is crucial because there is a limitation on the number of electrodes available, and once the inflammation sensor degrades after the acute inflammation period, it should remain usable as a regular electrode. In this work, conductive poly(3,4-ethylenedioxythiophene) polystyrenesulfonate-based MIPs were synthesized against biotin as a surrogate target marker. Specific biotin binding with MIPs was determined before and after degradation using electrochemical impedance spectroscopy (EIS) and compared with the control nonimprinted polymers (NIPs). Subsequently, MIPs were electrochemically degraded by EIS with different potentials, wherein a potential dependence was observed. With decreasing potential, fewer dissolved polymers and more monomer molecules were detected in the solution in which degradation took place. At a potential of 0.205 V a negligible amount of dissolved polymer in addition to the dissolved monomer molecules was measured, which can be defined as the limiting potential. Below this potential, only dissolved monomer molecules are obtained, which enables renal clearance. Biocompatibility testing revealed that both the polymer and the solution with dissolved monomer molecules do not exceed the ISO 10993-5 cytotoxicity threshold. Based on these findings, we have developed conductive, biocompatible, and controllably degradable MIPs capable of detecting biotin. This research work paves the way for the advancement of CIs, where inflammation can be detected using molecular imprinting technology without compromising the stability and biosafety of the product.

Exploring the effect of platelet-rich plasma on vascularization and survival of follicles in xenotransplanted human ovarian tissue

Research questionDo platelet-rich plasma (PRP) products, specifically human platelet lysate (hPL) and umbilical cord plasma, enhance vascularization and follicular survival in human ovarian tissue transplanted to immunodeficient mice? DesignHuman ovarian tissue was transplanted to subcutaneous pockets in nude mice, followed by daily injections for 6 days of PRP or saline at the transplantation sites. After a grafting period of 3 and 6 days, vascularization was assessed using CD-31 quantification, and gene expression of angiogenic markers (VEGF/Vegf) together with apoptosis-related genes (BAX/BCL-2), oxidative stress markers (HMOX-1/Hmox-1) and pro-inflammatory markers (Il-1β/Il-6/Tnf-α) was quantitively analysed. Follicle density was analysed in the grafts after 4 weeks. Additionally, a pilot study was conducted exploring the suitability of ultrasound scanning for assessing survival and vascularization in ovarian tissue xenografted to mice. ResultsAlthough there was a significant increase in the CD-31 area from day 3 to day 6 post-grafting, there were no significant differences between the hPL and control groups. Gene expression analysis revealed significant down-regulation of VEGF from day 3 to day 6 for both the hPL and control groups, and significant up-regulation of BAX/BCL-2 in the hPL group compared with the controls. The follicle density showed no significant differences in the hPL group and UCP groups compared with the controls. Furthermore, ultrasound biomicroscopy provided valuable insights into graft morphology, necrotic areas and blood flow, suggesting its potential as a monitoring tool. ConclusionsDespite the angiogenic properties of PRP, this study was unable to demonstrate a significant impact of hPL on vascularization or of hPL and UCP on follicular survival in xenotransplanted human ovarian tissue.

#715 Adiponectin / leptin ratio as an index to determine the risk of metabolic and rejection complications in patients after kidney transplantation

