Objective: Wingless/integrated (Wnt) signaling plays critical roles in maintaining environmental homeostasis and is also involved in the pathogenesis of inflammatory diseases. However, its role in macrophages during periodontitis is not well understood. The present study aims to investigate the interaction between Wnt signaling and macrophages in the context of periodontitis. Methods: Experimental periodontitis was induced in C57/BL6 mice using a Porphyromonas gingivalis (P.g)-associated ligature for 14 days. Immunohistochemistry was performed to study the expression of the pro-inflammatory cytokine tumor necrosis factor (TNF-α), the stabilization of β-catenin, and the macrophage marker F4/80 in the periodontal tissues. The effect of Wnt signaling on TNF-α was examined using Western blot analysis in Raw 264.7 murine macrophages stimulated by Wnt3a-conditioned medium, with or without Wnt3a antibody neutralization, and compared with primary cultured gingival epithelial cells (GECs). The effect of P.g lipopolysaccharide (LPS) on Wnt signaling was assessed by analyzing key components of the Wnt signaling pathway, including the activity of low-density lipoprotein receptor-related protein (LRP) 6 and nuclear accumulation of β-catenin in GEC and Raw 264.7 cells. Results: Over-expressions of TNF-α and activated β-catenin were presented in the macrophages in the gingiva from mice with P.g-associated ligature-induced periodontitis. The expression patterns of TNF-α and activated β-catenin were consistent with the expression of F4/80. In Raw 264.7 cells, activation of the Wnt signaling pathway led to an increase in TNF-α, but this effect was not observed in GEC. Additionally, treatment with LPS induced β-catenin accumulation and LRP6 activation in Raw 264.7 cells, which were blocked by the addition of Dickkopf-1(DKK1). Conclusions: Wnt signaling was aberrantly activated in the macrophages in experimental periodontitis. The activation of Wnt signaling in the macrophages may play a pro-inflammatory role in periodontitis. Targeting specific signaling pathways, such as the Wnt pathway, may hold promise for developing novel therapeutic interventions for periodontitis. Periodontitis is a chronic inflammatory disease that is initiated by reversible infectious gingivitis and develops to irreversible periodontal inflammation. It is a leading cause of jawbone loss and deep tissue damage . Among tThe GEC functions as a first line defense against the invasion of exogenous pathogens, protecting the periodontal tissues from biofilm contact. GEC also plays a critical role in triggering and modulating the immune response by expressing several pattern recognition receptors (PRRs) that recognize periodontal pathogens and activate the key inflammatory cytokines to trigger and regulate the immune response of macrophages ,5. GingiPorphyromonas gingivalis (P.g) is the most prominent. The virulence factor of P.g, lipopolysaccharide (LPS), plays the main pathogenic role in the initiation and enhancing of inflammation [Macrophages play a central role in initiating the immune response. A recent study showed that pro-inflammatory macrophages play a pathogenic role in the chronic inflammation associated with periodontitis and is a major resource of pro-inflammatory cytokines. Periodontitis-related inflammation is mainly driven by bacterial communities ,7, of whammation . The infammation , dysreguammation ,11,12,13Canonic Wnt signaling, also referred as the β-catenin-dependent pathway, is highly conserved and essential for development, tissue homeostasis, and disease . This paThe aberrant activation or dysregulation of the Wnt signaling has been shown to play a key causative mechanistic role in triggering and amplifying pathogenic inflammation ,17. In tAnimal: Wild-type (WT) C57BL/6 mice (Jackson Laboratory) were used for experiments at the age of 8–10 weeks old (body weight 20 g to 28 g) with equal numbers of male and female mice. For the in vitro study, a total 12 mice was used (6 per group). For the in vivo study, a total of 12 mice was used (6 per group). All mice were distributed into groups randomly with different cages/racks in the fully functional animal facility. The Institutional Animal Care and Use Committee approved all the animal experiments (IACUC-20-005). All the animal experiments complied with the ARRIVE guidelines.Animal model: P.g-associated ligature-induced experimental periodontitis was induced by ligation with silk  surrounding the maxillary second molars on the left side. The silk was soaked in the P.g (Strain ATCC 33277) suspension for 30 min before ligation. All the mice were anesthetized by intraperitoneal injection with ketamine/xylazine . The ligatures were checked every 2 days and replaced if missing. Both groups  were sacrificed on day 14, and samples were processed blinded.Immunohistochemistry staining: The maxillae from at least three animals in each treatment groups were harvested and fixed in 4% formaldehyde for 12 h. The samples were then decalcification in 10% EDTA for 2 weeks at 4 °C with agitation. After embedding with paraffin and sectioned along the long axis of the molars, these sections were deparaffinized and rehydrated, followed by antigen retrieval and blocking with 1% BSA  and 10% goat serum . Primary antibodies used in the study were rabbit anti-TNFα antibody , rabbit non-phospho (active) β-catenin at Ser33/37/Thr41 (D13A1) , and rabbit anti-mouse F4/80 . A rabbit-specific HRP/DAB kit  was used to show the antigen localizations. After hematoxylin counterstaining, images of gingival areas mesial and distal to the molars were taken under light microscope.Cell preparation and culture: Mouse gingival epithelial cells were isolated and cultured following a procedure described previously [P.g LPS  for 2 h before being pre-treated with control IgG  or anti-Wnt3a antibody  overnight, then cultured in a control medium or 50% Wnt3a-conditioned medium  for 48 h. To study the effect of P.g LPS on the Wnt pathway, gingival epithelial cells and Raw 264.7 murine macrophages were treated with 1 μg/mL P.g LPS and were harvested at 0, 1, 3, 6, 12, and 24 h for the Western blot analyses. For the immunohistochemistry analysis, GEC and Raw 264.7 murine macrophages were pretreated with DKK-1  for 24 h, followed by 1 μg/mL P.g LPS for 24 h.eviously . BrieflyNon-junctional β-catenin was isolated following a protocol previously described by Fagotto et al. . BrieflyRaw 264.7 cells and GEC cells were collected, washed, and suspended in a cytoplasmic extract buffer and incubated on ice for 40 min, and then the suspension was centrifuged at 12,000 rpm. The resulting supernatant containing the cytoplasmic extract was carefully collected and stored. The nuclear pellet was washed to remove any residual cytoplasmic material and then resuspended in a nuclear extraction buffer. This suspension was incubated on ice for 15 min and then centrifuged at 14,000 rpm. The resulting supernatant was collected, and the protein concentration of the nuclear extract was determined by a BCA protein assay kit  according to the manufacturer’s instructions. The nuclear extracts were then stored at −80 °C until use.Immunoblot analysis: The procedure was followed as described previously [eviously . BrieflyStatistical analysis: All the results were expressed as means ± SD. Unpaired Student’s t tests were used for comparisons of any two groups of data sets in the statistical analysis. Probability values less than 0.05 were considered statistically significant. All statistical analysis was performed by GraphPad Prism.TNF-α is known to be destructive and is implicated in bone loss during the disease, and there is a strong positive correlation between the over-expression of TNF-α in periodontal tissues and tissue destruction.P.g-associated ligature-induced periodontitis model. As depicted in To better understand the role of TNF-α in periodontitis, we examined its expression in a The stabilization and nuclear translocation of β-catenin represents the activation of Wnt signaling, which activates the nuclear transcriptional factor to promote Wnt target gene transcription. TNF-α can activate the Wnt/β-catenin pathway, while the activation of Wnt/β-catenin signaling augments TNF-α and induces NF-kB signaling and pro-inflammatory cytokine expression. Therefore, we examined the key Wnt signaling effector, non-phosphorylated β-catenin (np β-catenin), indicating the activation of the canonical Wnt pathway, in the periodontal tissues during periodontitis.As shown in These data suggested that the canonical Wnt pathway is likely activated in the gingiva during periodontitis and is consistent with the expression of the key pro-inflammatory cytokines of TNF-α. These results together suggested that canonical Wnt signaling is activated in gingiva, and this activation is involved in the inflammatory process during periodontitis.Given the important role that the Wnt signaling pathway plays in recruiting macrophages, we sought to determine whether the pathway is present in macrophages in the gingiva during periodontitis. To this end, we examined the expression of F4/80, a macrophage-specific marker. Our results, shown in To assess if the activation of the Wnt signaling in macrophages plays a pathogenic role in inflammation, we examined if activation of the canonical pathway alone could increase the expression of TNF-α in comparison to primary cultured gingival epithelial cells (GEC). We exposed GECs and Raw 264.7 cells to 50% Wnt3a-conditioned medium (CM), which induced mainly the canonical Wnt signaling pathway, resulting in the accumulation of β-catenin in the nucleus A,C. As sNext, upon confirmation of Wnt signaling activation, its effects on the expression of TNF-α were subsequently examined and compared, as depicted in To further test the hypothesis that the increase of TNF-α resulted from Wnt signaling activation, we examined whether Wnt-directed TNF-α secretion would be abolished or decreased when Wnt activation was inhibited in these cells. As shown in the representative Western blot in Overall, these findings support the hypothesis that activation of the Wnt pathway is likely associated with the recruitment and activation of macrophages in the gingiva during periodontitis, which may contribute to the pathogenesis of inflammation in periodontal diseases.P.g is lipopolysaccharide (LPS), which is important virulence factor in the mechanisms of periodontitis and can induce TNF-α secretion. Based on our previous findings, we hypothesized that LPS may also induce Wnt signaling activation in macrophages during periodontitis. To test this hypothesis, we examined the effects of LPS on key components of the Wnt pathway.The principal component from As shown in To further investigate the effect of LPS-induced Wnt signaling activation on TNF-α production, we used DKK1, a Wnt antagonist via the internalization of the key co-receptor LRP6 on the cell surface, to inhibit Wnt signaling in GEC and macrophages. As shown in Taken together, these results suggest that LPS functions as a strong activator of the Wnt pathway in both GEC and macrophages. However, Wnt signaling activation in macrophages, but not in GEC, likely plays a major pro-inflammatory role.P.g LPS ligature. We also demonstrated that the Wnt pathway has a direct pro-inflammatory effect on the production of TNF-α in macrophages in the gingival tissue. Furthermore, we found that LPS can activate the Wnt pathway, while inhibition of the Wnt signaling results in the reduction of TNF-α in macrophages.Periodontitis is a complex disease caused by the interaction between the oral microbiota and the host immune system, resulting in the destruction of the periodontal tissues and alveolar bone loss. In our present study, we focused on Wnt pathway activation and its relationship with TNF-α, a key pro-inflammatory cytokine in periodontitis. Our findings suggest that Wnt signaling is activated in the gingival tissue during periodontitis induced by The present study investigated the role of TNF-α and Wnt signaling in the pathogenesis of destructive periodontitis. The findings of this study are consistent with previous research that has shown TNF-α to be a potent early pro-inflammatory cytokine in destructive periodontitis, and that its activities in the periodontal tissues include degradation of the connective tissues and stimulation of osteoclast formation . PreviouP.g LPS activated Wnt signaling in macrophages, which likely induces M1 polarization based on its strong TNF-α induction effect. Wnt signaling has been shown to play a protective role in the development and maintenance of periodontal tissues and regeneration of the alveolar bone, periodontal ligament, and cementum [The study also found that cementum . TherefoAlthough the study did not find significant alteration of TNF-α expression upon activation of Wnt signaling, the present study did find interesting disparities in the accumulation of β-catenin and the phosphorylation of LRP6 during a time course, GEC displayed a rapid response to the stimulator, and the role of the Wnt signaling in GEC acting as an inducer or a messenger through the secretion of Wnt ligand or TNF-α for macrophages is unclear. The present study also showed that LPS acted as an activator of the Wnt pathway, likely through interfering with the binding of the Wnt ligand to the receptor or post-translational modification of the receptor. Further research is necessary to explore potential crosstalk between the Wnt and toll-like receptor signaling pathways, which both induce pro-inflammatory cytokines.The findings of this study contribute to our understanding of the complex interactions between TNF-α and Wnt signaling in periodontitis. Previous research has highlighted the significant role of TNF-α in the pathogenesis of destructive periodontitis, and our study found that activated Wnt signaling plays a complex and cell-type specific role in TNF-α expression, suggesting a modulating effect on inflammation during periodontitis.It is important to note that this study has some limitations. Firstly, we focused exclusively on the effects of Wnt signaling on TNF-α expression and did not investigate the potential influence of other inflammatory cytokines or signaling pathways. Given the complexity of periodontitis, it is likely other signaling pathways are involved and may cross-talk with Wnt signaling. Secondly, our experimental periodontitis mice model was limited to a 14-day period. While this time frame allowed us to observe significant changes in TNF-α expression, further research is needed to investigate the progression of periodontitis over shorter or longer periods. This would provide a more comprehensive understanding of the disease process. Additionally, we attempted to perform quantitative analysis of the images, but the complex and variable background made it impossible to obtain accurate and reproducible results. Therefore, we presented the data in a binary representation format, indicating either the presence or absence of staining, in order to provide a clear indication of the staining pattern, but it does have its limitations.In addition, in Overall, our findings shed new insights into the interplay between inflammation and Wnt signaling in macrophages during periodontitis and highlight the need for further research to fully understand the mechanism involved in this complex disease. Targeting specific signaling pathways, such as the Wnt pathway, may hold promise for developing novel therapeutic interventions for periodontitis. We hope our study will contribute the development of new treatments for this challenging condition.


