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In the healthy adult liver, most hepatocytes proliferate minimally. However, upon physical or chemical injury to the liver, hepatocytes proliferate extensively in vivo under the direction of multiple extracellular cues, including Wnt and pro-inflammatory signals. Currently, liver organoids can be generated readily in vitro from bile-duct epithelial cells, but not hepatocytes. Here, we show that TNFα, an injury-induced inflammatory cytokine, promotes the expansion of hepatocytes in 3D culture and enables serial passaging and long-term culture for more than 6 months. Single-cell RNA sequencing reveals broad expression of hepatocyte markers. Strikingly, in vitro-expanded hepatocytes engrafted, and significantly repopulated, the injured livers of Fah-/- mice. We anticipate that tissue repair signals can be harnessed to promote the expansion of otherwise hard-to-culture cell-types, with broad implications.

The inflammation response induced by brain trauma can impair recovery. This response requires several hours to develop fully and thus provides a clinically relevant therapeutic window of opportunity. Poly(ADP-ribose) polymerase inhibitors suppress inflammatory responses, including brain microglial activation. We evaluated delayed treatment with veliparib, a poly(ADP-ribose) polymerase inhibitor, currently in clinical trials as a cancer therapeutic, in rats and pigs subjected to controlled cortical impact (CCI). In rats, CCI induced a robust inflammatory response at the lesion margins, scattered cell death in the dentate gyrus, and a delayed, progressive loss of corpus callosum axons. Pre-determined measures of cognitive and motor function showed evidence of attentional deficits that resolved after three weeks and motor deficits that recovered only partially over eight weeks. Veliparib was administered beginning 2 or 24 h after CCI and continued for up to 12 days. Veliparib suppressed CCI-induced microglial activation at doses of 3 mg/kg or higher and reduced reactive astrocytosis and cell death in the dentate gyrus, but had no significant effect on delayed axonal loss or functional recovery. In pigs, CCI similarly induced a perilesional microglial activation that was attenuated by veliparib. CCI in the pig did not, however, induce detectable persisting cognitive or motor impairment. Our results showed veliparib suppression of CCI-induced microglial activation with a delay-to-treatment interval of at least 24 h in both rats and pigs, but with no associated functional improvement. The lack of improvement in long-term recovery underscores the complexities in translating anti-inflammatory effects to clinically relevant outcomes.

The vasculature system plays a critical role in inflammation processes in the body. Vascular inflammatory mechanisms are characterized by disruption of blood vessel wall permeability together with increased immune cell recruitment and migration. There is a critical need to develop models that fully recapitulate changes in vascular barrier permeability in response to inflammatory conditions. We developed a scalable platform for parallel measurements of trans epithelial electrical resistance (TEER) in 64 perfused microfluidic HUVEC tubules under inflammatory conditions. Over 250 tubules where exposed to Tumor necrosis factor alpha (TNFα) and interferon gamma (INF-γ) or human peripheral blood mononuclear cells. The inflammatory response was quantified based on changes TEER and expression of ICAM and VE-cadherin. We observed changes in barrier function in the presence of both inflammatory cytokines and human peripheral blood mononuclear cells, characterized by decreased TEER values, increase in ICAM expression as well changes in endothelial morphology. OrganoPlate 3-lane64 based HUVEC tubules provide a valuable tool for inflammatory studies in an automation compatible manner. Continuous TEER measurements enable long term, sensitive assays for barrier studies. We propose the use of our platform as a powerful tool for modelling endothelial inflammation in combination with immune cell interaction that can be used to screen targets and drugs to treat chronic vascular inflammation. [ABSTRACT FROM AUTHOR]

Sleep intensity is adjusted by the length of previous awake time, and under tight homeostatic control by protein phosphorylation. Here, we establish microglia as a new cellular component of the sleep homeostasis circuit. Using quantitative phosphoproteomics of the mouse frontal cortex, we demonstrate that microglia‐specific deletion of TNFα perturbs thousands of phosphorylation sites during the sleep period. Substrates of microglial TNFα comprise sleep‐related kinases such as MAPKs and MARKs, and numerous synaptic proteins, including a subset whose phosphorylation status encodes sleep need and determines sleep duration. As a result, microglial TNFα loss attenuates the build‐up of sleep need, as measured by electroencephalogram slow‐wave activity and prevents immediate compensation for loss of sleep. Our data suggest that microglia control sleep homeostasis by releasing TNFα which acts on neuronal circuitry through dynamic control of phosphorylation. Synopsis: Phosphorylation is a molecular determinant of homeostatic sleep control. Microglia, via the release of TNFα, control phosphorylation of proteins involved in sleep and regulate sleep homeostasis when sleep balance is challenged. Microglial TNFα modulates thousands of phosphorylation events in the cortex specifically during the light period, a phase of maximal sleep in mice.Substrates targeted by microglial TNFα comprise several phosphatases and kinases.Microglia‐specific deletion of TNFα in mice disrupts the phosphorylation of proteins important for sleep need and sleep duration at multiple phosphorylation sites.Microglial TNFα is required for the expression of sleep need and control of homeostatic sleep rebound following sleep deprivation. [ABSTRACT FROM AUTHOR]

Silver nanoparticles (AgNPs) have various applications such as their use in the medical field. As they have been reported to show antimicrobial and anti-tumor effects. The main purpose of the current study was to explore the anti-cancer effects of AgNPs administration alone or in a combination with cisplatin (CP) against hepatocellular carcinoma (HCC) in rats. Seventy-five rats (Sprague Dawley albino rats) were used in the present study. Rats were assigned to 5 groups. Group 1 served as normal control. Group 2 was injected intraperitoneally (IP) with a single dose (200 ml/kg) of diethylnitrosamine (DEN), then one week later carbon tetrachloride (CCl4) was injected IP (0.2 ml/kg) two times weekly for 14 successive weeks for the induction of HCC. Group 3 was treated with AgNPs (4 mg/kg) daily. Group 4 was treated with CP (6 mg/kg) once a week. Group 5 was treated with a combination of AgNPs (4 mg/kg/daily) and CP (2.5 mg/kg/weekly). Groups 3, 4, and 5 were treated for 3 successive weeks after induction of HCC. Hematological, biochemical, antioxidant activities, proinflammatory cytokines, and apoptotic genes were evaluated in the current study. Hematological results denoted normocytic normochromic anemia in all examined groups except group 2 which showed macrocytic hypochromic anemia, with thrombocytopenia and leukocytosis in all groups. Significant hypoproteinemia, hypoalbuminemia, and hypoglobulinemia were detected in all groups, with no significant changes (P < 0.05) in globulin in group 5. Significantly decreases in GSH and SOD were recorded in all groups. While the serum AST, ALT activities, and levels of total bilirubin, urea, creatinine, IL-6, TNF-α, AFP, VEGF, BAX, and caspase-3 were markedly elevated. The results revealed a remarkable improvement in group 5 than groups 3, and 4. The obtained results were supported by immunohistochemical and histopathological investigations of the liver tissue. [ABSTRACT FROM AUTHOR]

Supplemental Digital Content is Available in the Text. Oral cancer mouse models were used to demonstrate that sympathetic neurotransmission modulates oral cancer pain and tumor growth through adrenergic signaling in the tumor microenvironment. Head and neck squamous cell carcinoma (HNSCC) causes more severe pain and psychological stress than other types of cancer. Despite clinical evidence linking pain, stress, and cancer progression, the underlying relationship between pain and sympathetic neurotransmission in oral cancer is unknown. We found that human HNSCC tumors and mouse tumor tissue are innervated by peripheral sympathetic and sensory nerves. Moreover, β-adrenergic 1 and 2 receptors (β-ARs) are overexpressed in human oral cancer cell lines, and norepinephrine treatment increased β-AR2 protein expression as well as cancer cell proliferation in vitro. We have recently demonstrated that inhibition of tumor necrosis factor alpha (TNFα) signaling reduces oral cancer-induced nociceptive behavior. Norepinephrine-treated cancer cell lines secrete more TNFα which, when applied to tongue-innervating trigeminal neurons, evoked a larger Ca2+ transient; TNF–TNFR inhibitor blocked the increase in the evoked Ca2+ transient. Using an orthotopic xenograft oral cancer model, we found that mice demonstrated significantly less orofacial cancer-induced nociceptive behavior during systemic β-adrenergic inhibitory treatment with propranolol. Furthermore, chemical sympathectomy using guanethidine led to a significant reduction in tumor size and nociceptive behavior. We infer from these results that sympathetic signaling modulates oral cancer pain through TNFα secretion and tumorigenesis. Further investigation of the role of neurocancer communication in cancer progression and pain is warranted. [ABSTRACT FROM AUTHOR]

Aims: The pathophysiological mechanism(s) underlying non-alcoholic fatty liver disease (NAFLD) have yet to be fully delineated and only a single drug, peroxisome proliferator-activated receptor (PPAR) α/γ agonist saroglitazar, has been approved. Here, we sought to investigate the role of Regulator of G Protein Signaling 7 (RGS7) in hyperlipidemia-dependent hepatic dysfunction. Results: RGS7 is elevated in the livers of NAFLD patients, particularly those with severe hepatic damage, pronounced insulin resistance, and high inflammation. In the liver, RGS7 forms a unique complex with transcription factor ATF3 and histone acetyltransferase Tip60, which is implicated in NAFLD. The removal of domains is necessary for ATF3/Tip60 binding compromises RGS7-dependent reactive oxygen species generation and cell death. Hepatic RGS7 knockdown (KD) prevented ATF3/Tip60 induction, and it provided protection against fibrotic remodeling and inflammation in high-fat diet-fed mice translating to improvements in liver function. Hyperlipidemia-dependent oxidative stress and metabolic dysfunction were largely reversed in RGS7 KD mice. Interestingly, saroglitazar failed to prevent RGS7/ATF3 upregulation but it did partially restore Tip60 levels. RGS7 drives the release of particularly tumor necrosis factor α (TNFα) from isolated hepatocytes, stellate cells and its depletion reverses steatosis, oxidative stress by direct TNFα exposure. Conversely, RGS7 overexpression in the liver is sufficient to trigger oxidative stress in hepatocytes that can be mitigated via TNFα inhibition. Innovation: We discovered a novel non-canonical function for an R7RGS protein, which usually functions to regulate G protein coupled receptor (GPCR) signaling. This is the first demonstration for a functional role of RGS7 outside the retina and central nervous system. Conclusion: RGS7 represents a potential novel target for the amelioration of NAFLD. Antioxid. Redox Signal. 38, 137–159. [ABSTRACT FROM AUTHOR]