Abstract Background and Aims Adipose tissue, as an endocrine and paracrine organ, produces several hormones that are involved in the regulation of metabolic and inflammatory processes or immune system activity. While leptin is the main pro-inflammatory marker of adipose tissue, adiponectin is characterized by its anti-inflammatory effects. The adiponectin / leptin (A/L) ratio had been shown to correlate better with the occurrence of cardiometabolic risk factors than hormone concentrations alone. Results in the general population suggest that an A/L ratio &amp;gt; 1 can be considered normal, an A/L ratio of 0.5-1 indicates a moderate increase, and an A/L ration &amp;lt; 0.5 a high increase in cardiometabolic risk. The first aim of our study was to determine the risk of developing post-transplant diabetes mellitus (PTDM), prediabetes and other metabolic risk factors in relation to the A/L ratio in patients after kidney transplantation (KT). The second aim was to determine the risk of developing acute graft rejection in relation to the A/L ratio in patients after KT. Method A total of 104 patients were included in our prospective analysis after applying inclusion and exclusion criteria. Pre-transplantation and subsequently at 3, 6 and 12 months after KT, we recorded baseline characteristics of donors and recipients, including parameters characterizing graft function, metabolic (lipid profile, indices of glucose metabolism) and anthropometric parameters. At the same time, we examined serum adipocytokine concentrations (adiponectin, leptin) and calculated the A/L ratio. In the third month after KT, all patients underwent protocol graft biopsy and donor specific antibody (DSA) testing by Luminex method. Results We found that during the follow-up period, there was a significant increase in the A/L ratio in the control group (p = 0.0013) and, on the other hand, a significant decrease in the group who developed PTDM (p = 0.0003). Comparing the different subgroups divided on the A/L ratio one year after KT, we found that patients with an A/L &amp;lt; 0.5 were significantly longer in the dialysis program, had higher body mass index (BMI), waist circumference, worse graft function, and a higher prevalence of prediabetes and PTDM compared with those whose A/L ratio was &amp;gt; 1. Using logistic regression in multivariate analysis, we identified age at the time of KT [OR 1.0709, (p = 0.0337)], triglycerides level at 1 year after KT [OR 2.7735, (p = 0.446)] and A/L ratio &amp;lt; 0.5 [OR 3.1724, (p = 0.014)] as independent risk factors for the development of prediabetes and PTDM 1 year after KT. At the same time, after adjusting for differences in baseline donor and recipient characteristics, we identified the subgroup with an A/L ratio &amp;lt; 0.5 pre-transplant [HR 1.6126, (p = 0.0133)] and 3 months after KT [HR 1.3150, (p = 0.0172)] as an independent risk factor for the development of acute graft rejection. In the subsequent specification of the rejection episode, we found that A/L ratio &amp;lt; 0.5 pre-transplant [HR 2.2353, (p = 0.0357)] and 3 months after KT [HR 3.0954, (p = 0.0237)] is and independent risk factor for the occurrence of acute humoral rejection with DSA positivity. Conclusion In our study, we found that an A/L ratio &amp;lt; 0.5 is an independent risk factor for the development of prediabetes, PTDM 1 year after KT and acute humoral graft rejection with de novo DSA production in the third month after KT. This is the first study to investigate the correlation of the A/L ratio with the risk of developing metabolic and immunological complications in patients after KT.

#1918 L-NAME accelerates the progression of diabetic kidney disease in mice

Abstract Background and Aims Diabetic kidney disease (DKD) represents one of the major causes of end stage kidney disease worldwide. Despite DN having such a high incidence, mouse models of DN rarely mimic all aspects of the human disease, besides showing very slow progression and sometimes maintenance issues. For this reason, in this project we aimed to develop an accelerated, reproducible and accessible mouse model of DN. Method For our experiments we chose 9-week-old female and male of the leptin receptor knock-out (BKS.Cg-Dock7m +/+ Leprdb/J) mice as type 2 diabetes model. As these mice are still not prone to develop severe kidney injury despite their diabetic condition, we administered the eNOS inhibitor L-NAME for 6 weeks, in order to suppress nitric oxide production and therefore to aggravate kidney damage. Mice were divided in three groups: one vehicle control group, one receiving 40/mg/kg/day of L-NAME and one receiving 80/mg/kg/day of L-NAME dissolved in drinking water. Blood pressure and glomerular filtration rate (GFR) were measured at different time points, together with glycemia and albuminuria. Weight and water intake were also monitored throughout the study. At the end of the experiment, animals were killed and blood and organs collected for further analysis. Results Mice treated with 80 mg/kg/day of L-NAME showed an average increase of 10 mmHg in systolic blood pressure, with a peak of 30 mmHg increase at week 4 of treatment. On the other hand, 40 mg/kg/day of L-NAME did show any effect on blood pressure. Despite the different effect on blood pressure, three weeks of L-NAME administration caused a significant reduction in GFR at both doses, with 5% reduction for the 40 mg-group and 23% for the 80 mg-group. For the 80 mg-group we observed an even further GFR decline after 5 weeks of treatment, with a 28% reduction. No change in GFR was observed in the controls. This decrease in GFR was accompanied by a 10-fold increase in urinary albumin for both experimental groups already after 3 weeks of treatment, which after 4 weeks slightly declined but remained significantly higher than the controls (p &amp;lt; 0.0001). At a histological level, PAS staining of kidney sections revealed that in particular mice treated with 80 mg of L-NAME have enlarged Bowman capsule and glomerular tuft area, besides a reduced glomerular capillary density, coinciding with mesangial expansion. We also stained for overall collagen deposition with picrosirius red staining in order to detect possible fibrosis formation. We then observed that 80 mg of L-NAME accelerates interstitial fibrosis in the cortical area of the kidney, which also overlapped with an increased deposition of collagen I. This increase in collage I was also detected in kidney cortex of mice treated with 40 mg of L-NAME, although their overall collagen deposition and therefore fibrotic tissue did not differ from the control group. The medullar region of kidneys was also analyzed, but none of the two experimental groups revealed significant differences in terms overall collagen or collagen I deposition. Unlike mesangial expansion and fibrosis, L-NAME did not influence microphages infiltration, as revealed by CD68 staining in kidney sections. This suggests that L-NAME treatment does not accelerate kidney damage by triggering or accelerating inflammatory processes. This was also confirmed by qPCR analysis of kidney tissue, where the expression pro-inflammatory markers, such as IL1β, TNFα and MCP-1, did not change in the experimental groups. Conclusion Our results show that L-NAME considerably accelerates the progression of DKD in diabetic mice, with GFR declining and progressive albuminuria already after 3 weeks. Such disfunction is confirmed at 6 weeks by histological analysis, where mesangial expansion and fibrosis were detected. This model can be used to assess the therapeutic potential of novel interventions aimed to slow down DKD.