Scientific Reportshttps://doi.org/10.1038/s41598-017-10529-1, published online 04 September 2017Correction to: This Article contains errors.xmrk+ and Myc+ of the ‘Male’ group are partially overlapping.In Figure 2A, the panels xmrk+ of the ‘Male’ group is partially overlapping with panel Myc+ of the ‘Male’ group of Figure 6C.In addition, in Figure 6A, the panel The corrected Figures Finally, in the Methods section, under the subheading ‘Histological and immunocytological Analyses’ the following paragraph is omitted:2). For cortisol (Alexa Fluor 546)/HNF4a (Alexa Fluor 488) or Tgfb1a (Alexa Fluor 546)/HNF4a (Alexa Fluor 488) co-immunostainings, hepatocytes (HNF4a+) cells were firstly identified by manually annotating Alexa Fluor 488 (green)+ cells in the entire frame. Following which, % of hepatocytes with Alexa Fluor 546 (red) staining were counted for either cortisol or Tgfb1a.”“Images were captured from liver paraffin sections immunofluorescence stained for PCNA (Alexa Fluor 488), Caspase 3 (Alexa Fluor 488), cortisol (Alexa Fluor 546)/HNF4a (Alexa Fluor 488) or Tgfb1a (Alexa Fluor 546)/HNF4a (Alexa Fluor 488), followed by counterstaining for DAPI (405 nm). All images were captured with a Leica LSM 510 inverted confocal microscope. For PCNA and Caspase 3 staining images, number of positively stained cells were manually annotated by counting only DAPI-stained cells with Alexa Fluor 488 (green) signals for the entire frame and normalised to area (positive cell/mm


In recent decades, the global prevalence of brain illnesses has increased, as has the disease's socioeconomic impact. A rise in 5% of motor neuron disorders (MNDs) and neuromuscular degeneration is attributable to unidentified treatment, and no specific diagnostic biomarkers can prove the progression and prevention of these neuronal consequences  and blood serum of MND patients; nevertheless, they are not conclusive proof of the disease. These diagnostic criteria also aid in identifying clinically comparable motor neuron and neurodegenerative illnesses. As a result, the most significant challenge in today's MND treatment spectrum is the lack of accurate biomarkers to identify the appropriate medicines for motor neuron patients. As a result, researchers are establishing the role of many molecular markers in the diagnosis and their potential modulators to prevent neuronal demyelination (Minj et al., Preliminary research has found and shown the involvement of various molecular signaling targets, as well as their preventive measures, in MND and associated motor neuron disorders such as Alzheimer's, Huntington's, and Parkinson's disease. Scientists worldwide have begun to investigate and discover particular molecular targets as a biomarker in CSF and blood serum to diagnose and cure these neurological dysfunctions. These achievements will aid clinicians, individual drug development, and pharmaceutical research organizations identify therapy and diagnostic procedures.There is clinical and preclinical evidence that cellular and molecular signaling deregulation plays a role in the etiology of MND and similar neuromuscular degenerations. Based on laboratory study findings, researchers chose the following molecular targets as biomarkers and potential signaling modulators to prevent and diagnose MNDs. These cellular and molecular targets, as well as their modulators, can be investigated further in an MND and neuromuscular degenerative condition experimental paradigm as follows: SIRT-1 signaling activator solanesol; Nrf2/HO-1 signaling activator acetyl-11-keto-beta boswellic acid; IGF-1/GLP-1 signaling activator 4-hydroxyisoleucine; ERK1/2 signaling inhibitor alpha-mangostin; JAK-STAT signaling inhibitor and PPAR-⋎ signaling activator guggulsterone; PI3K/Akt/-mTOR signaling inhibitor chrysophanol; and c-JNK/p38MAPK signaling inhibitor Apigenin (Singh et al., This special issue aims to demonstrate the molecular biomarkers in CSF and blood serum as diagnostic tool. Additionally, to examine the neuroprotective potential of signaling modulators and to understand disease pathways experimentally by performing neurobehavioural, molecular, neurochemical, immunohistopathological, western blot analysis, and gross pathology abnormalities. This special issue also explores the efficacy of signaling modulators in conjunction with currently utilized MND medications and focuses solely on diagnosing motor neuron demyelination and identifying potential treatments to alleviate the neurocomplications associated with MND-like diseases.The author confirms being the sole contributor of this work and has approved it for publication.


In spite of their elevated presence in the laboratories, women are still underrepresented in the top ranks of the scientific careers, including biomedical disciplines. This Research Topic offered a platform to promote the work of women scientists across the field of Signaling. Studies from researchers from Australia, Spain, United States and France have addressed a variety of issues that keep in common signaling alterations and human diseases.Merechar et al. describe a new tool to test if gain-of-function Pik3ca mutations can cause vascular malformation syndromes. Expression of the most common post-zygotic activating mutation of Pik3ca in mice neural crest and related embryonic lineages allow the authors investigate the consequences of this mutation. Outcomes included macrocephaly, cleft secondary palate and more subtle skull anomalies. These murine phenotypes may aid discovery of new candidate human PRDs affecting craniofacial and vascular smooth muscle development as well as the reciprocal paracrine signaling mechanisms leading to tissue overgrowth.Inappropriate activation of the PI3K signaling pathway lies at the core of most human cancers. Missense mutations of the PIK3CA oncogene are also found in affected tissues of a distinct set of congenital tumors and malformations collectively termed PIK3CA-related disorders (PRDs). Chatterjee and Sing review the current understanding of EVs in retinal (patho) physiology including disease-associated EV alterations in specific retinal diseases. Identifying different sub-populations of EVs in the retina in normal versus diseased condition(s), understanding the consequence of cell-specific EV cargo  as well as understanding the mechanism behind the packaging of this cargo for retinal homeostasis would help to understand the mechanisms leading to retinal disease. The identification and characterization of cell specific EVs in body fluids like plasma, serum, VH, and aqueous humor in healthy subjects versus patients with retinal degenerative diseases will also be instrumental in development of biomarkers.Extracellular vesicles (EVs) secreted by cells have important functions in cellular homeostasis and cell-cell communication. Evolutionarily conserved from prokaryotes to eukaryotic cells, EVs play an important role in autocrine, paracrine, and endocrine signaling. Alterations in these mechanisms are implicated in multiple human disorders including prominent retinal degenerative diseases, like age related macular degeneration (AMD) and diabetic retinopathy (DR). Rincón-Ortega et al. use the follicular epithelium (FE) of the Drosophila ovary as a model system to study the role of cell-ECM interactions in stem cell proliferation and differentiation during development. Their studies point to a role for the integrin-mediated cell-BM interactions in the control of epithelial cell division and subsequent differentiation.Signaling by integrins is crucial for normal epithelial tissue growth and development, and continues to be critical for tissue homeostasis and regeneration throughout life. Defects in the correct control of stem cells proliferation and differentiation can lead to abnormal tissue and is a hallmark of cancer. Weerasinghe et al. Review bone-associated signaling pathways and the direct impact of antipsychotics on different receptors present in bone cells.An important aspect of signaling studies is that of investigate the potential effect side effects of medical treatments. Between 2005 and 2014, the prescription rate of antipsychotic use overall has increased in 10 out of 16 countries, and its use is associated with a number of side effects. A growing body of evidence suggests diminished bone mineral density (BMD) and increased fracture risk associated to antipsychotics.