The better understanding of the immunopathogenesis of psoriasis has led to the development of highly efficacious targeted therapies with favorable safety profiles. Among them, the class of Interleukin (IL)-17 antibodies are well established for the treatment of psoriasis, psoriatic arthritis and axial spondyloarthritis. Bimekizumab is a new antibody that simultaneously neutralizes IL-17A and IL-17F. We present two patients with psoriasis, who lost response to several biologics, among them IL-17 antagonists such as secukinumab, ixekizumab or brodalumab. Besides plaque-type psoriasis, patients also had psoriasis in hard-to-treat areas such as scalp and groins or psoriatic arthritis. Remarkably, both patients already responded to the therapy with bimekizumab 4 weeks after the first injection and, one year thereafter, both patients sustained PASI100. No side effects were observed. The fast response to bimekizumab emphasizes the crucial role of IL-17F in the pathogenesis of psoriasis. Besides, due to the new mechanism of action, non-responders to other anti-IL-17 therapies could benefit when switched to bimekizumab. [ABSTRACT FROM AUTHOR]

A global increase in older individuals creates an increasing demand to understand numerous healthcare challenges related to aging. This population is subject to changes in tissue physiology and the immune response network. Older individuals are particularly susceptible to infectious diseases, with one of the most common being urinary tract infections (UTIs). Postmenopausal and older women have the highest risk of recurrent UTIs (rUTIs); however, why rUTIs become more frequent after menopause and during old age is incompletely understood. This increased susceptibility and severity among older individuals may involve functional changes to the immune system with age. Aging also has substantial effects on the epithelium and the immune system that led to impaired protection against pathogens, yet heightened and prolonged inflammation. How the immune system and its responses to infection changes within the bladder mucosa during aging has largely remained poorly understood. In this review, we highlight our understanding of bladder innate and adaptive immunity and the impact of aging and hormones and hormone therapy on bladder epithelial homeostasis and immunity. In particular, we elaborate on how the cellular and molecular immune landscape within the bladder can be altered during aging as aged mice develop bladder tertiary lymphoid tissues (bTLT), which are absent in young mice leading to profound age-associated change to the immune landscape in bladders that might drive the significant increase in UTI susceptibility. Knowledge of host factors that prevent or promote infection can lead to targeted treatment and prevention regimens. This review also identifies unique host factors to consider in the older, female host for improving rUTI treatment and prevention by dissecting the age-associated alteration of the bladder mucosal immune system. • Aging is associated with increased susceptibility to rUTIs. • Aged mice develop bladder tertiary lymphoid tissues (bTLT). • bTLT are associated with significant increase in UTI frequency in mice and women. • Urothelial permeability and urobiome species change with age and menopausal status. • Multimodal therapies can lead to bTLT regression and improved rUTI outcomes. [ABSTRACT FROM AUTHOR]

Simple Summary: The nitric oxide donor, NCX4040, a non-steroidal anti-inflammatory-NO donor, is cytotoxic to a number of human tumors, including ovarian tumors cells. It has been shown to be safe in vivo. While the precise mechanism of action of NCX4040 is not clear at this time, NCX4040 has been reported to generate both reactive oxygen species (ROS) and reactive nitrogen species (RNS), causing significant DNA damage in tumor cells. In this study, we provide evidence that NCX4040 induces the differential induction of oxidative stress genes, inflammatory response genes (TNF, IL-1, IL-6 and COX2), DNA damage response and MAP kinase response genes in human ovarian tumor cells. Our studies strongly suggest that formation of ROS/RNS from both induction of NOX and CHAC1 which causes a significant depletion of GSH that results in oxidative stress. Furthermore, NCX4040 treatment induced TNF-dependent pathways which further increases oxidative stress in ovarian tumor cells, resulting in enhanced NCX4040-mediated cell death from apoptosis and/or ferroptosis. The nitric oxide donor, NCX4040 is a non-steroidal anti-inflammatory-NO donor and has been shown to be extremely cytotoxic to a number of human tumors, including ovarian tumors cells. We have found that NCX4040 is cytotoxic against both OVCAR-8 and its adriamycin-selected OVCAR-8 variant (NCI/ADR-RES) tumor cell lines. While the mechanism of action of NCX4040 is not entirely clear, we as well as others have shown that NCX4040 generates reactive oxygen species (ROS) and induces DNA damage in tumor cells. Recently, we have reported that NCX4040 treatment resulted in a significant depletion of cellular glutathione, and formation of both reactive oxygen and nitrogen species (ROS/RNS), resulting in oxidative stress in these tumor cells. Furthermore, our results indicated that more ROS/RNS were generated in OVCAR-8 cells than in NCI/ADR-RES cells due to increased activities of superoxide dismutase (SOD), glutathione peroxidase and transferases expressed in NCI/ADR-RES cells. Further studies suggested that NCX4040-induced cell death may be mediated by peroxynitrite formed from NCX4040 in cells. In this study we used microarray analysis following NCX4040 treatment of both OVCAR-8 and its ADR-resistant variant to identify various molecular pathways involved in NCX4040-induced cell death. Here, we report that NCX4040 treatment resulted in the differential induction of oxidative stress genes, inflammatory response genes (TNF, IL-1, IL-6 and COX2), DNA damage response and MAP kinase response genes. A mechanism of tumor cell death is proposed based on our findings where oxidative stress is induced by NCX4040 from simultaneous induction of NOX4, TNF-α and CHAC1 in tumor cell death. [ABSTRACT FROM AUTHOR]

A certain percentage of carpal tunnel syndrome (CTS) is associated with inflammatory conditions. Osteoarthritis (OA) increases the risk of CTS, and both diseases are common in the general population. Moreover, OA and CTS are often present in the same patients. Since inflammation and fibrosis are found in both conditions, the question is whether circulating inflammatory cytokines and cytokines involved in fibrosis in OA and CTS patients could serve as indicators of coexisting CTS and OA pathology. This investigation was performed on 31 CTS patients, 29 hip OA patients, and 15 healthy volunteers. Blood samples were collected, and serum levels of TGF-β1, BMP-7, IL-1β, and TNFα were measured using the ELISA method. The statistical analysis was performed to reveal the most significant differences in the serum levels of these cytokines. Statistical significance was set at p-values ≤   0.05. The serum level of TGF-β1 was the highest in CTS patients (16.36 pg/mL) and significantly different compared to OA and healthy control. Analysis of the cytokine serum level in the subdivided group revealed that serum levels of TGF-β1 and BMP-7 were significantly higher in CTS+/OA+ patients as well as BMP-7 in the OA+/CTS+ group. There was no significant difference in serum levels of the inflammatory cytokines TNFα and IL-1β among all groups. This study showed that in the end stage of CTS and OA, serum levels of inflammatory cytokines (IL1-β and TNFα) were not altered, while the serum levels of TGF-β1 and BMP-7 were significantly higher, especially in patients with coexisting OA and CTS. These findings suggest the possible values of TGF-β1 and BMP-7 as a predictive factor for the comorbidity of CTS and OA. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS), which are formed under the action of ionizing radiation and inflammation factors, react with lipids and lead to the formation of toxic metabolites; one of the most active among them is 4-hydroxy-2-nonenal (4-HNE). It modifies cell proteins by lysine, histidine, and cysteine residues and disrupts their functions. The purpose of this research is to assess the number of neurons containing 4-HNE-modified proteins two months after γ-irradiation of the head at doses of 2, 8, and 20 Gy and determine the development of neuroinflammation at this period. Mouse brain cells were isolated by centrifugation through Percoll solution after brain tissue treatment with accutase. The number of neurons and neurons containing 4-HNE-modified proteins, as well as the number of cells of resting and activated microglia, were analyzed using flow cytometry; the level of expression of proinflammatory cytokine TNFα gene in the hippocampus was analyzed using RT-PCR. It is shown that 14.3 ± 2.3% of neurons in the brains of control mice contain 4-HNE-modified proteins. Their number increased to 23.1 ± 2.6 and 34.8 ± 4.9% (p < 0.05) two months after irradiation of the head of mice at doses of 8 and 20 Gy. An increase in the proportion of activated microglia in isolated brain cells and the level of TNFα gene expression in the hippocampus in this period indicates the development of neuroinflammation. Therefore, in addition to direct damage during irradiation, neurons are exposed to ROS and toxic metabolites of lipid peroxidation, which are formed both during irradiation and during neuroinflammation developing in the long-term period after radiation exposure. The results of this research make it possible to consider the increase in the amount of 4-HNE-modified proteins in neurons as a marker of oxidative damage to these brain cells. [ABSTRACT FROM AUTHOR]