Impact of Prevalent Dietary Pattern on Serum Vitamin B12 Status and Its Association With Inflammation Among Reproductive Age Women.

Association between vitamin B12 deficiency (VB12D) and dietary patterns being well documented has bearing on obstetrics and neonatal outcomes. However, relationship between VB12D and serum inflammatory markers (IMs), particularly in vegetarian diet and Polycystic ovary syndrome (PCOS), remains elusive. This cross-sectional study assesses VB12D and IMs among reproductive age women consuming different diets. Nonvegetarian (PCOS, n = 104; healthy, n = 148) and vegetarian women (PCOS n = 112; healthy, n = 186) are for evaluated clinical, biochemical, hormonal assessment, inflammatory, and four vitamin B 12 (VB12) markers. VB12D is defined by Fedosov's wellness quotient (4cB12). Using 4cB12, prevalence of VB12D is discerned in 54.4% (PCOS: 72.1%; healthy 36.5%) and 93.4% (PCOS: 95.9%; healthy: 91.9%) among nonvegetarians and vegetarians, respectively. Vegetarian PCOS women depict lowest median (interquartile range [IQR]) of serum B12 76.2(72.6) pgmL-1, holotranscobalamine (HTC) 37.9(11.3) and highest homocysteine (HCY) 40.32(6.0) µmolL-1, methylmalonic acid (MMA) 352.26(156.7) nmolL-1 with highest Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) and IMs (Monocyte chemoattractant protein 1 (MCP-1), High sensitivity C-reactive protein (hs-CRP), Tumour Necrosis Factor alpha (TNF-α)and Interleukin 6 (IL-6)). Significant correlation of serum hs-CRP, TNF-α, and IL-6 with VB12 markers is observed. The VB12D is rampant among reproductive age women that gets exacerbated by PCOS or vegetarian diet. It is directly correlated with magnitude of proinflammatory markers. The results carry substantial implications for public health policies aimed at improving preconception maternal VB12 status for better future pregnancy and offspring outcomes.

Evaluation of the Clinical Effectiveness of the Drug Laennec in Complex Rehabilitation of Patients with Post-COVID Syndrome: a Randomized Trial

INTRODUCTION. The development of post-COVID syndrome is accompanied by an increase in markers of systemic inflammation, a violation of the detoxification function of the liver, caused by both direct viral damage to hepatocytes and an increased iatrogenic load on the hepatobiliary system due to polypharmacy. AIM. Studying the use of the drug Laennec in the rehabilitation period as a hepatoprotector and immunomodulator in patients with post-COVID syndrome. MATERIALS AND METHODS. The study included 40 patients with post-COVID syndrome aged 30 to 60 years, with steatosis and steatohepatitis (increased levels of liver transaminases). The clinical effectiveness of Laennec was assessed based on the dynamics of pro-inflammatory biomarkers, liver metabolism indicators, as well as functional tests and psychological questionnaires. RESULTS. The inclusion of Laennec in the rehabilitation program for patients with post-COVID syndrome leads to a more pronounced improvement in lipid metabolism parameters, correction of liver metabolism parameters, a decrease in the level of pro-inflammatory biomarkers, and an improvement in the quality of life of patients than in the control group. DISCUSSION. Significantly significant positive dynamics in the severity of complaints of general weakness and sleep disturbance, as well as indicators of memory impairment and impaired concentration in the main group could indicate the achievement of anabolic, neurotrophic and bioenergetic effects of the Laennec drug. A significantly significant decrease in the level of liver transaminases in the main group indicates the hepatoprotective effect of the drug Laennec, due to the amino acids, vitamins and microelements included in the drug, which support the reactions of phases 1 and 2 of liver detoxification. Normalization of elevated pro-inflammatory markers (ferritin, IL-6, CRP) in patients of the main group may indicate the presence of anti-inflammatory and immunoregulatory effects of the Laennec drug. CONCLUSIONS. A course prescription of the drug Laennec for the purpose of correcting pro-inflammatory markers and hepatoprotection can be recommended for use in complex non-drug rehabilitation to increase its clinical effectiveness, as well as improve subjective indicators of the quality of life of patients with post-COVID syndrome.