Inflammation is a response of the organism to an external factor that disrupts its natural homeostasis, and it helps to eliminate the cause of tissue injury. However, sometimes the body’s response is highly inadequate and the inflammation may become chronic. Thus, the search for novel anti-inflammatory agents is still needed. One of the groups of natural compounds that attract interest in this context is lichen metabolites, with usnic acid (UA) as the most promising candidate. The compound reveals a broad spectrum of pharmacological properties, among which anti-inflammatory properties have been studied both in vitro and in vivo. The aim of this review was to gather and critically evaluate the results of the so-far published data on the anti-inflammatory properties of UA. Despite some limitations and shortcomings of the studies included in this review, it can be concluded that UA has interesting anti-inflammatory potential. Further research should be directed at the (i) elucidation of the molecular mechanism of UA; (ii) verification of its safety; (iii) comparison of the efficacy and toxicity of UA enantiomers; (iv) design of UA derivatives with improved physicochemical properties and pharmacological activity; and (v) use of certain forms or delivery carriers of UA, especially in its topical application. Inflammation is a dynamic response of the organism to an external factor that disrupts its natural homeostasis—most commonly apathogenic microorganisms or physical agents. Generally, this process helps to eliminate the cause of tissue injury, but in some diseases, the body’s response is highly inadequate. The unfolding chronic inflammation can result in cellular destruction and damage to tissues, or even promote the development of some serious diseases, such as cancer . The visSalix sp. One of the groups of natural compounds that attract interest in this context is lichen metabolites. Lichens are composite organisms, that are primarily formed by the symbiotic co-existence of algal and/or cyanobacterial units and fungi, with the participation of basidiomycete yeasts and some bacterial communities [Usnea, Alectoria, Cladonia, Lecanora, Ramalina, and Flavocetraria. Usnic acid (UA) was first isolated in 1844 and, since then, its biological properties have been intensively studied, focusing mainly on antimicrobial, cytotoxic, antioxidant, and anti-inflammatory activities. It should be mentioned that this is a chiral compound . A literature search was conducted in the PubMed, Google Scholar, and Scopus databases, covering reports up to December 2022. Initially, the search term “usnic acid” was used, but it was too general and gathered papers on all of the different activities of this compound. For example, Scopus found 1313 articles containing this keyword. Accordingly, the following search terms were refined: “usnic acid anti-inflammatory”, “usnic acid antiinflammatory”, “usnic acid inflammation”, and “anti-inflammatory effects of usnic acid”. An additional criterion was the English language of the articles. After checking the titles and abstracts of the papers, 72 articles were selected. Then, after a deeper analysis of the full text, 23 duplicates and 26 studies were excluded. Further reports were found by checking the reference lists of previously identified scientific publications. Of the remaining 23 papers, 5 review articles were excluded, resulting in a total of 18 original studies used to prepare this review. The flow chart of the search method is shown in Several in vitro studies described the anti-inflammatory activity of usnic acid in an attempt to discover the potential mechanism at the cellular level. The published studies involved experiments on leukocytes or platelets isolated from blood, referring to the production of an eicosanoid inflammatory mediator, but also on RAW 264.7 macrophages stimulated by LPS, where NO or a different cytokine release was measured. Details of the experiments published to date and their results are shown in 50 value of 42 ± 2.2 μM, whereas the values for the reference substances were 0.4 ± 0.21 μM (nordihydroguaiaretic acid) and 37 ± 4.6 μM  [Kumar and Müller investigated the effect of (+)-UA on leukotriene B4 (LTB4) synthesis from bovine polymorphonuclear leukocytes. The compound was shown to have only a weak inhibitory effect on LTB4 biosynthesis, with an IC50 values of 12.8 µM and 4.7 µM, respectively. TNF-α mRNA expression was also inhibited. Western blot assay showed that UA suppressed LPS-induced inducible nitric oxide synthase (iNOS) protein synthesis and NF-κB p65 nuclear translocation in the cells tested. The degradation of I-κBα, a protein that inhibits NF-κB by masking the nuclear localization signals of NF-κB proteins and keeping them sequestered in an inactive state in the cytoplasm, was inhibited [In the study on LPS-stimulated RAW 264.7 macrophages, significant reductions in the TNF-α level and NO production were observed after UA treatment at doses of 0.5–400 µM, with ICIn a study by Huang et al. (2014), in the same cellular model, a similar decrease in the production of pro-inflammatory factors TNF-α, IL-1β, IL-6, and NO after treatment with UA at the concentrations of 1.5 and 10 µg/mL was noted. The observed decrease in the expression of TNF-α mRNA, COX-2 mRNA, and iNOS mRNA confirmed the activity of the compound at the cellular transcriptional and translational levels. At the same time, the mRNA levels of anti-inflammatory IL-10 and anti-inflammatory mediator heme oxygenase-1 (HO-1) increased significantly. Furthermore, a reduction in NF-κB activation was observed. These results indicated a dual effect of UA in reducing inflammation by stimulating the secretion of anti-inflammatory factors and inhibiting pro-inflammatory factors .In our own studies on LPS-stimulated RAW 264.7 cells, the effects of both UA enantiomers at concentrations of 10 and 25 µg/mL were compared. A significant reduction in NO production was found for both concentrations, irrespective of the enantiomer used. In the case of IL-6, only the 25 µg/mL dose of both enantiomers had a significant effect on its release, whereas TNF-α production decreased only slightly, with no significant differences compared to control cells treated with LPS. In addition, the effect of both enantiomers on the expression of pro-inflammatory proteins: toll-like receptor 4 (TLR4), cytosolic phospholipase A2 (cPLA2), and cyclooxygenases  was also assessed. The inhibitory effect on TLR4 was observed at UA concentrations of 10 and 25 µg/mL, irrespective of the enantiomer used. Both UA enantiomers significantly and dose-dependently reduced cPLA2 synthesis in comparison to LPS-stimulated macrophages, with the strongest effect observed for (+)-UA at a concentration of 10 µg/mL. A dose-dependent decrease in COX-1 protein levels was observed for both enantiomers, but for (+)-UA only the higher dose made the effect significantly different from LPS-stimulated macrophages. Both UA enantiomers significantly decreased COX-2 protein levels, but for (−)-UA, the effect was dose-independent. Surprisingly, (+)-UA slightly increased COX-2 synthesis at the higher dose. The study showed a slight pro-inflammatory effect of (+)-UA, as the compound increased cPLA2 and COX-2 expression at the higher dose of 25 µg/mL, whereas no such effect was observed for (−)-UA [A recent study tested the effect of UA on a broad panel of cytokines produced by unstimulated human breast cancer MCF-7 cells. In a concentration range of 0.62–15.64 µM, the compound significantly reduced the release of NO, vascular endothelial growth factor (VEGF), prostaglandin E2 (PGE2), cytokines , TNF-α, IL-6) in the cells, in a dose-dependent manner, compared to control cells. The compound also reduced the expression levels of COX-2 and iNOS genes .It is noteworthy that UA was also tested in vivo in several models involving wound-healing, neurodegenerative, or lung diseases. Nevertheless, only one of these studies compared the impact of both enantiomers. Details of the experiments published to date and their results are shown in In probably the first published study, the anti-inflammatory potential of (+)-UA was evaluated in a rat model of induced chronic and acute inflammation; the compound’s activity was comparable to ibuprofen, which was used as a reference substance . After aHard-to-heal wounds are a major health care problem. Inflammation is one of the natural stages of wound healing, forming an immune barrier against microbes. In many chronic wounds, there is clinically significant wound infection and/or excessive inflammation. The interesting efficacy of UA, in liposome form, in the treatment of burn wounds has been demonstrated in two experiments by the same research group . In animSome recent studies, probably inspired by the lipophilic properties of UA and the proven ability of (−)-UA to cross the blood–brain barrier in vitro , have atThe neuroprotective effect of UA, resulting from its anti-inflammatory properties, was also suggested by the results of another study, with Parkinson’s-like brain changes induced by MPTP  in mice . UA suppOne hypothesis for Alzheimer’s disease is that amyloid-β protein (Aβ) is deposited as amyloid fibers or non-fibrous amorphous aggregates in senile plaques, resulting in impaired neuronal transmission . Aβ1−42 2O2 were significantly reduced, while the observed increase in SOD and GSH activities indicated the antioxidant properties of the compound [The anti-inflammatory properties of UA have also been assessed for acute lung injury and acute respiratory distress syndrome, inflammatory diseases characterized by lung infiltrates, pulmonary edema, or hypoxemia, but also by a rapid overproduction of pro-inflammatory cytokines and chemokines, with a mortality rate of up to 40%. Both diseases are serious, with a long course for which there is still no effective treatment . Zu-Qingcompound . In an ecompound .Due to the documented anti-inflammatory effects of usnic acid, attempts have also been made to modify its structure in order to obtain synthetic derivatives, with improved physicochemical and anti-inflammatory properties. The studies were mainly conducted within in vitro models on LPS-stimulated cells of various origins, but two in vivo experiments have also been described. Details of the experiments published to date and their results are shown in 50 values ranging from 1.40 to 5.70 μM compared to an IC50 > 100 μM for the parent compound.Vanga et al. , synthes50 (1.40 and 1.88 μM) were most similar to the values obtained for prednisolone (IC50 0.52 μM), used as a reference drug. Interestingly, two of the intermediates tested  ranged from 5.3 μM to 7.5 μM and were many times lower compared to the parent UA (>100 μM), while for dexamethasone the IC50 values were 1.5 and 2.9 μM, respectively. The authors noted that the introduction of an enamine group at the C-2 position of (+)-UA, as present in the derivatives 5, 6, and 13, significantly increased the assessed anti-inflammatory activity, compared to the parent compound. Moreover, these most active derivatives were also characterized by the presence of electron-withdrawing groups in the phenacyl moiety, such as chloro, nitro, and bromo groups, while compounds bearing aromatic or heteroaromatic substituents (4 and 8–12) were significantly less active [In a subsequent study, the same group of authors synthesized ten new (+)-UA imidazolium salts, which were evaluated for the in vitro anti-inflammatory potential of TNF-α and IL-1β on the LPS-stimulated human lymphoma U937 cell line. The three most active synthesized derivatives inhibited the release of TNF-α and IL-1β in 80.1 and 25.4% -UA structure, retaining its anti-inflammatory properties, with additional properties to inhibit tau protein aggregation (an important element in the pathogenesis of Alzheimer’s disease). Twenty-five enamine derivatives and twenty-five hydrazines and hydrazides of (+)-UA were synthesized, but due to their better water solubility, the sodium salt of usnic acid  elucidation of the molecular mechanism of UA’s anti-inflammatory activity; (ii) verification of UA’s hepatotoxic properties, especially at the higher doses used; (iii) comparison of the efficacy and toxicity of UA enantiomers; (iv) design of UA derivatives, with improved physicochemical properties  and pharmacological activity, as well as high safety; and (v) use of certain forms or delivery carriers of UA, especially in its topical application.