Schizophrenia (SCZ) is a chronic neurodevelopmental psychotic disorder. The immune system and neuroinflammation seem to play a central role in the pathophysiology of SCZ. Clozapine is an effective atypical antipsychotic used for treatment-resistant SCZ. Life-threatening side effects, such as myocarditis, limit its use. We investigated the immunomodulatory effects of clozapine in an astroglial model of neuroinflammation. We thus assessed the effect of clozapine on the production of inflammatory mediators in human-derived astroglial (A172) cells, stimulated with a cytokine mix (TNFα, IL-1β, IFNγ). RT-PCR and ELISA analyses demonstrated that clozapine suppressed gene expression and production of TNFα, IL-1β and IL-8 and increased COX2 mRNA 24 h after stimulation. Clozapine inhibited Akt phosphorylation induced by the cytokine mix at 10 min and 40 min, as assessed by Western blot analysis with anti-pT308Akt antibody. Pretreatment with the Akt inhibitor MK-2206 increased COX2 gene expression in cytokine-stimulated cells, suggesting that Akt inhibition may be involved in COX2 gene expression upregulation. Clozapine may possess dual beneficial effects: inhibiting astroglial production of proinflammatory cytokines, thus attenuating neuroinflammation, and upregulating COX2 expression that may be relevant to improvement of neural functioning while accounting for some of its detrimental effects. Patients with TRS and neuroinflammatory markers may benefit particularly from clozapine treatment. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) occurs not only in the knee but also in peripheral joints throughout the whole body. Previously, we have shown that the expression of cellular communication network factor 3 (CCN3), a matricellular protein, increases with age in knee articular cartilage, and the misexpression of CCN3 in cartilage induces senescence-associated secretory phenotype (SASP) factors, indicating that CCN3 promotes cartilage senescence. Here, we investigated the correlation between CCN3 expression and OA degenerative changes, principally in human femoral head cartilage. Human femoral heads obtained from patients who received total hip arthroplasty were categorized into OA and femoral neck fracture (normal) groups without significant age differences. Gene expression analysis of RNA obtained from femoral head cartilage revealed that CCN3 and MMP-13 expression in the non-weight-bearing part was significantly higher in the OA group than in the normal group, whereas the weight-bearing OA parts and normal cartilage showed no significant differences in the expression of these genes. The expression of COL10A1, however, was significantly higher in weight-bearing OA parts compared with normal weight-bearing parts, and was also higher in weight-bearing parts compared with non-weight-bearing parts in the OA group. In contrast, OA primary chondrocytes from weight-bearing parts showed higher expression of CCN3, p16, ADAMTS4, and IL-1β than chondrocytes from the corresponding normal group, and higher ADAMTS4 and IL-1β in the non-weight-bearing part compared with the corresponding normal group. Acan expression was significantly lower in the non-weight-bearing group in OA primary chondrocytes than in the corresponding normal chondrocytes. The expression level of CCN3 did not show significant differences between the weight-bearing part and non-weight-bearing part in both OA and normal primary chondrocytes. Immunohistochemical analysis showed accumulated CCN3 and aggrecan neoepitope staining in both the weight-bearing part and non-weight-bearing part in the OA group compared with the normal group. The CCN3 expression level in cartilage had a positive correlation with the Mankin score. X-ray analysis of cartilage-specific CCN3 overexpression mice (Tg) revealed deformation of the femoral and humeral head in the early stage, and immunohistochemical analysis showed accumulated aggrecan neoepitope staining as well as CCN3 staining and the roughening of the joint surface in Tg femoral and humeral heads. Primary chondrocytes from the Tg femoral head showed enhanced expression of Ccn3, Adamts5, p16, Il-6, and Tnfα, and decreased expression of Col2a1 and -an. These findings indicate a correlation between OA degenerative changes and the expression of CCN3, irrespective of age and mechanical loading. Furthermore, the Mankin score indicates that the expression level of Ccn3 correlates with the progression of OA. [ABSTRACT FROM AUTHOR]

Rotator cuff tendon (RCT) disease results from multifactorial mechanisms, in which inflammation plays a key role. Pro-inflammatory cytokines and tendon stem cell/progenitor cells (TSPCs) have been shown to participate in the inflammatory response. However, the underlying molecular mechanism is still not clear. In this study, flow cytometry analyses of different subpopulations of RCT-derived TSPCs demonstrate that after three days of administration, TNFα alone or in combination with IFNγ significantly decreases the percentage of CD146+CD49d+ and CD146+CD49f+ but not CD146+CD109+ TSPCs populations. In parallel, the same pro-inflammatory cytokines upregulate the expression of CD200 in the CD146+ TSPCs population. Additionally, the TNFα/IFNγ combination modulates the protein expression of STAT1, STAT3, and MMP9, but not fibromodulin. At the gene level, IRF1, CAAT (CAAT/EBPbeta), and DOK2 but not NF-κb, TGRF2 (TGFBR2), and RAS-GAP are modulated. In conclusion, although our study has several important limitations, the results highlight a new potential role of CD200 in regulating inflammation during tendon injuries. In addition, the genes analyzed here might be new potential players in the inflammatory response of TSPCs. [ABSTRACT FROM AUTHOR]

As a widely prescribed anti-diabetic drug, metformin has been receiving novel attention for its analgesic potential. In the study of the complex etiology of neuropathic pain (NeP), male and female individuals exhibit quite different responses characterized by higher pain sensitivity and greater NeP incidence in women. This "gender gap" in our knowledge of sex differences in pain processing strongly limits the sex-oriented treatment of patients suffering from NeP. Besides, the current investigation of the analgesic potential of metformin has not addressed the "gender gap" problem. Hence, this study focuses on metformin and sex-dependent analgesia in a murine model of NeP induced by chronic constriction injury of the sciatic nerve. We investigated sexual dimorphism in signaling pathways involved by 7 days of metformin administration, such as changes in AMP-activated protein kinase and the positive regulation of autophagy machinery, discovering that metformin affected in a sexually dimorphic manner the immunological and inflammatory response to nerve lesion. These effects were complemented by morphological and adaptive changes occurring after peripheral nerve injury. Altogether these data can contribute to explaining a number of potential mechanisms responsible for the complete recovery from NeP found in male mice, as opposed to the failure of long-lasting recovery in female animals. [ABSTRACT FROM AUTHOR]

Background: In the recent years, the focus on taking out biologically active molecules from animals' byproducts, especially from marine organisms have been increased. These sources play a major role in extracting collagen to be used as a biomaterial for medical applications and in the food industry. However, collagen-producing firms can be found all around the world, but not all of them kind 100% pure collagen; instead, they make gelatin hydrolyzed collagen. These businesses lack additional collagen development. Therefore, extracting collagen from fish scales as a potential raw material and using it to study high-value applications might potentially produce an economic potential for anti-inflammatory protein. Methodology: Isolation and characterization methods were undertaken when fish scales (450gm) were procured from a local market, then washed with distilled water several times to remove any dirt, dust, and unwanted materials from the scales. Then, they were soaked with (0.2 M) of NaOH2 for 12 hours to get rid of non-collagen proteins. For demineralization purposes, they were immersed with (0.5 M) of HCl for 12 hours. Later, for digestion to be carried out (0.5 M) of acetic acid was added and drenched for about 36 hours with stirring performed at 4 ºC. after the digestions step, the samples were filtered. Moreover, collagen was detected and characterized by the size exclusion chromatography (SEC) assay. Furthermore, the extracted collagen was used in vivo to detect fish-tumor necrosis factor (TNFα) to notice the effect of extracted collagen as an anti-inflammatory protein as a part of the medical application method. Results: The SEC assay revealed that Col-I is purified and separated as soluble monomeric protein (53 min) fractions which correspond to a molecular weight of roughly 300 kDa. Also, the result indicated a mild significant decrease of collagen on TNFα (down-regulation). Conclusion: In summary, we conducted to use collagen for biomedical and pharmaceutical purposes as an antiinflammatory. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is associated with brain amyloid‐β (Aβ) peptide accumulation and neuroinflammation. Currants, a low glycemic index dried fruit, and their components display pleiotropic neuroprotective effects in AD. We examined how diet containing 5% Corinthian currant paste (CurD) administered in 1-month-old 5xFAD mice for 1, 3, and 6 months affects Aβ levels and neuroinflammation in comparison to control diet (ConD) or sugar-matched diet containing 3.5% glucose/fructose (GFD). No change in serum glucose or insulin levels was observed among the three groups. CurD administered for 3 months reduced brain Aβ42 levels in male mice as compared to ConD and GFD, but after 6 months, Aβ42 levels were increased in mice both on CurD and GFD compared to ConD. CurD for 3 months also reduced TNFα and IL-1β levels in male and female mouse cortex homogenates compared to ConD and GFD. However, after 6 months, TNFα levels were increased in cortex homogenates of mice both on CurD and GFD as compared to ConD. A similar pattern was observed for TNFα-expressing cells, mostly co-expressing the microglial marker CD11b, in mouse hippocampus. IL-1β levels were similarly increased in the brain of all groups after 6 months. Furthermore, a time dependent decrease of secreted TNFα levels was found in BV2 microglial cells treated with currant phenolic extract as compared to glucose/fructose solution. Overall, our findings suggest that a short-term currant consumption reduces neuroinflammation in 5xFAD mice as compared to sugar-matched or control diet, but longer-term intake of currant or sugar-matched diet enhances neuroinflammation. [ABSTRACT FROM AUTHOR]

Background: Uncertain effects of probiotics and/or prebiotics have been reported in experimental and clinical colitis. This study aims to examine the effects of a synbiotic combination comprising Bacillus licheniformis DSM 17236 and Saccharomyces cerevisiae cell wall extract on dextran sulfate sodium (DSS)-induced colitis in Sprague Dawley rats. Methods: Acute colitis was induced in rats by oral administration of DSS 3.5% for 7 days. Fifty rats were divided equally into five groups; one control group and the other groups were induced with colitis and treated with or without the tested synbiotic, mixed with diet, for 28 days and sulfasalazine (100 mg/kg) via intragastric tube once daily for 14 days. Results: Symptomatically, the synbiotic administration raised the disease activity index (DAI) to comparable scores of the DSS group, specially from the 2nd to 7th days post DSS intoxication. It also induced a significant (p < 0.05) amplification of WBCs, myeloperoxidase (MPO), malondialdehyde (MDA), nuclear factor kappa B (NF-kB) expression and proinflammatory cytokines tumor necrosis factor alpha (TNFα), interferon gamma (INFγ), and interleukin-1 beta (IL-1β) while depressed the antioxidant enzymes glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD) when compared with the DSS and control groups. The DSS intoxicated and Synbiotic+DSS groups showed desquamations of the covering epithelium, noticeable diffuse leukocytic infiltrations, sever catarrhal enteritis, ischemic colitis with diffuse coagulative necrosis of the entire colonic mucosa. Contrarily, sulfasalazine proved to be effective in the reduction of the tested inflammatory markers and the pathological degenerative changes of the DSS ulcerative colitis. Conclusion: The examined synbiotic did not ameliorate but aggravated the DSS-induced colitis, so it should be subjected to intensive experimental and clinical testing before their use in animals and human. [ABSTRACT FROM AUTHOR]