Mitochondria targeted esculetin administration improves insulin resistance and hyperglycemia-induced atherosclerosis in db/db mice.

The development and progression of hyperglycemia (HG) and HG-associated atherosclerosis are exacerbated by mitochondrial dysfunction due to dysregulated mitochondria-derived ROS generation. We recently synthesized a novel mitochondria-targeted esculetin (Mito-Esc) and tested its dose-response therapeutic efficacy in mitigating HG-induced atherosclerosis in db/db mice. In comparison to simvastatin and pioglitazone, Mito-Esc administration resulted in a considerable reduction in body weights and improved glucose homeostasis, possibly by reducing hepatic gluconeogenesis, as indicated by a reduction in glycogen content, non-esterified free fatty acids (NEFA) levels, and fructose 1,6-bisphosphatase (FBPase) activity. Interestingly, Mito-Esc treatment, by regulating phospho-IRS and phospho-AKT levels, greatly improved palmitate-induced insulin resistance, resulting in enhanced glucose uptake in adipocytes and HepG2 cells. Also, and importantly, Mito-Esc administration prevented HG-induced atheromatous plaque formation and lipid accumulation in the descending aorta. In addition, Mito-Esc administration inhibited the HG-mediated increase in VACM, ICAM, and MAC3 levels in the aortic tissue, as well as reduced the serum pro-inflammatory cytokines and markers of senescence. In line with this, Mito-Esc significantly inhibited monocyte adherence to human aortic endothelial cells (HAECs) treated with high glucose and reduced high glucose-induced premature senescence in HAECs by activating the AMPK-SIRT1 pathway. In contrast, Mito-Esc failed to regulate high glucose-induced endothelial cell senescence under AMPK/SIRT1-depleted conditions. Together, the therapeutic efficacy of Mito-Esc in the mitigation of hyperglycemia-induced insulin resistance and the associated atherosclerosis is in part mediated by potentiating the AMPK-SIRT1 axis. KEY MESSAGES: Mito-Esc administration significantly mitigates diabetes-induced atherosclerosis. Mito-Esc improves hyperglycemia (HG)-associated insulin resistance. Mito-Esc inhibits HG-induced vascular senescence and inflammation in the aorta. Mito-Esc-mediated activation of the AMPK-SIRT1 axis regulates HG-induced endothelial cell senescence.

Cannabidiol prevents LPS-induced inflammation by inhibiting the NLRP3 inflammasome and iNOS activity in BV2 microglia cells via CB2 receptors and PPARγ

Neuroinflammation stands as a critical player in the pathogenesis of diverse neurological disorders, with microglial cells playing a central role in orchestrating the inflammatory landscape within the central nervous system. Cannabidiol (CBD) has gained attention for its potential to elicit anti-inflammatory responses in microglia, offering promising perspectives for conditions associated with neuroinflammation. Here we investigated whether the NLRP3 inflammasome and inducible nitric oxide synthase (iNOS) are involved in the protective effects of CBD, and if their modulation is dependent on cannabinoid receptor 2 (CB2) and PPARγ signalling pathways. We found that treatment with CBD attenuated pro-inflammatory markers in lipopolysaccharide (LPS)-challenged BV2 microglia in a CB2- and PPARγ-dependent manner. At a molecular level, CBD inhibited the LPS-induced pro-inflammatory responses by suppressing iNOS and NLRP3/Caspase-1-dependent signalling cascades, resulting in reduced nitric oxide (NO), interleukin-1β (IL-1β), and tumour necrosis factor-alpha (TNF-α) concentrations. Notably, the protective effects of CBD on NLRP3 expression, Caspase-1 activity, and IL-1β concentration were partially hindered by the antagonism of both CB2 receptors and PPARγ, while iNOS expression and NO secretion were dependent exclusively on PPARγ activation, with no CB2 involvement. Interestingly, CBD exhibited a protective effect against TNF-α increase, regardless of CB2 or PPARγ activation. Altogether, these findings indicate that CB2 receptors and PPARγ mediate the anti-inflammatory effects of CBD on the NLRP3 inflammasome complex, iNOS activity and, ultimately, on microglial phenotype. Our results highlight the specific components responsible for the potential therapeutic applications of CBD on neuroinflammatory conditions.