The isolated rapid-eye-movement sleep behavior disorder (iRBD) is a prodromal condition of Lewy body disease including Parkinson's disease and dementia with Lewy bodies (DLB). We aim to investigate the longitudinal evolution of DLB-related cortical thickness signature in a prospective iRBD cohort and evaluate the possible predictive value of the cortical signature index in predicting dementia-first phenoconversion in individuals with iRBD.We enrolled 22 DLB patients, 44 healthy controls, and 50 video polysomnography-proven iRBD patients. Participants underwent 3-T magnetic resonance imaging (MRI) and clinical/neuropsychological evaluations. We characterized DLB-related whole-brain cortical thickness spatial covariance pattern (DLB-pattern) using scaled subprofile model of principal components analysis that best differentiated DLB patients from age-matched controls. We analyzed clinical and neuropsychological correlates of the DLB-pattern expression scores and the mean values of the whole-brain cortical thickness in DLB and iRBD patients. With repeated MRI data during the follow-up in our prospective iRBD cohort, we investigated the longitudinal evolution of the cortical thickness signature toward Lewy body dementia. Finally, we analyzed the potential predictive value of cortical thickness signature as a biomarker of phenoconversion in iRBD cohort.R = − 0.55, P = 0.024 and R = − 0.56, P = 0.036, respectively) as well as visuospatial impairment . The longitudinal trajectory of DLB-pattern revealed an increasing pattern above the cut-off in the dementia-first phenoconverters  but no significant change in parkinsonism-first phenoconverters . The mean value of the whole-brain cortical thickness predicted phenoconversion in iRBD patients with hazard ratio of 9.33 [1.16–74.12]. The increase in DLB-pattern expression score discriminated dementia-first from parkinsonism-first phenoconversions with 88.2% accuracy.The DLB-pattern was characterized by thinning of the temporal, orbitofrontal, and insular cortices and relative preservation of the precentral and inferior parietal cortices. The DLB-pattern expression scores correlated with attentional and frontal executive dysfunction (Trail Making Test-A and B: Cortical thickness signature can effectively reflect the longitudinal evolution of Lewy body dementia in the iRBD population. Replication studies would further validate the utility of this imaging marker in iRBD.The online version contains supplementary material available at 10.1186/s40035-023-00356-y. Isolated rapid eye movement (REM) sleep behavior disorder (iRBD) is associated with synucleinopathy and in most cases, evolves into Lewy body disease (LBD) , 2. DemeDLB patients have progressive cognitive decline with characteristic cognitive fluctuations, vivid hallucination, parkinsonism and RBD as core clinical features . SignifiTo develop an imaging biomarker predicting Lewy body dementia in an iRBD population, we set out to characterize magnetic resonance imaging (MRI)-driven whole-brain cortical signature from DLB and apply it in our prospective iRBD cohort. With longitudinal data, we analyzed the evolution of the cortical signature and the relationship between the imaging marker change and clinical phenoconversion in individuals with iRBD.18F-florbetaben PET . We. We37]. Consistent with a previous longitudinal study in a large cohort , the meaIn this study, the low value of mean cortical thickness at baseline was found to significantly predict overall phenoconversion. This result is in agreement with a previous study by Pereira et al., showing that the mean cortical thickness in ROIs that differed between converters and non-converters, significantly predicted overall phenoconversion in iRBD . The basIn an effort to stratify LBD subtypes in iRBD patients, Rahayel et al. derived a clinical-imaging signature from iRBD patients that could predict dementia-first phenoconversion . The cliIn our study, none of the clinical biomarkers were able to discriminate dementia-first from parkinsonism-first conversion Table . These fThe current study has some limitations. First, the MRI-driven DLB-related cortical thickness pattern needs to be reproduced in large multicenter cohorts. Second, large-sample studies with longer follow-ups would be valuable to evaluate the relationship between the cortical signature and various prodromal markers comprehensively. Nevertheless, the sample size in this study was acceptable for analyzing the predictive performance of our imaging markers because of the relatively high HRs of these markers . Third, The MRI-driven DLB-pattern reflects prodromal clinical features and longitudinal evolution of Lewy body dementia in individuals with iRBD. The MRI-based index consisting of the mean cortical thickness values and the DLB-pattern expression score would help predict and monitor DLB-type phenoconversion in individual patients with iRBD.Additional file 1: Fig. S1. Stable regions of interestin DLB-related cortical thickness pattern. Fig. S2. Direct comparison of the cortical thickness for iRBD versus DLB. Fig. S3. Disease-free survival in iRBD patients with Kaplan-Meier analysis. Fig. S4. Correlation between the DLB-pattern and the AD-pattern. Fig. S5. Longitudinal change of DLB-pattern in the non-converter iRBD group. Table S1. List of ROIs for cortical thickness analysis.


The surface structure, morphology, crystalline quality, and chemical composition were determined using a variety of analytical techniques, including powder X-ray diffraction (XRD), scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HR-TEM), and X-ray photoelectron spectroscopy (XPS). The electrochemical characterization by CV revealed a linear range of UA from 0.1 mM to 8 mM, with a detection limit of 0.005 mM and a limit of quantification (LOQ) of 0.008 mM. A study of the sensitivity of NiCo2O4 nanostructures modified on the surface to UA detection with amperometry has revealed a linear range from 0.1 mM to 4 mM for detection. High stability, repeatability, and selectivity were associated with the enhanced electrochemical performance of non-enzymatic UA sensing. A significant contribution to the full outperforming sensing characterization can be attributed to the tailoring of surface properties of NiCo2O4 nanostructures. EIS analysis revealed a low charge-transfer resistance of 114,970 Ohms that offered NiCo2O4 nanostructures prepared with 5 mL of radish white peel extract, confirming an enhanced performance of the presented non-enzymatic UA sensor. As well as testing the practicality of the UA sensor, blood samples from human beings were also tested for UA. Due to its high sensitivity, stability, selectivity, repeatability, and simplicity, the developed non-enzymatic UA sensor is ideal for monitoring UA for a wide range of concentrations in biological matrixes.The ability to measure uric acid (UA) non-enzymatically in human blood has been demonstrated through the use of a simple and efficient electrochemical method. A phytochemical extract from radish white peel extract improved the electrocatalytic performance of nickel–cobalt bimetallic oxide (NiCo As NiCo2O4 nanostructures exhibit poor electrochemical performance due to their limited surface properties, they have been utilized in composites with other nanostructured materials, such as Fe2O3 @ NiCo2O4 [2/NiCo2O4 [3O4/NiCo2O4 [2O4 @ graphene [2O4 systems exhibit enhanced charge transfer between electrode and analyte. Several morphologies of NiCo2O4 have been prepared, including nanotubes [2O4’s diverse architecture, however, the material fails to perform as expected [2O4 in order to develop more sensitive non-enzymatic UA sensors. Recent years have seen a great deal of interest in green chemistry due to its simplicity, eco-friendliness, low cost, and environmental friendliness [2O4 nanostructured materials from biomass wastes. The present study examines for the first time the effect of radish white peel extract (Raphanus sativus) on the morphology, crystal defect, and surface properties of NiCo2O4 that will be used for fabricating a non-enzymatic UA sensor. Radish white peel extract contains a wide range of phytochemicals, including alkaloids, glucosinolates, phenolic compounds, organic acids, anthocyanins, and isothiocyanates. [A purine-based compound called alkaloids is used by our bodies to produce uric acid (UA) ,2. UA co NiCo2O4 , MnO2/Ni/NiCo2O4 , Co3O4/N/NiCo2O4 ,38, and graphene . It has anotubes , nanorodanotubes , nanosphanotubes , and nananotubes . In spitexpected ,36,38. Andliness ,40,41,422O4 nanostructures.The radish white peel extract possesses major phytochemicals, like as 4-hydroxycinnamic acid, caffeic acid, ferulic acid, and isothiocyanates, which were used as stabilizing, capping, and reducing agents, and played a significant role toward enhanced electrochemical properties of NiCo2O4 nanostructures by hydrothermal methods. The volume of extracts from peels was examined in order to determine the ideal concentration at which NiCo2O4 nanostructures could be scaled up for high performance production. An extensive linear range of UA detection was detected using surface-modified NiCo2O4 nanostructures ranging from 0.1 mM to 8 mM.In this study, we have used radish white peel extracts for the surface engineering of NiCo2·6H2O), nickel chloride hexahydrate (NiCl2·6H2O), glucose, lactic acid, uric acid, sodium chloride, potassium chloride, ascorbic acid, sodium hydroxide, urea, hydrochloric acid, disodium phosphate, and monopotassium phosphate were applied without pretreatment. We obtained all analytical grade chemical reagents from Sigma Aldrich, Karachi, and Sindh Pakistan. UA detection was performed by preparing the desired solutions in deionized water, followed by preparing a buffer solution containing 0.1 M phosphate at pH 7.0 for UA detection.In this study, cobalt chloride hexahydrate  using an accelerating voltage of 10 kV. We assessed the crystal quality of radish white peel extract-assisted NiCo2O4 nanostructures using powder X-ray diffraction (XRD) at 45 kV and 45 mA using CuK radiation (λ = 1.5418 Å) as an X-ray source. The localized nanoscale structure was studied using high-resolution transmission electron microscopy (HRTEM) at a voltage of 200 kV. Utilizing energy dispersive spectroscopy, we were able to quantify the elemental mapping. The chemical composition of the surface was determined using X-ray photoelectron spectroscopy (XPS) in high vacuum. As a reference binding energy, we calibrated the XPS data using C1s at 284.6 eV and deconvolved the XPS features using a Shirley-type background and Voigt curves.An assessment of the morphology of NiCo2O4 nanostructures was conducted using a three-electrode setup. Three electrodes were used: silver–silver chloride  as a reference electrode, platinum sheet as a counter electrode, and glassy carbon electrode (GCE) as a working electrode. Prior to modification of the GCE, it was polished with an alumina paste of (0.3 µM) and silicon paper and then cleaned with deionized water. The material ink was prepared by dispersing 10 mg of NiCo2O4 nanostructures in 2.5 m of deionized water and 0.5 mL of Nafion (5%) in 2.5 m of deionized water. We achieved homogeneous material ink after 15 min of an ultrasonic bath. NiCo2O4 nanostructures were applied to the surface of GCE using a drop-cast method. A 10 mM stock solution of UA was prepared in a 0.1 M phosphate buffer solution of pH 7.0. Prior to dissolving the phosphate buffer solution, UA was dissolved in propanol. The selectivity of the non-enzymatic UA sensor was determined with 0.1 mM interfering species, like urea, lactic acid, glucose, ascorbic acid, potassium ions, and sodium ions, in the presence of the same concentration of UA. The linear range of the UA sensor was determined by CV and chronoamperometry methods using various UA concentrations dissolved in 0.1 M phosphate buffer solution (PBS) at pH 7.0. Using this method, it was possible to estimate the low limit of detection of the non-enzymatic sensor [A variety of electrochemical methods have been used to characterize the non-enzymatic UA sensor, including cyclic voltammetry, amperometry, electrochemical impedance spectroscopy, and linear sweeping voltammetry. The electrochemical evaluation of NiCoc sensor .2O4 nanostructures that reveal their crystal quality. Furthermore, we investigated the effects of phytochemicals present in the white peel extract of radish on the crystal quality of NiCo2O4 nanostructures. The reference card number 00-020-0781 confirms that the typical reflections of NiCo2O4 nanostructures are well matched with the cubic phase of NiCo2O4. The diffraction patterns of NiCo2O4 nanostructures have been identified as (111), (220), (311), (222), (400), (511), and (440), respectively, at 19.40, 31.30, 36.880, 38.590, 44.850, 59.410, and 65.300. The morphology of NiCo2O4 nanostructures prepared with radish peel extract was examined by SEM. This was performed to compare them with pristine NiCo2O4 nanostructures, as shown in 2O4 nanostructures with a high degree of heterogeneity exhibit a typical nanorod-like orientation as shown in 2O4 nanostructures toward short-range nanoparticles, confirming the influence of phytochemicals on surface modification. As well as being much smaller in size, nanostructures have a size range between 50 and 100 nm, which is characteristic of nanoparticles. 