Simple Summary: Breast cancer is the most prominent cancer among women. Therapeutic resistance and reoccurrence of cancer after various therapies, such as chemotherapy, radiotherapy etc., is the major cause of death. In various studies, it has been shown that dysregulation or defect in the cell death mechanisms such as apoptosis and autophagy is the key reason behind the issues. An increase in the level of inhibitors of apoptosis (IAPs) proteins and autophagy is founded in various cancers. Besides, the SMAC proteins that inhibit the action of IAPs are also found to be downregulated. In this study, BV6, a SMAC mimetic compound, not only induces apoptosis via inhibiting the action of IAPs but also downregulates autophagy. In addition, promising anticancer results were obtained after treating the breast cancer cells with BV6 in combination with death ligands such as TNF-related apoptosis-inducing ligand (TRAIL) and Tumor necrosis factor-α (TNF-α). It is an investigational study, and to the best of our knowledge, our study, for the first time, demonstrated the effect of BV6 on autophagy. This study presents the significance of SMAC mimetic compounds in the downregulation of factors responsible for cancer resistance and reoccurrence. The study may well provide a potent therapeutic target for the development of novel anti-cancer therapy using SMAC mimetics. Cancer is the utmost common disease-causing death worldwide, characterized by uncontrollable cell division with the potential of metastasis. Overexpression of the Inhibitors of Apoptosis proteins (IAPs) and autophagy correlates with tumorigenesis, therapeutic resistance, and reoccurrence after anticancer therapies. This study illuminates the role and efficacy of smac mimetic compound BV6 alone and in co-treatment with death ligands such as TRAIL and TNFα in the regulation of cell death mechanisms, i.e., apoptosis and autophagy. In this study, MTT assays, wound healing assays, and cellular and nuclear morphological studies were done. DAPI staining, AO/EtBr staining and AnnexinV/PI FACS was done to study the apoptosis. The expression of IAPs and autophagy biomarkers was analyzed using Real time-PCR and western blotting. Meanwhile, TEM demonstrated autophagy and cellular autophagic vacuoles in response to the BV6. The result shows a promising anti-cancer effect of BV6 alone as well as in combinational treatment with TRAIL and TNFα, compared to the lone treatment of TRAIL and TNFα in both breast cancer cell lines. The smac mimetic compound might provide an alternative combinational therapy with conventional anticancer therapies to tackle their inefficiency at the advanced stage of cancer, cancer resistance, and reoccurrence. Also, IAPs and autophagic proteins could act as potent target molecules for the development of novel anti-cancer drugs in pathogenesis and the betterment of regimens for cancer. [ABSTRACT FROM AUTHOR]

IFNγ is traditionally known as a proinflammatory cytokine with diverse roles in antimicrobial and antitumor immunity. Yet, findings regarding its sources and functions during the regeneration process following a sterile injury are conflicting. Here, we show that natural killer (NK) cells are the main source of IFNγ in regenerating muscle. Beyond this cell population, IFNγ production is limited to a small population of T cells. We further show that NK cells do not play a major role in muscle regeneration following an acute injury or in dystrophic mice. Surprisingly, the absence of IFNγ per se also has no effect on muscle regeneration following an acute injury. However, the role of IFNγ is partially unmasked when TNFα is also neutralized, suggesting a compensatory mechanism. Using transgenic mice, we showed that conditional inhibition of IFNGR1 signaling in muscle stem cells or fibro-adipogenic progenitors does not play a major role in muscle regeneration. In contrast to common belief, we found that IFNγ is not present in the early inflammatory phase of the regeneration process but rather peaks when macrophages are acquiring an anti-inflammatory phenotype. Further transcriptomic analysis suggests that IFNγ cooperates with TNFα to regulate the transition of macrophages from pro- to anti-inflammatory states. The absence of the cooperative effect of these cytokines on macrophages, however, does not result in significant regeneration impairment likely due to the presence of other compensatory mechanisms. Our findings support the arising view of IFNγ as a pleiotropic inflammatory regulator rather than an inducer of the inflammatory response. [ABSTRACT FROM AUTHOR]

Background: Complications of non-alcoholic fatty liver disease (NAFLD) include 67% of the criteria for metabolic syndrome. Acalypha indica L., (AI) which is one of a herbal plant had been known as anti-oxidant and anti-inflammatory effects. The effect of AI for therapy investigated by looking of the immune defense mechanisms. This researched was assessed by molecular docking approached on TLR9, NFκB, TNFα expression and liver morphological changes. Methods: Animal models of steatohepatitis were collected from high-fructose and cholesterol diet (HFCD) of Sprague-Dawley rats for 12 weeks and followed by therapy for 8 weeks. There were 5 groups from twenty five researched rats, include normal group (K1), HFCD group (K2), HFCD group supplemented with 400 mg Acalypha indica L. (K3), combination between 400 mg AI+Gemfibrozil (Gem) 31 mg (K4) and Gem 31 mg/kg (K5) in kgBW, respectively. Results: The results of molecular docking were carried out by assessing the interaction between hydrogen molecules of AI compounds and amino acid residues in TLR9, NFκB, TNFα. Morphological changes were assessed by scoring system. Statistical analyzed used Kruskall Wallis with post hoc Mann Whitney test continued by Spearman correlation test. Conclusion: The molecular docking analysis showed that, an alkaloid compounds were found besides the flavonoid compounds that can bind to the binding pocket of inflammatory markers with the best binding energies. Other compounds, there are dasycarpidan-1-methanol, acetate (ester), fenofibrate and quinine. Supplementation of AI would reduced hypertrophy (p=0.031), macrovesicular steatosis (p=0.018), inflammation foci (p=0.005) and also decreased of TLR9 (p=0.009), NFκB (p=0.009), TNFα (p=0.009) expression, but not as good as the combination of AI+Gem. [ABSTRACT FROM AUTHOR]

Metformin is the approved medication for managing global health issues concerning type 2 diabetes mellitus (T2DM). Using natural bioactive compounds as an alternative therapy is crucial to managing several metabolic diseases. Therefore, due to recent limited studies that detected the role of bee venom (BV) in improving diabetic conditions in Iraq, the current study was designed to identify the potential therapeutic role of BV and metformin in diabetic mice. Twenty male mice (Balb/c) aged about 60 days with an average weight of 26.55±2.70 g were randomly divided into four groups (n=5). The animals were placed in plastic cages for acclimatization for one week of access to food and water ad libitum. Overnight fasting was applied to 15 mice which were then injected with 95 mg/kg body weight of prepared alloxan. The mice were supplemented with glucose fluid for 3 days. On day 4, the blood was collected from the tail to measure the circulating glucose level. When blood glucose levels exceed 200 mg/dl, the animals are considered diabetic. After induction of diabetes, the animals were divided as follows: Control group: included five mice that were not subjected to diabetes induction; the animals in this group did not receive any medications. Diabetic group: including five mice confirmed with diabetes without receiving any treatments. Metformin group: including five diabetic mice exposed to a single oral dose of 150 mg/kg of metformin for 30 days. Bee Venom group: including five diabetic mice exposed to a single intraperitoneal dose of 1 mg/kg Bee Venom for 30 days. After 30 days of treatment, blood was drained, and serum was obtained to detect the levels of glucose, insulin, TNFα, IL6, and IL10 by using precise enzyme linked immunosorbent assay (ELIZA) kits. Also, the pancreas was collected from all mice for histopathological investigation. The result displayed significantly elevated glucose concentration in diabetic mice, while metformin and BV significantly reversed these increases. A significant decline in insulin concentration was seen in diabetic mice, whereas metformin and BV significantly enhanced this reduction in insulin concentration. Furthermore, mice treated with alloxan exhibited remarkable increases in TNFα and IL6 compared to control mice, while supplemented metformin and BV significantly reduced these high concentrations. Moreover, the level of IL10 markedly declined in diabetic mice, which was reversed significantly in response to metformin and BV. Histological detection of the pancreas in diabetic mice showed significant changes in the shape and size of islets associated with the arrangement and number of beta cells with a reduction of islets covering connective tissue. Metformin slightly restored these alterations; however, significant and remarkable restoring of histological changing was promoted by BV. Thus, BV could be a potential agent for managing metabolic disorders including diabetes. [ABSTRACT FROM AUTHOR]