2O4 nanostructures. According to 2O4 nanostructures were examined with HRTEM images and Fourier transform (FFT) patterns in order to analyze the deep morphological features and to calculate atomic d-spacing. 2O4 nanostructures. According to 2O4 nanostructures have a d-spacing of 0.39 nanometers. Using elemental mapping. 2O4 nanostructures, as shown in 2O4 nanostructures , and physic/chemisorbed water on the surface of the material. The XPS study has revealed that green organic reducing, capping, and stabilizing agents from radish white peel extract have a relatively high concentration of oxygen ions (O−) and Co3+ compared to pristine NiCo2O4 nanostructures. These surface properties are highly desirable for a catalytic reaction [3+/Co2+ and Ni2+/Ni3+ metallic positively charged ions in the surface of NiCo2O4 nanostructures. Consequently, these metallic ions have offered a high density of catalytic sites for the favorable electrocatalytic reaction of UA.The presence of green organic recuing, capping, and stabilizing agents in the radish white peel extract have the capability to tailor the surface properties of nanostructured materials ,42. Usined peaks ,44. Furt studies ,44 have reaction ,44. Furt2O4 nanostructures were characterized electrochemically using a three-electrode cell configuration in order to detect UA. 2O4 nanostructures for the oxidation of UA. A CV curve was measured at 50 mV/s using both pristine NiCo2O4 nanostructures and NiCo2O4 nanostructures, which had been surface-modified with 5 mL and 10 mL of radish white peel extract in the absence or presence of UA. A CV curve, such as that shown in 2O4 nanostructures placed in 0.5 mM UA in a phosphate buffer solution at pH 7.0. When comparing NiCo2O4 nanostructures prepared with 5 mL of radish white peel extract with pristine and surface-modified NiCo2O4 nanostructures soaked in 10 mL of radish white peel extract, it is evident that the electrocatalytic properties of these nanostructures are well described when compared to pristine and surface-modified NiCo2O4 nanostructures soaked in 10 mL of radish white peel extract. This is due to the enhanced conductivity of the material and the enriched surface sites on the surface. Due to the poor catalytic properties and the electrical conductivity of NiCo2O4, the CV curve shows that the peak current is relatively low when UA is oxidized on pristine NiCo2O4 nanostructures. However, the electrochemical performance of NiCo2O4 nanostructures was strongly dependent on the volume of radish white peel extract, as evidenced by the limited electrochemical signal in 2O4 nanostructures that are highly desirable for electrochemical applications, 5mL of radish white peel extract appears to provide the best conditions. Moreover, NiCo2O4 nanostructures prepared using 5 mL of radish white peel extract and bare glassy carbon electrodes were tested in the presence of 0.5 mM UA and only in the presence of a phosphate buffer solution at pH 7.0, as shown in 2O4 nanostructures; however, the bare glassy carbon electrode, as shown in 2O4 nanostructures synthesized with radish white peel extract were only effective for driving UA oxidation in the phosphate buffer solution, thus full non-enzymatic UA sensor characterization was carried out.NiCo2O4 nanostructures, electrons are transferred from UA to Co3+ and Ni3+ ions and reduced to Co2+ and Ni2+. In addition to altering the size and surface properties of NiCo2O4 nanostructures, phytochemicals in radish white peel extract enhanced the charge transfer between the electrode and analyte solution. 2O4. As shown in 2O4 nanostructure-based electrodes have been found to have a high scan rate in recent studies. According to CV analysis, UA oxidation is accompanied by two electron and proton transfers [2—0.99) supports the analytical aspects of an enzyme-free UA sensor for precise and accurate quantification of UA. Therefore, surface adsorption and electrochemistry were responsible for controlling the UA oxidation reaction.When UA is oxidized on NiCoransfers ,45,46,472O4 nanostructures prepared with 5 mL of radish white peel extract. As a result of the pH change of 0.5 mM of UA, CV curves for NiCo2O4 nanostructures are illustrated in 2O4 nanostructures are strongly regulated by the pH of the analyte solution when it comes to their activity. Such aspects have previously been examined in a study [2O4 nanostructures are limited in their ability to function under different pH conditions of analyte solutions. Consequently, the catalytic sites are diminishing, the material is unstable, and the surfaces are etched [2O4 nanostructures’ electrochemical activity. UA oxidation occurs when two protons are lost and is suppressed by UA’s low pH of 7.0. However, at higher pH levels, NiCo2O4 nanostructures are likely to be etched, resulting in poor performance.Our study examined the effect of pH on the oxidation process of NiCo a study . NiCo2O4e etched . As a re2O4 nanostructures prepared with 5 mL of radish white peel extract. The linear range of a non-enzymatic UA sensor was evaluated by varying electrochemical modes in order to maintain the sensing range of each electrochemical mode. The linear range of UA was first investigated by CV at 50 mV/s in a phosphate buffer solution at pH 7.0 at different concentrations of UA. The linear range of UA was determined to be between 0.1 mM and 8 mM, and the peak current for UA oxidation increased linearly with increasing UA concentration, as shown in Analyses of the linear range and limit of detection of UA were conducted using NiCo2O4 nanostructures with radish white peel extract presented outstanding linearity and high sensitivity. The SEM, XRD, HRTEM, and XPS measurements demonstrated tailored morphology, excellent crystal quality, a significant amount of surface vacancies, and an abundance of Co(III) and Ni(II) chemical states, which led to the superior performance of NiCo2O4 nanostructures prepared with 5 mL of radish white peel extract. In order to evaluate the selectivity of a UA sensor before implementing it in actual sample analysis, we tested the proposed UA sensor in the presence of possible interfering species during detection of UA in real blood and urine samples. It was determined that the selectivity of the interfering compounds could be monitored by preparing NiCo2O4 nanostructures with radish white peel extract containing glucose, ascorbic acid, urea, lactic acid, mannose, sodium ions, chloride ions, potassium ions, and calcium ions, as shown in 2O4 nanostructures with phytochemicals from radish white peel extract, which enabled it to detect specific electroactive molecules.A linear fitting of the UA detection resulted in excellent performance with strong analytical features. The results confirm the algorithm’s promising performance for the detection of UAs and its application in real-world sample analysis. Due to the high density of catalytic sites, enhanced electrical conductivity, and biomimetic compatibility of nanostructured material with the surface of GCE, UA detection on surface-modified NiCo2O4 nanostructures can be used for long-term applications, since the material does not change surface features under ambient conditions. However, we examined the stability of NiCo2O4 nanostructures in 0.5 mM UA solution by measuring the amperometric response over a period of 1000 s. This is depicted in We examined the repeatability of the modified UA sensor electrode by measuring 20 CV cycles at a scan rate of 50 mV/s in 0.5 mM. It was found that the device could be reused, as shown in 2O4 nanostructures, electrochemical impedance spectroscopy (EIS) was performed using a sweeping frequency range of 100 kHz to 1 Hz, amplitude of 10 mV and biasing potential of 0.6 V. The Nyquist plots were measured for the three samples of NiCo2O4 nanostructures, including pristine sample 1 and sample 2 in 0.5 mM UA, and the EIS data were fitted with Z-view software as illustrated in 2O4 nanostructures and the 0.5 mM analyte solution [2O4 nanostructures, sample 1, and sample 2 in the presence of analyte were as 125,100 Ohm, 114,970 Ohm, and 181,270 Ohm, respectively. The EIS study has verified that sample 1 of NiCo2O4 nanostructures prepared with 5 mL of radish white peel extract has more favorable charge transport over the pristine and sample 2-based NiCo2O4 nanostructures; hence, we observed better electrochemical measurements of sample 1 toward the quantification of a sensitive UA signal.Our results in terms of wide linear range, low limit of detection, excellent selectivity, stability, and repeatability confirmed the superior performance compared to other UA sensors/biosensors reported in the existing literature, as shown in solution . The estThe non-enzymatic UA sensor configuration presented in 2O4 nanostructures. It has been combined with phytochemicals derived from radish white peel. It was determined that a volume of 5 mL of radish white peel extract was optimal for the large-scale synthesis of NiCo2O4 nanostructures. The CV exhibited a linear range from 0.1 mM to 8 mM, the LSV showed 0.1 mM to 7 mM, and the amperometric signal for the UA sensor showed a linear range from 0.1 mM to 4 mM. We found that the LOD and LOQ of the present non-enzymatic UA sensor were 0.005 mM and 0.008 mM, respectively. Furthermore, the present UA sensor was evaluated in terms of its selectivity, stability, repeatability, and sensitivity. Consequently, a wide linear range and a low limit of detection were achieved. Several factors contribute to the sensor’s performance, including large surface vacancies, abundant chemical states of Co(III), Ni(II), tailored surface morphology, fast charge-transfer rate, and outstanding crystal quality. It has been demonstrated that there is a satisfactory level of detection of UA in human blood samples .An easy and rapid method has been developed for quantifying UA in human blood and urine samples by using surface-modified NiCo


Introduction: Mathematical model can be used to model complex biological processes, and have shown potential in describing apoptosis in chondrocytes.Method: In order to investigate the regulatory mechanisms of TNF signaling pathway in regulating chondrocyte apoptosis, a fractional-order differential equation model is proposed to describe the dynamic behavior and mutual interaction of apoptosis-related genes under the activation of TNF signaling pathway. Compared with the traditional molecular biology techniques, the proposed mathematical modeling has advantages to providing a more comprehensive understanding of the regulatory mechanisms of TNF signaling pathway in chondrocyte apoptosis.Result: In this paper, differentially expressed genes induced by IL-1β in human chondrocyte apoptosis are screened using high-throughput sequencing. It is found that they were significantly enriched in the TNF signaling pathway. Therefore, a mathematical model of the TNF signaling pathway is built. Using real-time PCR experiments, mRNA data is measured and used to identify the model parameters, as well as the correlation coefficient. Finally, the sensitivity of the model parameters is discussed by using numerical simulation methods, which can be used to predict the effects of different interventions and explore the optimal intervention strategies for regulating chondrocyte apoptosis.Discussion: Therefore, fractional-order differential equation modeling plays an important role in understanding the regulatory mechanisms of TNF signaling pathway in chondrocyte apoptosis and its potential clinical applications. Osteoarthritis (OA) is a chronic degenerative joint disease characterized by articular cartilage destruction, subchondral bone remodeling, osteophyte formation, and inflammatory changes in periarticular tissues, which seriously affects the quality of life of patients. According to statistics, the incidence of osteoarthritis is 50% in people over 50 years old, 80% in people over 60 years old, and the disability rate is as high as 53% . OsteoarHowever, for the treatment of OA, the current goal is mainly to relieve pain and improve joint function, and there is no specific drug. Therefore, exploring an intervention that can effectively delay the progress of OA and improve the OA condition in the early stage is considered to be a very potential treatment strategy that can effectively reduce the development process of OA patients . StudiesThe signal transduction pathway of chondrocyte apoptosis is very complex, and the signal pathways involved mainly include TNF signaling pathway . MAPK siIn the IL-1β-induced primary human chondrocytes, baicalin downregulated the mRNA and protein expression of MMP1 and MMP13, and promoted the expression of collagen type II and Aggrecan. Baicalin significantly inhibits cartilage degradation in DMM-induced OA mice, suggesting that breviscapine may be a potential drug for OA treatment . In addiStudies have found that , OA chonIn summary, the signaling pathways involved in the regulation of articular chondrocyte apoptosis are diverse and extremely complex, but the most significant signaling pathway regulating chondrocyte apoptosis is still unclear, and the most critical signaling molecules in the signaling pathway are also unclear. Therefore, it is urgent to screen the signaling pathways that most significantly affect chondrocyte apoptosis and discover the most critical signaling molecules in this signaling pathway. The mathematical modeling of signaling pathways through high-throughput sequencing is the key to solve the above problems.Dynamic modeling and analysis of biological systems can simplify the biological system into a mathematical model for analysis and numerical simulation, thus replacing the actual complex, long-term, expensive and even impossible experiments, greatly improving the research efficiency, and studying the influence of artificially imposed control conditions on the operation process of biological systems. For example, infectious disease modeling  and signDrosophila large intestine boundary cells, used Hybrid functional Petri net (HFPN) for modeling. In 2009, Drosophila germ layer was further understood. Immediately following this, In 2003, Drosophila large intestine border cell formation relying on Delta-Notch signaling pathway . Written informed consent was obtained from all patients. The study has been approved by the Shenzhen Second People’s Hospital , China. The method to deal with the cartilage in order to obtain the proper chondrocytes is as follows:After scraping the cartilage tissue with a blade, rinse it three times with physiological saline containing 3% PS. Use a two-step digestion method to separate the articular chondrocytes. Transfer the cartilage tissue to a sterile culture dish and wash it three times with PBS containing 3% PS. Carefully remove any attached soft tissue or synovial fluid, and use ophthalmic scissors to crush it into pieces smaller than 1mm3. Transfer the crushed tissue to a new 50 mL centrifuge tube and add 5 times the volume of 0.25% trypsin. Digest for 20 min and then wash the remaining tissue three times with low glucose DMEM medium. Add 5 times the volume of a solution containing 0.2% type II collagenase and digest gently on a shaking bed at 37°C for 4-5 times, 25 min each time. After each digestion, collect the cells in the supernatant and culture the cells using low glucose DMEM medium containing 20% fetal bovine serum.In the early stage, normal chondrocytes were stimulated with IL-1β at a concentration of 10 ng/mL. The results showed that chondrocytes began to apoptosis at 6h, reached the peak at 18h, and then the degree of apoptosis decreased .Therefore, in this study, the experiments are divided into three groups: 1) normal chondrocytes served as blank control, 2) normal chondrocytes were stimulated with IL-1β (10 ng/mL) for 6 h, and 3) normal chondrocytes were stimulated with IL-1β(10 ng/mL) for 18 h. The cells were collected and subjected to high-throughput sequencing.p = 3.21e-09 for TNF signaling pathway, p = 5.76e-05 for NF-κβ signaling pathway, p = 1.23e-04 for Cytokine-cytokine receptor interaction signaling pathway. The significant difference value for T-cell receptor signaling was p = 1.77e-04. The detailed results are shown in The results of sequencing showed that the significant difference value was The results of the above high-throughput sequencing suggest that the most significant signaling pathway regulating chondrocyte apoptosis under IL-1β-stimulated conditions is the TNF signaling pathway. Therefore, this project proposes a scientific hypothesis: Through the mathematical modeling of IL-1β/TNF signaling pathway screened by high-throughput sequencing, the regulatory effect of signal molecules in the IL-1β/TNF signaling pathway on chondrocyte apoptosis can be systematically analyzed, and new targets for the treatment of osteoarthritis can be further explored through the sensitivity analysis of parameters.According to the signaling molecular transduction mechanism of TNF signaling pathway regulating chondrocyte apoptosis, we use differential equations to model the dynamics of chondrocyte apoptosis. Firstly, the signal molecule transduction map of TNF signaling pathway regulating chondrocyte apoptosis was drawn by Portable Pathway Builder Tool 2.0  Pre-processing of samples(2) Add 200 μL chloroform, vigorously shake the tube several times, and let it stand at room temperature for 5 min.(3) Centrifuge at 4°C and 12,000 rpm for 15 min to separate the sample into three phases: the upper phase (RNA), the middle phase (protein), and the lower phase (DNA).(4) Transfer the upper aqueous phase (approximately 400 μL) to a new 1.5 mL EP tube, add 400 μL isopropanol, mix thoroughly, and let it stand at room temperature for 10 min.(5) Centrifuge at 4°C and 12,000 rpm for 10 min, and a white RNA pellet should be visible at the bottom of the tube.(6) Discard the supernatant, add 1 mL 75% ethanol without RNase, vortex, and centrifuge at 4°C and 10,000 rpm for 5 min.(7) Repeat step 6 once.(8) Discard the supernatant, air-dry the RNA pellet for 5–10 min, and dissolve the pellet in 20 μL DEPC water.−3.(9) Measure the OD260, OD280, and OD260/OD280 values of the RNA using a micro spectrophotometer, and calculate the purity and concentration of the RNA. Estimate the RNA quality based on the OD260/OD280 ratio, which should be between 1.8 and 2.0 for experimental requirements. Calculate the total RNA concentration (μg/μL) using the following formula: Total RNA concentration = OD260 × 40×10(10) Store the total RNA at −80°C for future use.Cell: Add 1 mL Trizol reagent, mix thoroughly by pipetting, transfer to a RNase-free 1.5 mL EP tube, and incubate for 10 min to lyse the cells.Melt the RNA template, NASe-free Water, and 10× gDNA Remover Buffer on ice. In a nuclease-free micro centrifuge tube, prepare a reaction system (10 μL) on ice according to the table below. The reaction system is shown in Subsequently, the mixed system was blown and briefly centrifuged using a pipettor, and the reaction was carried out on a PCR instrument. The procedure is given as follows:The cells were incubated at 42°C for 2 min and at 60°C for 5 min.Following the preceding reaction, the system was rapidly chilled on ice and subsequently subjected to a brief centrifugation. The addition of specific components was then carried out based on the desired detection index .Subsequently, employing a liquid motion beat blending system and brief centrifugation, the sample was added to the PCR reaction with the following program: incubation at 25°C for 10 min, followed by incubation at 50°C for 15 min and then further incubated at 85°C for an additional 5 min. The reverse transcription product was subsequently placed on ice or refrigerated as required.The Real-time PCR reaction system is shown in -△△Ct method , we obtained simulation results of 19 genes, as shown in The correlation coefficient (R) was calculated to compare the experimental findings with model stimulation. The results, as presented in Based on the accurate mathematical model obtained, the influence of model parameter changes on the system will be studied next, that is, the sensitivity of parameters in the model will be analyzed. Given the positive correlation between chondrocyte apoptosis and osteoarthritis severity, this project aims to employ gene interference experiments to silence related genes while making subtle adjustments to model parameters that significantly impact output. The ultimate goal is to reduce chondrocyte apoptosis and identify new targets and strategies for treating osteoarthritis. In this section, sensitivity of molecules based on the mathematical model was analyzed by numerical simulations. Because the severity of chondrocyte apoptosis is positively correlated with the severity of osteoarthritis, this project aims to greatly change the output of the model by making subtle adjustments to the sensitive parameters of the model, that is, to reduce chondrocyte apoptosis, and to provide new targets and strategies for the treatment of osteoarthritis. Therefore, in this study, the degree of change in the mRNA expression of the gene Caspase-3 was used as an indicator of parameter sensitivity. The simulation results show that Similarly, an insensitive parameter , TNF-α (x3), TRADD (x5), RIP1(x7), NIK (x8), TAK1 (x9), IKK-β (x10), MKK4(x11), FADD (x12), ITCH (x15), Caspase-8 (x18) and Caspase-3 (x19) at 0 h, 6 h, 12 h, 18 h, 24 h, 30 h, 36 h, 42 h, 48 h, 54 h, 60 h, 66 h, and 72 h. The 62 parameters of the model are estimated by using the collected experimental data. Since the process of chondrocyte apoptosis is time-dependent, the parameter estimation was divided into three stages. The effectiveness of parameter estimation is evaluated by calculating the correlation coefficient between the experimental data and the mathematical model, with a maximum correlation coefficient of 0.969 (See Since most of the current research on the regulation of signal pathways in chondrocyte apoptosis is based on molecular biology methods, it can only focus on studying the signaling molecules in a specific pathway, making it difficult to understand the mechanism of signal transduction at a systemic level. In this study, high-throughput sequencing is firstly used to screen for differentially expressed genes involved in IL-1β-induced apoptosis in human chondrocytes. The results show significant enrichment in the TNF signaling pathway. Therefore, mathematical modeling approach is adopted to quantitatively analyze the mechanism of TNF signaling pathway in regulating chondrocyte apoptosis. Next, qPCR experiments are performed to measure the mRNA expression levels of IRAK1 (.969 See . Finally