The complex relationship between inflammation, its effects on neuronal excitability and the ensuing plasticity of dorsal root ganglion (DRG) sensory neurons remains to be fully explored. In this study, we have employed a system of experiments assessing the impact of inflammatory conditioned media derived from activated immune cells on the excitability and activity of DRG neurons and how this relates to subsequent growth responses of these cells. We show here that an early phase of increased neuronal activity in response to inflammatory conditioned media is critical for the engagement of plastic processes and that neuronal excitability profiles are linked through time to the structural phenotype of individual neurons. Pharmacological blockade of neuronal activity was able to abolish the growth-promoting effects of inflammatory media. Our results suggest that targeting the activity of DRG neurons may provide a novel therapeutic avenue to manipulate their growth status and potential for plasticity in response to inflammation. Importantly, the same pharmacological blockade in vivo abolished pain responses in a mouse model of multiple sclerosis. While further studies are needed to fully elucidate the underlying mechanisms of the relationship between neural activity and growth status, a more complete understanding of this relationship may ultimately lead to the development of new treatments for neuropathic pain in disorders associated with heightened immune responses such as rheumatoid arthritis and multiple sclerosis., (© 2024 The Author(s). Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Overproduction of uric acid caused by increased expression and/or enhanced activity of xanthine oxidase (XO) is one of the major etiologies of hyperuricemia, which had a significant sex differences. As an important enzyme involved in production of reactive oxygen species and uric acid, activity of XO is highly correlated with hyperuricemia and its complications. However, the mechanisms underlying XO dysregulation remain unclear, and sex difference in the prevalence of hyperuricemia has been well known. To explore the potential role of intestinal farnesoid X receptor (FXR) on XO regulation and production, and the mechanisms of sex differences in this pathological process. Two hundred and sixty-one dyslipidemia participants and intestine-specific FXR-knockout mice were used to study the relationship between the intestinal FXR and the serum uric acid level. Western blotting, quantitative real-time PCR, and dual-luciferase reporter assay, were applied to clarify the regulatory role of FXR deficiency on XO. Special inhibitors, agonists, siRNA, sex hormones were used to investigate the mechanism of sex difference in FXR deficiency induced hyperuricemia in cell and animal model. Serum fibroblast growth factor 19 (FGF19) levels were lower in hyperuricemia patients in a sex difference manner. Increased local TNFα level driven by intestinal FXR deficiency/inhibition induced overexpression and hyperactivity of intestinal XO, leading to elevated intestinal uric acid synthesis, and subsequently resulting in hyperuricemia. We found that estrogens inhibited XO expression and activity, whereas androgens enhanced XO activity, leading to the sex difference in FXR deficiency induced hyperuricemia. Infliximab treatment eliminated the sex difference in uric acid levels in intestinal FXR-knockout mice. This study demonstrated the role of intestinal FXR in the pathogenesis of hyperuricemia, and partially elucidated the mechanisms underlying the sex differences of hyperuricemia., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Microglia are the most important immune cells in the central nervous system (CNS), which can defend against external pathogens and stimuli. Dysregulation of microglia releases excessive proinflammatory cytokines and leads to neuroinflammation, which is fundamental to the pathophysiology of multiple neurological diseases. However, the molecular mechanisms underlying the regulation of proinflammatory cytokines in microglia are still not well-understood. Here, we identified that inhibitor of DNA binding protein 2 (Id2) was a negative regulator of tumor necrosis factor-α (TNFα) in cultured microglia. Knockdown of Id2 significantly increased the expression of TNFα in microglia, while overexpression of Id2 inhibited TNFα expression. Furthermore, by interacting with the p65 subunit of nuclear factor kappa-B (NF-κB), Id2 suppressed the transcription activation of NF-κB and inhibited TNFα expression. Interestingly, in lipopolysaccharides (LPS)-treated microglia, Id2 increased and underwent a cytoplasmic relocation. Immunoprecipitation and immunostaining results showed that by binding to the LIM domain of Id2, a scaffold protein PDZ and LIM 5 (PDLIM5) involved in the Id2 cytoplasmic relocation, which inactivated Id2 and resulted in higher TNFα expression in LPS-treated microglia. Collectively, our data delineate a novel effect of Id2 on TNFα regulation in microglia, which may shed a light on the proinflammatory cytokines regulating in microglia associated neuroimmune disorders., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Podocytes express large-conductance Ca 2+ -activated K + channels (BK channels) and at least two different pore-forming KCa1.1 subunit C-terminal splice variants, known as VEDEC and EMVYR, along with auxiliary β and γ subunits. Podocyte KCa1.1 subunits interact directly with TRPC6 channels and BK channels become active in response to Ca 2+ influx through TRPC6. Here, we confirmed that Ca 2+ influx through TRPC channels is reduced following the blockade of BK channels by paxilline. The overall abundance of KCa1.1 subunits, as well as that of β4 and γ3 subunits, were increased in glomeruli isolated from Sprague Dawley rats during chronic puromycin aminonucleoside (PAN) nephrosis. Exposing cultured mouse podocytes for 24 h to recombinant TNFα, a circulating factor implicated in pediatric nephrotic syndromes, did not affect the total abundance of KCa1.1, but did evoke significant increases in both β4 and γ3. However, TNFα evoked a marked increase in the surface abundance of KCa1.1 subunits, similar to that of its previously reported effects on TRPC6 channels. The effect of TNFα on the surface expression of KCa1.1 was eliminated following siRNA knockdown of the β4 subunits, suggesting a role for this subunit in KCa1.1 trafficking to the cell surface. By contrast, treating podocytes with suPAR did not affect the total or surface expression of KCa1.1. The coordinated activation of KCa1.1 channels may promote Ca 2+ influx through TRPC channels during normal and abnormal podocyte function by maintaining a membrane potential that allows for the efficient permeation of divalent cations through TRPC pores.

Diabetes mellitus type 2 (DMT2) promotes Achilles tendon (AS) degeneration and exercise could modulate features of DMT2. Hence, this study investigated whether tenocytes of non DMT2 and DMT2 rats respond differently to normo- (NG) and hyperglycemic (HG) conditions in the presence of tumor necrosis factor (TNF)α or cyclic stretch. AS tenocytes, isolated from DMT2 (fa/fa) or non DMT2 (lean, fa/+) adult Zucker Diabetic Fatty (ZDF) rats, were treated with 10 ng/mL TNFα either under NG or HG conditions (1 g/L vs. 4.5 g/L glucose) and were exposed to cyclic stretch (14%, 0.3 Hz, 48 h). Tenocyte survival, metabolic activity, gene and/or protein expression of tendon extracellular matrix component collagen type 1, alpha smooth muscle actin (αSMA, Acta2), the stress defense enzyme heme oxygenase-1 (Hmox1) as well as suppressors of cytokine signaling (Socs)1 and Socs3 were analyzed. Tenocyte vitality remained high, but metabolic activity was slightly impaired by HG conditions irrespectively of cell origin. Collagen type 1 alpha protein and gene expression was suppressed by TNFα, but only in cells of non DMT2 animals in NG culture medium. Higher amounts of αSMA were visualized in tendons/tenocytes of diabetic rats or those exposed to TNFα. Cyclic stretch caused cell alignment in zero stretch direction. In addition, it led to a significant reduction of cell perimeters, particularly in cells of DMT2 donor rats under HG conditions. Hmox1, Socs1 and Socs3 were induced by HG, but only in tenocytes of diabetic rats (4 h). Stretch induced significantly Hmox1 transcriptional activity under NG conditions and Socs3 under HG conditions especially in tenocytes of DMT2 rats. The response of tenocytes to TNFα and cyclic stretch depends on glucose supply and origin suggesting their irreversible impairment by DMT2., (© 2024 The Author(s). Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.)

Background: Ginkgo biloba leaf extract EGb 761® has shown clinical efficacy in patients with mild cognitive impairment and dementia. However, the pharmacological action of EGb 761® in Alzheimer's disease (AD) remains unclear and molecular mechanisms targeted in the brain are not completely understood., Hypothesis/purpose: We aimed to investigate 1) the potential sex-dependent effects of oral administration of EGb 761® in 5xFAD mice, an AD mouse model, and 2) the underlying microglial subtype responsible for the observed anti-inflammatory effects in the brain., Methods: Eight-week old 5xFAD and wild type mice received EGb 761®-supplemented diet or control diet for eight weeks. The study investigated changes in cognitive function as well as amyloid plaque load, expression of AD-related genes, and anti-inflammatory effects. Moreover, we used organotypic brain slices for confirmation and assessment of concentration-dependency of the observed EGb 761® effects and performed single cell RNA sequencing on the prefrontal cortex of male mice with focus on microglia., Results: We demonstrate that EGb 761® treatment improves cognitive function in 5xFAD mice in several behavioral tests. Analysis of the brain tissue from these animals indicated a reduction in amyloid plaque load in the prefrontal cortex (PFC). This brain area was further investigated to assess the molecular changes that occurred following EGb 761® intake. Alterations in the expression of genes related to AD were highly sex-specific with effects on the cholinergic system, the γ-secretase complex, and neuroinflammation. Anti-inflammatory effects of EGb 761® with a particularly pronounced reduction of the TNFα-response could be shown for the PFC but also peripherally in the serum of 5xFAD mice of both sexes. Single-cell RNA sequencing revealed that EGb761® mainly affected disease-associated microglia stage 2 (DAM2), which are thought to have a detrimental role in AD., Conclusions: EGb 761® shows efficacy in the treatment of cognitive deficits in the 5xFAD mouse model via multimodal activity, including sex-specific and sex-unrelated mechanisms including the normalization of neuroinflammatory parameters., Competing Interests: Declaration of competing interest S.W. and M.D.L. are employees of Dr. Willmar Schwabe GmbH & Co. KG, a producer of medicinal products containing the special extract from Ginkgo biloba leaves (EGb 761®). The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kristina Endres reports financial support and equipment, drugs, or supplies were provided by Dr Willmar Schwabe GmbH und Co KG Kristina Endres reports financial support was provided by MWG Rhineland-Palatinate. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier GmbH.)

Dental caries is one of the most common health issues worldwide arising from the complex interactions of bacteria. In response to harmful stimuli, desirable outcome for the tooth is the formation of tertiary dentin, a protective reparative process that generates new hard tissue. This reparative dentinogenesis is associated with significant inflammation, which triggers the recruitment and differentiation of dental pulp stem cells (DPSCs). Previously, we have shown that brain-derived neurotrophic factor (BDNF) and its receptor TrkB, key mediators of neural functions, are activated during the DPSC-mediated dentin regeneration process. In this study, we further define the role of inflammation in this process and apply stem cell engineering to enhance dentin regeneration in injured teeth. Our data show that TrkB expression and activation in DPSCs rapidly increase during odontogenic differentiation, further amplified by inflammatory inducers and mediators such as TNFα, LTA, and LPS. An in vivo dentin formation assessment was conducted using a mouse pulp-capping/caries model, where CRISPR-engineered DPSCs overexpressing BDNF were transplanted into inflamed pulp tissue. This transplantation significantly enhanced dentin regeneration in injured teeth. To further explore potential downstream pathways, we conducted transcriptomic profiling of TNFα-treated DPSCs, both with and without TrkB antagonist CTX-B. The results revealed significant changes in gene expression related to immune response, cytokine signaling, and extracellular matrix interactions. Taken together, our study advances our understanding of the role of BDNF in dental tissue engineering using DPSCs and identifies potential therapeutic avenues for improving dental tissue repair and regeneration strategies., Competing Interests: Conflicts of Interest: The authors declare no conflict of interest.

Obesity has become a global epidemic and is associated with comorbidities, including diabetes, cardiovascular, and neurodegenerative diseases, among others. While appreciable insight has been gained into the mechanisms of obesity-associated comorbidities, effects of age, and duration of obesity on the female brain remain obscure. To address this gap, adolescent and mature adult female mice were subjected to a high-fat diet (HFD) for 13 or 26 weeks, whereas age-matched controls were fed a standard diet. Subsequently, the expression of inflammatory cytokines, neurotrophic/neuroprotective factors, and markers of microgliosis and astrogliosis were analyzed in the hypothalamus, hippocampus, and cerebral cortex, along with inflammation in visceral adipose tissue. HFD led to a typical obese phenotype in all groups independent of age and duration of HFD. However, the intermediate duration of obesity induced a limited inflammatory response in adolescent females' hypothalamus while the hippocampus, cerebral cortex, and visceral adipose tissue remained unaffected. In contrast, the prolonged duration of obesity resulted in inflammation in all three brain regions and visceral adipose tissue along with upregulation of microgliosis/astrogliosis and suppression of neurotrophic/neuroprotective factors in all brain regions, denoting the duration of obesity as a critical risk factor for neurodegenerative diseases. Importantly, when female mice were older (i.e., mature adult), even the intermediate duration of obesity induced similar adverse effects in all brain regions. Taken together, our findings suggest that (1) both age and duration of obesity have a significant impact on obesity-associated comorbidities and (2) early interventions to end obesity are critical to preserving brain health., (© 2024 The Author(s). Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Obesity is associated with a low-grade chronic inflammatory process characterized by higher circulating TNFα levels, thus contributing to insulin resistance. This study evaluated the effect of silybin, the main bioactive component of silymarin, which has anti-inflammatory properties, on TNFα levels and its impact on glucose uptake in the adipocyte cell line 3T3-L1 challenged with two different inflammatory stimuli, TNFα or lipopolysaccharide (LPS). Silybin's pre-treatment effect was evaluated in adipocytes pre-incubated with silybin (30 or 80 µM) before challenging with the inflammatory stimuli (TNFα or LPS). For the post-treatment effect, the adipocytes were first challenged with the inflammatory stimuli and then post-treated with silybin. After treatments, TNFα production, glucose uptake, and GLUT4 protein expression were determined. Both inflammatory stimuli increased TNFα secretion, diminished GLUT4 expression, and significantly decreased glucose uptake. Silybin 30 µM only reduced TNFα secretion after the LPS challenge. Silybin 80 µM as post-treatment or pre-treatment decreased TNFα levels, improving glucose uptake. However, glucose uptake enhancement induced by silybin did not depend on GLUT4 protein expression. These results show that silybin importantly reduced TNFα levels and upregulates glucose uptake, independently of GLUT4 protein expression.

The noble gas argon is one of the most promising neuroprotective agents for hypoxic-reperfusion injuries of the brain. However, its effect on traumatic injuries has been insufficiently studied. The aim of this study was to analyze the effect of the triple inhalation of the argon-oxygen mixture Ar 70%/O 2 30% on physical and neurological recovery and the degree of brain damage after traumatic brain injury and to investigate the possible molecular mechanisms of the neuroprotective effect. The experiments were performed in male Wistar rats. A controlled brain injury model was used to investigate the effects of argon treatment and the underlying molecular mechanisms. The results of the study showed that animals with craniocerebral injuries that were treated with argon inhalation exhibited better physical recovery rates, better neurological status, and less brain damage. Argon treatment significantly reduced the expression of the proinflammatory markers TNFα and CD68 caused by TBI, increased the expression of phosphorylated protein kinase B (pAKT), and promoted the expression of the transcription factor Nrf2 in intact animals. Treatment with an argon-oxygen breathing mixture after traumatic brain injury has a neuroprotective effect by suppressing the inflammatory response and activating the antioxidant and anti-ischemic system.

Obesity promotes metabolic diseases such as type 2 diabetes and cardiovascular disease. PKCδI is a serine/threonine kinase which regulates cell growth, differentiation, and survival. Caspase-3 cleavage of PKCδI releases the C-terminal catalytic fragment (PKCδI&#95;C), which promotes inflammation and apoptosis. We previously demonstrated an increase in PKCδI&#95;C in human obese adipose tissue (AT) and adipocytes. Subsequently, we designed a small molecule drug called NP627 and demonstrated that NP627 specifically inhibited the release of PKCδI&#95;C in vitro. Here, we evaluate the in vivo safety and efficacy of NP627 in a diet-induced obese (DIO) mouse model. The results demonstrate that NP627 treatment in DIO mice increased glucose uptake and inhibited the cleavage of PKCδI&#95;C in the AT as well as in the kidney, spleen, and liver. Next, RNAseq analysis was performed on the AT from the NP627-treated DIO mice. The results show increases in ADIPOQ and CIDEC, upregulation of AMPK, PI3K-AKT, and insulin signaling pathways, while inflammatory pathways were decreased post-NP627 administration. Further, levels of lncRNAs associated with metabolic pathways were affected by NP627 treatment. In conclusion, the study demonstrates that NP627, a small-molecule inhibitor of PKCδI activity, is not toxic and that it improves the metabolic function of DIO mice in vivo.

Introduction: Bacteremia is a life-threatening condition that can progress to sepsis and septic shock, leading to significant mortality in the emergency department (ED). The standard diagnostic method, blood culture, is time-consuming and prone to false positives and false negatives. Although not widely accepted, several clinical and artificial intelligence-based algorithms have been recently developed to predict bacteremia. However, these strategies require further identification of new variables to improve their diagnostic accuracy. This study proposes a novel strategy to predict positive blood cultures by assessing sepsis-induced immunosuppression status through endotoxin tolerance assessment., Methods: Optimal assay conditions have been explored and tested in sepsis-suspected patients meeting the Sepsis-3 criteria. Blood samples were collected at ED admission, and endotoxin (lipopolysaccharide, LPS) challenge was performed to evaluate the innate immune response through cytokine profiling., Results: Clinical variables, immune cell population biomarkers, and cytokine levels (tumor necrosis factor [TNFα], IL-1β, IL-6, IL-8, and IL-10) were measured. Patients with positive blood cultures exhibited significantly lower TNFα production after LPS challenge than did those with negative blood cultures. The study also included a validation cohort to confirm that the response was consistent., Discussion: The results of this study highlight the innate immune system immunosuppression state as a critical parameter for sepsis diagnosis. Notably, the present study identified a reduction in monocyte populations and specific cytokine profiles as potential predictive markers. This study showed that the LPS challenge can be used to effectively distinguish between patients with bloodstream infection leading to sepsis and those whose blood cultures are negative, providinga rapid and reliable diagnostic tool to predict positive blood cultures. The potential applicability of these findings could enhance clinical practice in terms of the accuracy and promptness of sepsis diagnosis in the ED, improving patient outcomes through timely and appropriate treatment., Competing Interests: EH-J, AU, XP, and JS are employees/advisors of the spin-off company Loop Diagnostics related to this biomarker. EH-J, EP-M, XP and JS are shareholders of a spin-off company Loop Diagnostics related to this biomarker. AL and EM have received honoraria from Loop Diagnostics. JS and EH-J are the authors of a PCT patent application to protect against the use of the method and kit for the early detection of sepsis WO2021245025A1. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Hernández-Jiménez, Plata-Menchaca, Berbel, López de Egea, Dastis-Arias, García-Tejada, Sbraga, Malchair, García Muñoz, Larrad Blasco, Molina Ramírez, Pérez Fernández, Sabater Riera and Ulsamer.)

Significance: Electronic cigarettes (e-cigarettes) have become a popular way to smoke all over the world. Chronic exposure to e-cigarette aerosol may influence lung health. This study uses an animal model to explore the time course of the effect of exposure to e-cigarette aerosols on the lung. Methods: Lung samples were collected after exposure of Balb/c mice to e-cigarette aerosols for 1 h/day (6 times/week) for 1, 2 and 4 weeks and compared to sham-exposed controls. Examined biomarkers including inflammatory cells, tumor necrosis factor α (TNFα), interleukin-6 (IL-6), interleukin-10 (IL-10), reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione peroxidase (GPx), catalase, superoxide dismutase (SOD), and Thiobarbituric acid reactive substances (TBARS). Results: Exposure of animals to e-cigarette aerosols induced significant increases (P

Abstract Background High glucose concentration increases the glycation process which leads to oxidative stress and inflammation, that can cause complications in diabetes. Several medicinal plants have been used in the treatment of diabetes and its complications. One of them is Kappaphycus alvarezii, an algae that has known antidiabetic abilities. This study aimed to examine the effect of K. alvarezii active fraction on plasma hydrogen peroxide (H2O2) and Tumor Necrosis Factor α (TNFα) levels, renal NADPH oxidase 4 (NOX4) and Nuclear Factor κ B (NFκB) gene expressions. Methods Active fraction was obtained from bioassay-guided fractionation with antiglycation ability. In vivo study was performed on twenty Wistar male rats. The level of H2O2 was measured using H2O2 Assay Kit, the Optical Density value measured using spectrophotometer at a wavelength of 405 nm. Plasma TNFα level was measured using ELISA. Renal NOX4 and NFκB gene expression was analyzed using qPCR. Results Active fraction significantly reduced plasma H2O2 but not TNFα levels. Furthermore, renal NOX4 gene expression was lower in the diabetic rat group treated with active fraction compared to the untreated group but not NFκB gene expression. Conclusions K. alvarezii active fraction has an activity to reduce plasma H2O2 as well as renal NOX4 gene expression. Therefore, this fraction could be developed as a potential candidate for diabetes treatment through oxidative stress mechanisms.