Antitumor efficacy was increased when combined with tumor targeting antibodies. Here we report the development of TAEK-VAC-HerBy (TVH), a first-in-class human tumor antibody enhanced killing (TAEK) vaccine based on the non-replicating MVA-BN viral vector. It encodes the membrane bound form of human CD40L, HER2 and the transcription factor Brachyury. TVH is designed for therapeutic use in HER2- or Brachyury-expressing cancer patients in combination with tumor targeting antibodies. To preclude possible oncogenic activities in infected cells and to prevent binding of vaccine-encoded HER2 by monoclonal antibodies trastuzumab and pertuzumab, genetic modifications of HER2 were introduced in the vaccine. Brachyury was genetically modified to prevent nuclear localization of the protein thereby inhibiting its transcriptional activity. CD40L encoded in TVH enhanced human leukocyte activation and cytokine secretion in vitro. Lastly, TVH intravenous administration to non-human primates was proven immunogenic and safe in a repeat-dose toxicity study. Nonclinical data presented here highlight TVH as a first-in-class immunotherapeutic vaccine platform currently under clinical investigation.The induction of antiviral innate immunity by systemic immunization with live virus can be employed to positively impact the response to therapeutic vaccination. We previously demonstrated that systemic immunization with a non-replicating MVA encoding CD40 ligand (CD40L) enhances innate immune cell activation and function, and triggers potent antitumor CD8 Live viral vectors represent an excellent vaccine platform to deliver TAAs and stimulate the immune system. Viral infection, recognition and death of infected cells trigger the activation of antiviral defense mechanisms that modulate antitumor innate and adaptive immune responses.Therapeutic cancer vaccines are designed to generate de novo and expand pre-existing CD41. Furthermore, the recombinant MVA based Ebola vaccine MVABEA® (MVA-BN Filo) administered in combination with Janssen's Ebola vaccine ZABDENO® (Ad26.ZEBOV) was recently approved by the European Medicines Agency (EMA)2. MVA is currently being employed in clinical trials against infectious diseases3 and in patients with cancer4. Hence, MVA-BN’s excellent safety profile in humans6, the maintained capability to induce strong and durable cellular and humoral immune responses7, and the capacity to express numerous transgenes make it an attractive vaccine platform.Modified Vaccinia Ankara (MVA)-BN is a highly attenuated vaccinia strain, replication deficient in humans and other mammals, including immunocompromised mice and non-human primates (NHPs), and is approved as a third-generation smallpox and monkeypox vaccine in USA (JYNNEOS™), Canada (IMVAMUNE®) and the European Union (IMVANEX®)9. These tumors have a distinctive molecular signature which is associated with aggressive biological behavior and poor clinical outcome. HER2 is considered a driver oncogene promoting cancer cell growth. Accordingly, inhibition of the HER2 signaling pathway translates into clinical benefit. In addition to the fact that HER2 is a target for cell signaling inhibition, HER2 is also a potential target for immunotherapy10 in the context of combination therapies11. However, a substantial fraction of patients is primarily resistant or will become resistant to HER2 targeting drugs and ultimately will end up dying of cancer12.Human epidermal growth factor receptor 2 (HER2) amplification and overexpression play a key role in many epithelial tumor types including breast and gastric cancer14. Brachyury is a driver of epithelial-to-mesenchymal transition (EMT). EMT is a reversible process during which cells switch from a polarized, epithelial phenotype into a highly motile, mesenchymal phenotype15. It has been demonstrated that overexpression of Brachyury in human carcinoma cell lines is able to drive a switch from an epithelial to a mesenchymal-like phenotype16. In addition, Brachyury is highly overexpressed in the rare bone sarcoma chordoma17. Together with differential Brachyury expression mostly in tumor tissues18, these findings make Brachyury an attractive target for cancer vaccine development. MVA vector-based vaccines targeting Brachyury are currently under clinical evaluation20.Brachyury is a member of the T-box family of transcription factors, characterized by a highly conserved DNA-binding domain designated as T domain+ T cells `license´ DCs to prime CD8+ T cell responses21. Our previous work showed that systemic immunization with a non-replicating MVA encoding CD40L (MVA-CD40L) enhanced antitumor immune responses in different tumor models and mouse strains22. In addition, immunization with MVA-CD40L modulated the production of proinflammatory cytokines and expanded and activated natural killer (NK) cells systemically. Indeed, therapeutic combination of MVA-CD40L with antibodies targeting TAAs significantly improved antitumor responses against established, aggressive carcinomas, relying on a NK cell- and Fc receptor-dependent mechanism.The TNF receptor family member CD40 is considered a costimulatory master-switch of dendritic cells (DCs). CD40-CD40 ligand (CD40L)-based interaction of DCs with activated CD4In this study we describe the development of a novel human tumor antibody enhanced killing (TAEK) vaccine (VAC) encoding the membrane bound form of human CD40L, HER2 (Her) and the human transcription factor Brachyury (By), . It is designed for intravenous administration in patients with advanced HER2-or Brachyury-expressing cancers and to be combined with HER2-targeting therapeutic antibodies.TVH carries genetic modifications of HER2 to prevent possible oncogenic activities in infected cells. Additionally, vaccine encoded HER2 cannot be bound by the monoclonal antibodies trastuzumab and pertuzumab due to modifications in the respective binding epitopes. Brachyury was genetically modified to prevent translocation of the protein into the nucleus and thereby inhibits its transcriptional activity. TVH infection in vitro led to potent human leukocyte and DC activation and cytokine secretion. In addition, TVH administered intravenously to NHPs was immunogenic and showed a favorable safety profile in a repeat-dose toxicity study. Nonclinical data presented here highlight TVH as a first-in-class immunotherapeutic vaccine which currently undergoes clinical testing (NCT04246671).TVH is a recombinant immunotherapy vaccine based on the non-replicating MVA-BN viral vector, which itself is licensed as smallpox and monkeypox vaccine under the trade name JYNNEOS®. It encodes the human HER2, the human transcription factor Brachyury and the membrane bound form of human CD40L in the Intergenic Region (IGR) 88/89 of the MVA-BN genome Fig. a. Expres23. To avoid potential issues caused by the function of Brachyury as a transcription factor its nuclear localization signal was deleted in TVH to retain it in the cytoplasm24. Moreover, Brachyury antigen cross-presentation might conceivably be more efficient with cytoplasmic-expressed Brachyury when compared to Brachyury that is localized in the nucleus. To analyze subcellular localization of Brachyury, BALB/3T3 clone A31 cells were infected with TVH, lacking the potential nuclear localization signal (NLS) of Brachyury (ΔNLS), or with a recombinant MVA encoding Brachyury still containing its NLS (MVA-Brachyury) for comparison. Subcellular distribution of Brachyury-ΔNLS encoded by TVH was confined to the cytoplasm, as the nucleus was devoid of staining . The rationale behind TVH involves administration of MVA with trastuzumab and/or pertuzumab. Consequently, binding of these antibodies to the MVA-encoded HER2 must be prevented. Likewise, any adverse effects which might result from HER2 overexpression by MVA in targeted cells need to be avoided.Two humanized monoclonal antibodies targeting HER2, trastuzumab and pertuzumab, have been developed and approved for treatment of HER2-overexpressing malignancies31, leading to a loss of binding of vaccine-encoded HER2 by trastuzumab . Hence, a total of eight amino residues were mutated to alanine: H267, F279, V308, S310, L317, H318, K333, P337 which completely abolished binding of pertuzumab to TVH-expressed HER2  and pertuzumab (Perjeta®) binding sites were mutated . Interaction of trastuzumab with HER2 requires 3 loops in the juxtamembrane region of HER2 formed by amino acids 578–582 (loop1), 591–594 (loop2), as well as 614–624 (loop3). Based on structure analysis and relevant literature search the key residues E580, F595 as well as K615 were mutated to alanine in TVH21. Hence, we next tested whether human CD40L encoded in TVH would induce activation and maturation of human DCs. Transduction of human monocyte-derived DCs from healthy donors using TVH resulted in surface expression of HER2 and CD40L . Increased mean fluorescence intensity (MFI) of CD86, MHC-II and CD70 was observed upon overnight incubation of infected DCs with TVH and depending on TCID50 used for infection  would result in immune cell activation. PBMCs were incubated with TVH or ConA, which was used as a positive control. Supernatants were analyzed in terms of cytokine secretion profile. TVH infection induced IL-12p70, IFN-γ, IL-6 and TNF-α and to a higher level than in response to ConA  as well as the surface presentation of immunogenic HLA/peptide complexes are key steps in the generation of adaptive immune responses by vaccines. To test whether TVH-derived antigens are presented on APC upon infection, we performed the ProPresent® antigen presentation assay with TVH infected human macrophages. We first differentiated the human monocytic cell line THP-1 into macrophages using Phorbol-12-Myristate-13-Acetate (PMA). As control, cellular markers of differentiation were analyzed  and Tris-buffered saline (TBS) was administered as control as summarized in Table 9 Inf.U/animal, when administered intravenously three times in 3-week intervals.To interrogate the safety and immunogenicity profile of systemic TVH administration, we performed a repeated-dose toxicity study in male and female cynomolgus monkeys. Two different doses of TVH were tested by IV injection . We first analyzed the presence of cytokines in serum after each immunization. Interestingly, IFN-γ, IL-6, IL-18, CCL2 and CCL4 were detected between 4 and 24 h after TVH immunization  and after  the first TVH administration. NK cells were identified as outlined in the gating strategy . Analysis of NK cells revealed a reduction in frequency even in TBS treated animals  monocytes, CD16+/CD14+ (CD16/CD14 DP) monocytes and CD16-/CD14+ (CD14 SP) monocytes, similar to their human counterparts41. Activation of APCs was measured according to increased expression levels of the co-stimulatory markers CD40, CD80 and CD86. Of note, cDC1 were not detectable in any TVH dose 48 h after treatment. The remaining DC and monocyte subsets showed increased expression levels of the co-stimulatory markers CD80  seems to be involved in breast cancer progression and the ability of cells to switch between different phenotypes, known as epithelial-mesenchymal plasticity, is associated with resistance to targeted therapies50. Several FDA approved monoclonal antibodies mediate ADCC, including trastuzumab51 and pertuzumab, however, not all patients respond to ADCC-based therapies. Compounding this issue is the lack of NK cell infiltration into the tumor and inherently low efficacy observed with ADCC52. Thus, rational combination therapies that leverage ADCC would improve the outcome of antibody-based therapies. For instance, innate immune stimulation by TLR ligands leading to efficient activation of NK cells has been reported to improve their ADCC function53. Furthermore, systemic infection using different virus strains is described to induce NK cell proliferation and trafficking not only to the infected sites but also to other organs in a cytokine-dependent manner55. In this line, we demonstrated that intravenous application of TVH results in strong activation of NK cells both in NHPs and mice22. Specifically, PBMC analysis of TVH immunized NHPs revealed the upregulation of CD69 as well as Ki67 in NK cells, indicative of potent activation and proliferation in a relevant species for clinical studies. Likewise, infection of human PBMCs with TVH resulted in the upregulation of those markers on CD16-CD56bright and CD16+CD56dim human NK cells, suggesting a broad ability of our platform to activate NK cells across species.The main rationale of the cancer vaccine TVH is to combine it with tumor-targeting antibodies in order to deploy its full therapeutic capacity. Tumor-targeting antibodies have the ability to specifically recognize TAA and direct various components of the immune system against the tumor. ADCC is one of the central mechanisms in antibody-mediated cancer immunotherapy, involving NK cells56. Among them IL-12p70 and IL-18 are cytokines typically induced by CD40L signaling in DCs and are potent activators of IFN-γ expression in NK cells58. Indeed, IL-12p70 and IL-18 were readily detected after TVH infection of human monocyte-derived DCs and PBMCs. IFN-γ was also produced by TVH-infected human PBMCs and in NHPs 4 and 24 h after TVH immunization. IL-12p70 is key to enhance activation of NK cells resulting in killing of tumor cells that have downregulated HLA37, as well as tumor cells coated by TAA targeting antibodies by ADCC38. In line with this, TVH infection of PBMCs enhanced NK cell activation by means of CD69 expression and enhanced NK cell cytotoxic activity against K562 target cells.We have previously reported that DCs play a central role for MVA-CD40L-mediated immune activation. CD40L signaling leads to activation and maturation of DCs accompanied