Accumulating evidence from both animal and human studies suggests that activation of beige fat increases cellular energy expenditure, ultimately reducing adiposity. Here, we report the central role of adipocyte-derived lysyl oxidase (Lox) in the formation of thermogenic beige fat. Mice exposed to cold or a β3 agonist showed drastically lower Lox expression in thermogenically activated beige fat. Importantly, inhibition of Lox activity with BAPN stimulated biogenesis of beige fat in inguinal white adipose tissue (iWAT) under housing conditions and potentiated cold-induced adaptive thermogenesis and beiging in both iWAT and epididymal white adipose tissue (eWAT). Notably, white adipocytes with Lox repression undergo transdifferentiation into beige adipocytes which can be suppressed by tumor necrosis factor-α (TNFα) via ERK activation. This work provides new insight into the molecular control to expand beige fat by Lox inhibition and suggest the potential for utilizing inhibitor of Lox to treat the emerging epidemics of obesity and diabetes.

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

One of the main causes of acute kidney injury is ischemic reperfusion injury (IRI). Inflammatory response, apoptotic damages, and oxidative stress-related injuries are all involved in the pathogenesis of IRI. Toll-like receptors (TLR) are strongly associated with IRIs, especially TLR4, which is markedly induced in response to IRI. Accordingly, the current study aimed to investigate the potential renoprotective effect of ultrapure lipopolysaccharide from Rhodobacter sphaeroides (ULPS-RS) at two doses in an animal model of bilateral IRI. A total of 30 adult male rats were divided randomly into five equal groups of control (laparotomy plus bilateral renal IRI), vehicle (same as the control group, but pretreated with the vehicle), sham (laparotomy only), ULPS-RS (same as the control group, but pretreated with 0.1 mg/kg of ULPS-RS), and ULPS-RSH (same as the control group, but pretreated with 0.2 mg/kg of ULPS-RS). Subsequent to 30 min of ischemia and 2 h of reperfusion, serum samples were collected for measuring urea, creatinine, and neutrophil gelatinase-associated lipocalin. Afterward, tissue samples were obtained from all animals to measure inflammatory mediators (interleukin 6, interleukin 1β, and tumor necrosis factor α), oxidative stress marker (8-isoprostane), apoptosis mediators (B cell lymphoma 2 [Bcl2]), and Bcl2-associated X protein (Bax). In the control group, all of the measured parameters were significantly elevated in response to IRI, except for Bcl2, which decreased significantly. On the other hand, exactly opposite effects were observed in the ULPS-RS treated groups indicating the nephroprotective effect of this compound against IRI at both tested doses. The findings reveal for the first time that ULPS-RS has the therapeutic potential of attenuating the renal dysfunction induced by IRI.

Migraine (Mg) is a multifaceted neurovascular disorder caused by genetic and several environmental etiologies. We have implemented a case-control study of TNFα gene polymorphism in 212 Mg patients and 218 healthy controls utilizing the ARMS-PCR technique, followed by Sanger sequencing. Besides, we have conducted a meta-analysis of different genetic models (five genetic models) to combine and summarize the available data from 11 studies (including this present research). The strength of genetic associations in the meta-analysis used to assess by the pooled odds ratio (OR) and 95% confidence intervals (CI). The results of this case-control study discovered a significant relationship with Mg in recessive and homozygous genotype with OR = 2.35 (95% CI [0.96–5.74]), p-value = 0.045. Also, the outcomes of meta-analysis suggested an irrelevant relationship between TNFα gene (rs1800629) polymorphism and Mg susceptibility in the five genetic models. However, subgrouping based on ethnic background showed a significant association in the allelic genetic model with OR = 1.53 (95% CI [1.02–2.31]), p = 0.040 respectively. The meta-analysis results of TNFα gene polymorphism may represent a risk factor for Mg among Asians. In the future, large scale, multicentric case-control study by classification of patients with Mg with or without aura can be performed worldwide to identify the potential genetic risk factors leading to Mg pathogenesis.

Background: Alzheimer's disease (AD) is a neurodegenerative disorder marked by brain inflammation resulted in structural damage and brain dysfunction. Tumor necrosis factor α (TNFα) is a cytokine that plays an important role in inflammation. Dates fruit may help to fight oxidative stress and inflammation in the brain. Objective: To determine the effect of date fruit extracts on blood TNFα levels and brain weight of alzheimer’s model rats. Methods: This research is a laboratory experimental study by post-test only with control group design using alzheimer model rats. This study used 6 treatment groups with simple randomization. Each treatment group was represented by 8 Sprague Dawley rats. The normal control group (KN) was not induced by Hcy and was not given date palm extract, the negative control group (K-) was the Alzheimer's experimental rats which was not given the date palm extract, the positive group was the Alzheimer's experimental rats which was given the Donepezil (K+). Groups P1, P2, P3 were Alzheimer's experimental rats that were given date palm extract at a dose of 200, 400,800 mg / kgBW / day. The effect of date palm extract dosage on TNFα levels and brain weight were analyzed using the One Way Anova test followed by Tukey's post hoc test. Results: The difference in TNFα levels between groups showed a significant difference (p = 0.00). Meanwhile there was no significant difference in brain weight among all groups (p > 0,05). Conclusion: Date palm extract at doses of 200, 400, 800 mg / kgBW can decrease blood TNFα levels of Alzheimer’s model rats.

Traumatic brain injury is the most common cause of death and disability in young people including sport athletes and soldiers, people under 45 years of age in the industrialized countries, representing a growing health problem in developing countries, as well as in aging communities. Treatment of the latter is a serious challenge for modern medicine. This type of injury leads to many kinds of disorders and, quite often, to disability. These issue require development of new methods for brain trauma treatment. The new approach to brain trauma treatment was studied in murine experiments. In particular, sodium salt of deoxyribonucleic acid (DNA) was used. This preparation is a drug known as a mixture of peptides with immunomodulatory effect which is widely used for different kinds of therapy. Derinat, a sodium salt of DNA, isolated from the caviar of Russian sturgeon, is a proven immunomodulator for treatment of diseases associatd with reactive oxygen species (ROS), including brain ischemia-reperfusion (IR) injury. Here we show that treatment with Derinat exert neuroprotective, anti-oxidative, and anti-inflammatory effects in experimental model of traumatic brain injury (TBI) in rats. Intraperitoneal injection of Derinat several times over 3 days after TBI showed less pronounced damage of the injured brain area. Immunohistochemical study showed that the Derinat-induced morphological changes of microglia in cerebral cortex and hippocampus 7 days after TBI. TBI-induced accumulation of 8-oxoguanine (8-oxoG), the marker of oxidative damage, was significantly attenuated by Derinat administration, both on 7th and 14th day after TBI. To investigate cellular mechanism of anti-inflammatory effects, the primary cultures of murine microglia supplied with ATP (50 M and 1 mM), as a substance released at injured site, were used to mimic the in vitro inflammatory response. Derinate treatment caused an increase of glial levels of mRNAs encoding neurotrophic factor (GDNF) and nerve growth factor (NGF) in the presence of ATP, whereas tissue plasminogen activator (tPA) mRNA was inhibited by ATP with or without Derinat. Interleukin-6 (IL-6) mRNA expression was not affected by ATP but was increased by Derinat. Both mRNA and protein levels of ATP-induced TNFα production were significantly inhibited by Derinat. These results partially contribute to understanding mechanisms of immunomodulatory effects of DNA preparations in traumatic brain injury.

Abstract Background Gentamicin (GM) is a low-cost, low-resistance antibiotic commonly used to treat gram-negative bacterial diseases. Cisplatin (Csp) is a platinum-derived anti-neoplastic agent. This experiment aimed to identify the early signs of gentamicin and cisplatin-induced nephrotoxicity in rats. Thirty Wistar rats were divided into three groups of 10: a control group, which received no treatment; a gentamicin group administered by a dose of (100 mg/kg, IP) for 7 consecutive days, and a cisplatin group was administered intraperitoneal in a dose of (1.5 mg/kg body weight) repeated twice a week for 3 weeks. Results Both experimental groups exhibited increased levels of creatinine, urea, and uric acid, with the cisplatin-treated group showing higher levels than the gentamicin group. Experimental groups also exhibited significantly increased Malondialdehyde (MDA), reduced glutathione (GSH), and glutathione peroxidase (GSH-Px) with more pronounced effects in the cisplatin-treated group. Further, both experimental groups exhibited significant up-regulation of Tumor Necrosis Factor α (TNF-α), caspase-3, and Bax and down regulation of Bcl-2. Conclusion These findings confirm the use of necrotic, apoptotic genes as early biomarkers in the detection of tubular kidney damage. Further, cisplatin was shown to have a greater nephrotoxic effect than gentamicin; therefore, its use should be constrained accordingly when co-administered with gentamicin.

Abstract Background Amyotrophic lateral sclerosis (ALS) is a fatal multifactorial neurodegenerative disease characterized by the selective death of motor neurons. Cytosolic phospholipase A2 alpha (cPLA2α) upregulation and activation in the spinal cord of ALS patients has been reported. We have previously shown that cPLA2α upregulation in the spinal cord of mutant SOD1 transgenic mice (SOD1G93A) was detected long before the development of the disease, and inhibition of cPLA2α upregulation delayed the disease’s onset. The aim of the present study was to determine the mechanism for cPLA2α upregulation. Methods Immunofluorescence analysis and western blot analysis of misfolded SOD1, cPLA2α and inflammatory markers were performed in the spinal cord sections of SOD1G93A transgenic mice and in primary motor neurons. Over expression of mutant SOD1 was performed by induction or transfection in primary motor neurons and in differentiated NSC34 motor neuron like cells. Results Misfolded SOD1 was detected in the spinal cord of 3 weeks old mutant SOD1G93A mice before cPLA2α upregulation. Elevated expression of both misfolded SOD1 and cPLA2α was specifically detected in the motor neurons at 6 weeks with a high correlation between them. Elevated TNFα levels were detected in the spinal cord lysates of 6 weeks old mutant SOD1G93A mice. Elevated TNFα was specifically detected in the motor neurons and its expression was highly correlated with cPLA2α expression at 6 weeks. Induction of mutant SOD1 in primary motor neurons induced cPLA2α and TNFα upregulation. Over expression of mutant SOD1 in NSC34 cells caused cPLA2α upregulation which was prevented by antibodies against TNFα. The addition of TNFα to NSC34 cells caused cPLA2α upregulation in a dose dependent manner. Conclusions Motor neurons expressing elevated cPLA2α and TNFα are in an inflammatory state as early as at 6 weeks old mutant SOD1G93A mice long before the development of the disease. Accumulated misfolded SOD1 in the motor neurons induced cPLA2α upregulation via induction of TNFα.

Silver nanoparticles (AgNPs) have various applications such as their use in the medical field. As they have been reported to show antimicrobial and anti-tumor effects. The main purpose of the current study was to explore the anti-cancer effects of AgNPs administration alone or in a combination with cisplatin (CP) against hepatocellular carcinoma (HCC) in rats. Seventy-five rats (Sprague Dawley albino rats) were used in the present study. Rats were assigned to 5 groups. Group 1 served as normal control. Group 2 was injected intraperitoneally (IP) with a single dose (200 ml/kg) of diethylnitrosamine (DEN), then one week later carbon tetrachloride (CCl4) was injected IP (0.2 ml/kg) two times weekly for 14 successive weeks for the induction of HCC. Group 3 was treated with AgNPs (4 mg/kg) daily. Group 4 was treated with CP (6 mg/kg) once a week. Group 5 was treated with a combination of AgNPs (4 mg/kg/daily) and CP (2.5 mg/kg/weekly). Groups 3, 4, and 5 were treated for 3 successive weeks after induction of HCC. Hematological, biochemical, antioxidant activities, proinflammatory cytokines, and apoptotic genes were evaluated in the current study. Hematological results denoted normocytic normochromic anemia in all examined groups except group 2 which showed macrocytic hypochromic anemia, with thrombocytopenia and leukocytosis in all groups. Significant hypoproteinemia, hypoalbuminemia, and hypoglobulinemia were detected in all groups, with no significant changes (P < 0.05) in globulin in group 5. Significantly decreases in GSH and SOD were recorded in all groups. While the serum AST, ALT activities, and levels of total bilirubin, urea, creatinine, IL-6, TNF-α, AFP, VEGF, BAX, and caspase-3 were markedly elevated. The results revealed a remarkable improvement in group 4 than groups 3, and 5. The obtained results were supported by immunohistochemical and histopathological investigations of the liver tissue.

Introduction: Non-alcoholic fatty liver disease (NAFLD) is a condition where excess fat accumulates in the liver (hepatic steatosis) and there is no history of alcohol abuse or other secondary causes of chronic liver disease. NAFLD is a very common disorder, occurring in 25% of the global population. NAFLD is now the most common chronic liver disorder in Western countries. Liver biopsy is the gold standard for NAFLD diagnosis and staging; however, this is invasive, costly and not without risk. Biomarkers that could diagnose and stage disease would reduce the need for biopsy and allow stratification of patients at risk of progression to non-alcoholic steatohepatitis (NASH).Methods: One hundred and thirty-five patients were involved in the study [N = 135: n = 34 controls; n = 26 simple steatosis; n = 61 NAFLD/NASH, and n = 14 alcoholic liver disease (ALD)]. Clinically diagnosed (ICD-10) patient serum samples were obtained from Discovery Life Sciences (US) along with clinical history. Samples were run in duplicate using high-sensitivity cytokine array I, immunoassays and ELISAs. In total, n = 20 individual biomarkers were investigated in this pilot study.Results: Thirteen/20 (65%) biomarkers were identified as significantly different between groups; IFNγ, EGF, IL-1β, IL-6, IL-8, IL-10, TNFα, FABP-1, PIIINP, ST2/IL-33R, albumin, AST and ALT. Five/20 (25%) biomarker candidates were identified for further investigation; namely, three biomarkers of inflammation, IL-6, IL-8, and TNFα, and two biomarkers of fibrosis, PIIINP and ST2/IL-33R.Discussion: Single biomarkers are unlikely to be diagnostic or predictive at staging NAFLD due to the complex heterogeneity of the disease. However, biomarker combinations may help stratify risk and stage disease where patients are averse to biopsy. Further studies comparing the 5 biomarkers identified in this study with current diagnostic tests and fibrotic deposition in liver tissue are warranted.

Objective: Type 1 diabetes (T1D) is characterized by autoimmune-associated β-cell loss, insulin insufficiency, and hyperglycemia. Although TNFα signaling is associated with β-cell loss and hyperglycemia in non-obese diabetic mice and human T1D, the molecular mechanisms of β-cell TNF receptor signaling have not been fully characterized. Based on work in other cell types, we hypothesized that receptor interacting protein kinase 1 (RIPK1) and receptor interacting protein kinase 3 (RIPK3) regulate TNFα-induced β-cell death in concert with caspase activity. Methods: We evaluated TNFα-induced cell death, caspase activity, and TNF receptor pathway molecule expression in immortalized NIT-1 and INS-1 β-cell lines and primary mouse islet cells in vitro. Our studies utilized genetic and small molecule approaches to alter RIPK1 and RIPK3 expression and caspase activity to interrogate mechanisms of TNFα-induced β-cell death. We used the β-cell toxin streptozotocin (STZ) to determine the susceptibility of Ripk3+/+ and Ripk3−/− mice to hyperglycemia in vivo. Results: Expression of TNF receptor signaling molecules including RIPK1 and RIPK3 was identified in NIT-1 and INS-1 β cells and isolated mouse islets at the mRNA and protein levels. TNFα treatment increased NIT-1 and INS-1 cell death and caspase activity after 24–48 h, and BV6, a small molecule inhibitor of inhibitor of apoptosis proteins (IAPs) amplified this TNFα-induced cell death. RIPK1 deficient NIT-1 cells were protected from TNFα- and BV6-induced cell death and caspase activation. Interestingly, small molecule inhibition of caspases with zVAD-fmk (zVAD) did not prevent TNFα-induced cell death in either NIT-1 or INS-1 cells. This caspase-independent cell death was increased by BV6 treatment and decreased in RIPK1 deficient NIT-1 cells. RIPK3 deficient NIT-1 cells and RIPK3 kinase inhibitor treated INS-1 cells were protected from TNFα+zVAD-induced cell death, whereas RIPK3 overexpression increased INS-1 cell death and promoted RIPK3 and MLKL interaction under TNFα+zVAD treatment. In mouse islet cells, BV6 or zVAD treatment promoted TNFα-induced cell death, and TNFα+zVAD-induced cell death was blocked by RIPK3 inhibition and in Ripk3−/− islet cells in vitro. Ripk3−/− mice were also protected from STZ-induced hyperglycemia and glucose intolerance in vivo. Conclusions: RIPK1 and RIPK3 regulate TNFα-induced β-cell death in concert with caspase activity in immortalized and primary islet β cells. TNF receptor signaling molecules such as RIPK1 and RIPK3 may represent novel therapeutic targets to promote β-cell survival and glucose homeostasis in T1D.

Purpose: There has been growing evidence that inflammatory markers play a role in the development as well as severity of Type 2 diabetes mellitus (T2DM). This study has been designed to decipher the involvement of C-Reactive Protein (CRP), Tumor Necrosis Factor (TNFα), Interleukin-6 (IL-6) and Interleukin-10 (IL-10) in the etiopathogenesis of T2DM. Basic procedures: A total of 480 T2DM cases and 540 healthy controls were recruited for the study. Blood samples were collected from each study subject to measure the serum levels of CRP, TNFα, IL-6 and IL-10. Main findings: We found that serum levels of CRP in mg/dl (4.2 ± 0.9), TNFα in pg/ml (34.5 ± 8.8), IL-6 in pg/ml (19.2 ± 7.2) in T2DM patients were significantly high as compared to control participants (CRP; 1.4 ± 0.6, TNFα; 12.7 ± 3.4, IL-6; 3.1 ± 1.4; P < 0.0001). The serum levels of IL-10 in pg/ml were lower in T2DM cases compared to controls (4.35 ± 1.2 vs. 9.6 ± 1.2). In addition, we observed a significant association of CRP levels with insulin resistance, obesity and dyslipidemia. Increased TNFα levels were strongly associated with female gender, Poor glycemic control and strong family history of diabetes. Poor glycemic control was significantly associated with elevated IL-6 levels. Moreover, significantly reduced IL-10 levels were found in T2DM patients with sedentary lifestyle; low educational and rural background. Conclusions: This study showed a strong relationship between TNFα, IL-6, CRP, IL-10 and T2DM patients of Kashmiri ethnicity, treated at SMHS Hospital. Thus, supporting other studies and showing that cytokines may be good markers for T2DM development.

Milk-derived peptides are known to confer anti-inflammatory effects. We hypothesised that milk-derived cell-penetrating peptides might modulate inflammation in useful ways. Using computational techniques, we identified and synthesised peptides from the milk protein Alpha-S1-casein that were predicted to be cell-penetrating using a machine learning predictor. We modified the interpretation of the prediction results to consider the effects of histidine. Peptides were then selected for testing to determine their cell penetrability and anti-inflammatory effects using HeLa cells and J774.2 mouse macrophage cell lines. The selected peptides all showed cell penetrating behaviour, as judged using confocal microscopy of fluorescently labelled peptides. None of the peptides had an effect on either the NF-κB transcription factor or TNFα and IL-1β secretion. Thus, the identified milk-derived sequences have the ability to be internalised into the cell without affecting cell homeostatic mechanisms such as NF-κB activation. These peptides are worthy of further investigation for other potential bioactivities or as a naturally derived carrier to promote the cellular internalisation of other active peptides.