by the expression of a wide range of proinflammatory chemokines and cytokines59. Interestingly, CCL2 and CCL4 were detected 4 h after each TVH immunization of NHPs, which is consistent with our previous observations in mice22. Hence, this highlights the ability of TVH to repeatedly activate NK cells after each vaccine administration.Cytolytic activity of NK cells can also be augmented by chemokines such as CCL2, CCL3, CCL4, CCL5, CCL10, and CXC3L161. Interestingly, we found both costimulatory receptors CD80 and CD70 upregulated upon overnight incubation of TVH infected DCs. In conclusion, TVH is a potent activator of DCs and NK cells in NHPs and human, showing similar features as our preclinical candidate in mouse models.Apart from soluble factors such as IL-12p70 and IL-18, activation of NK cells by DCs can also be promoted by direct cell–cell interaction via surface molecules. It has been reported that NK cells resemble T cells in their expression pattern of several co-stimulatory molecules such as CD28 or CD27. Blockage of CD28-CD80/86 or CD27-CD70 interaction between NK cells and DCs has been shown to reduce NK cell activation and cytotoxicity22. Furthermore, the great potential of MVA as a T cell activator in cancer patients was also proven in clinical trials. Intravenous but also subcutaneous treatment with MVA expressing Brachyury or HER2 led to the induction of tumor specific CD8 T cells in treated patients62. Intravenous application of a live virus expressing the immunostimulatory molecule CD40L might raise some safety concerns. MVA-BN, which is the backbone used for TVH, is a highly attenuated vaccinia virus strain that cannot replicate in human cells as its replication ability is largely restricted to embryonic avian cells63. MVA was approved by the FDA (JYNNEOS®) as a non-replicating vaccine against smallpox and monkeypox1 and its safety64 has been extensively proven. Despite the excellent safety profile of MVA-BN, addition of CD40L could have an impact on toxicity. Agonist anti-CD40 antibody treatment has been associated with toxicity in the clinic leading to dose limitation and, consequently, minimal clinical responses. Treatment-induced cytokine release syndrome can occur within hours and in some cases, lymphopenia and hepatotoxicity was observed that can persist for up to several weeks67. Agonist CD40 antibody-related side effects have been linked to multiple factors including CD40-induced uncontrolled activation of immune cells as well as FcyR-mediated ablation of cells. In contrast to the excessive availability of antibody in the blood after systemic administration of anti-CD40 antibodies, TVH relies on the expression of the membrane-bound natural ligand CD40L by infected cells. This ensures CD40L-dependent activation of cells in a more controlled manner and eliminates any side effects driven by antibody Fc recognition. Nevertheless, a careful dose escalation is needed in the clinic to mitigate any risks of toxicity. In addition, as for other immune therapeutics, IL-6R blocking antibodies or corticosteroids needs to be available for rapid intervention in patients with onset of first CRS symptoms.Even though we were not able to measure T cell activity in the current study due to the lack of material, we gained strong evidence for the great potential of TVH to activate CD8 T cells from previous studies in mice. We could show that intravenous injection of MVA expressing a tumor antigen together with murine CD40L led to the activation and expansion of tumor-specific CD8 T cells which was shown to be crucial for therapeutic efficacy9 Inf.U of TVH administered IV in three-week intervals to NHPs as a cancer immunotherapy demonstrated an acceptable safety profile and was locally well tolerated in animals. None of the clinical observations or the findings in clinical or anatomic pathology were considered severe as many findings were reversible and none of them caused harm to the animals by impairing their physical capacity.This assumption was confirmed by the repeat-dose toxicity study in NHPs. The application of three doses of up to 6.75 × 10In summary, this study confirmed the mechanism of action previously proposed by our mouse data with MVA-CD40L. Intravenous application of TVH led to systemic activation of innate immunity ultimately resulting in enhanced NK cell activation and function. Enhanced ADCC function of NK cells can synergize with tumor targeting antibodies and thus leverage efficacy of antibody-based therapies. Our results highlight the safety and therapeutic potential of TVH. A Phase 1 open label trial has been initiated in patients with advanced Brachyury- and/or HER2-expressing cancer treated intravenously with TVH vaccine (NCT04246671).Macaca fascicularis) of Vietnamese origin were selected to provide 20 healthy animals of each sex. Animals of the same sex were housed in pairs or groups. Tap water was provided ad libitum. Twice daily, each pair or group of animals was offered a certified lab diet (LabDiet 5048). Animals regularly received fresh fruit and vegetables as food supplement. Animals were housed in a climate-controlled room, with a minimum of eight air changes/hour and with a cycle of 12 h light and 12 h dark.The NHP study was carried out in accordance with the approved guidelines for animal experiments at Covance Laboratories GmbH . The experimental protocol was approved by the government (LANUV) of North Rhine-Westphalia. All experiments were performed in compliance with the ARRIVE guidelines . The repeat-dose toxicity study was conducted in compliance with OECD Principles of good laboratory practice (GLP), as revised in 1997. Purpose-bred naïve cynomolgus monkeys . HeLa cells were cultured in RPMI-1640 Glutamax medium supplemented with 10% FCS and 0.1 mg/ml Gentamicin. THP-1 cells were cultured in RPMI-1640 Glutamax medium supplemented with 10% FCS, 1% NEAA, 1% Sodium Pyruvate and 1% Penicillin/Streptomycin (RPMI-10). K562 cells were cultured in IMDM supplemented with 10% FCS, 1% NEAA, 1% Sodium Pyruvate and 1% Penicillin/Streptomycin (IMDM-10). All cell culture reagents were obtained from Gibco. All cells were grown in incubators at 37 °C and 5% CO2.The BALB/3T3 clone A31, THP-1, K562, CT26.WT and HeLa cell lines were obtained from ATCC or the European Collection of Cell Cultures. The CT26 murine colon carcinoma cell line expressing human HER2 (CT26.HER2) was licensed from the Regents University of California69. Cells were further cultured in RPMI-10. To generate monocyte-derived DCs from healthy donors, monocytes were isolated from buffy coats according to manufacturer´s instructions using StraightFrom™ Buffy Coat CD14 MicroBead Kit (Miltenyi Biotec). Differentiation of monocytes into monocyte-derived DCs was performed using Mo-DC Differentiation Medium (Miltenyi Biotech) according to manufacturer´s instructions. Monocyte-derived DCs were further cultured in RPMI supplemented with 10% FCS, 1% Penicillin/Streptomycin and beta-mercaptoethanol (Sigma-Aldrich). Cryopreserved Peripheral Blood Mononuclear cells (PBMCs) were obtained from CTL Europe GmbH. Cryopreserved human and NHP PBMCs were thawed using CTL Anti-Aggregate Wash Solution according to manufacturer´s instructions (CTL Europe GmbH).THP-1 cells were differentiated into macrophages in the presence of 200 ng/ml Phorbol-12-myristate-13-acetate (PMA) for 3 days followed by 2 days of rest70. TAEK-VAC-HerBy (TVH) encodes the full-length membrane-bound form of human CD40 ligand  and two tumor-associated antigens (TAAs): human HER2  and the human transcription factor Brachyury .MVA-BN® was developed by Bavarian Nordic and is deposited at the European Collection of Cell Cultures (ECACC) (V00083008). Recombinant viruses were generated using standard methods31. To abrogate pertuzumab binding to TVH-encoded HER2, residues H267, F279, V308, S310, K333, P337, L317 and H318 were mutated to alanine. To avoid any potential adverse effects upon HER2 overexpression by TVH (i) the extracellular dimerization domain was mutated, (ii) the tyrosine kinase activity was inactivated, and (iii) potential phosphorylation sites were eliminated. The introduced mutations close to the above described pertuzumab binding site will already affect heterodimer formation. To further prevent dimerization, residues D277 and E280 were mutated to arginine and lysine, respectively. Kinase activity was ablated by substituting residue K753 in the tyrosine kinase domain to methionine. To eliminate phosphorylation at known intracellular tyrosine residues amino acids 1139 to 1248 were deleted, and Y1023 was mutated to alanine.To abrogate trastuzumab binding to TVH-encoded HER2, residues E580, F595 as well as K615 were mutated to alanine71, respectively. All three promoters belong to the E1.1 and immediate early (IE) groups of poxviral promoters, driving very early gene expression73. Transgenes with their respective promoters were inserted into the intergenic region (IGR) 88/89 of MVA-BN. Genetically pure stocks were used for production of research grade material in chicken embryo fibroblasts, and infectious titers were determined as tissue culture infectious dose 50 (TCID50) per mL.In Brachyury, a potential nuclear localization signal (aa 286–293) was deleted to prevent localization of the protein into the nucleus and thereby inhibit its transcriptional activity. CD40 ligand encoded by TVH was left unmodified. The cDNAs of all three transgenes were codon optimized and adapted to avoid large stretches of identity or repeated sequences in or between the genes and synthesized by GeneArt . The HER2, CD40L, and Brachyury genes were under control of the MVA version of the vaccinia virus B2R promoter, designated as Pr1328, the MVA1-40 K promoter derived from the MVA095R gene (vaccinia virus H5R gene), and the Pr13.5long promoter6 HeLa cells were infected with TVH using 4 Inf.U per cell overnight. To assess TVH encoded antigen loading into HLA, THP-1 derived macrophages cultured in 100 mm2 cell culture dishes were infected using 4 Inf.U per cell. To address TVH induced activation and maturation of DCs, monocyte-derived DCs from healthy donors were infected overnight with TVH at the indicated Inf.U. Supernatants were collected afterwards for cytokine/chemokine analysis. To address the activation of human PBMCs, cells were infected with TVH using 0.5 Inf.U per cell overnight. 3.75 µg/ml Concanavalin A  was used as positive control. Supernatants were collected afterwards for cytokine/chemokine analysis. All cells were further processed for flow cytometry analysis.For the assessment of transgene expression, 1 × 102. Next day, cells were harvested and analyzed by flow cytometry.To address NK cell killing, PBMCs were infected with TVH or activated with ConA as described above. NK cells were magnetically sorted using MojoSort Human NK Cell Isolation Kit according to manufacturer´s instructions (Biolegend). K562 target cells were fluorescently labelled using Cell Proliferation Dye 670 (CPD670). Effector and target cells were co-cultured in 96 well plates at a ratio of 1:1 overnight in an incubator at 37 °C and 5% CO9 Inf.U; High = 6.75 × 109 Inf.U) to cynomolgus monkeys  was assessed in 3-week intervals . As control tris-buffered saline (TBS) was used. An assessment of delayed onset toxicity and/or reversibility of toxicity was made during a 4-week recovery period . Assessment of toxicity was based on clinical observations, body weights, body temperature, food consumption, assessment of feces consistency, ophthalmology, electrocardiography, and clinical and anatomic pathology evaluations. Complete necropsies were performed on all animals, with a recording of macroscopic abnormalities for all tissues. Organ weights and microscopic examinations were conducted.Toxicity of TAEK-VAC-HerBy, following three intravenous bolus administrations of two different doses  were collected from all animals once during the predose phase and on Days 4, 8, 25, 43, and 46 of the dosing phase and from all surviving animals during the last week of the recovery phase.Statistical analyses were performed as described in the figure legends using GraphPad Prism version 8.4.3 for Windows . For immunological data, data shown are the Mean and Standard Error of the Mean (SEM). Analysis of variance (one or two-way ANOVA) with multiple comparisons tests were used to determine statistical significance between treatment groups.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.