Immunofluorescence Analysis Research Articles

Characterization of Neutrophil Functional Responses to SARS-CoV-2 Infection in a Translational Feline Model for COVID-19

There is a complex interplay between viral infection and host innate immune response regarding disease severity and outcomes. Neutrophil hyperactivation, including excessive release of neutrophil extracellular traps (NETs), is linked to exacerbated disease in acute COVID-19, notably in hospitalized patients. Delineating protective versus detrimental neutrophil responses is essential to developing targeted COVID-19 therapies and relies on high-quality translational animal models. In this study, we utilize a previously established feline model for COVID-19 to investigate neutrophil dysfunction in which experimentally infected cats develop clinical disease that mimics acute COVID-19. Specific pathogen-free cats were inoculated with SARS-CoV-2 (B.1.617.2; Delta variant) (n = 24) or vehicle (n = 6). Plasma, bronchoalveolar lavage fluid, and lung tissues were collected at various time points over 12 days post-inoculation. Systematic and temporal evaluation of the kinetics of neutrophil activation was conducted by measuring markers of activation including myeloperoxidase (MPO), neutrophil elastase (NE), and citrullinated histone H3 (citH3) in SARS-CoV-2-infected cats at 4 and 12 days post-inoculation (dpi) and compared to vehicle-inoculated controls. Cytokine profiling supported elevated innate inflammatory responses with specific upregulation of neutrophil activation and NET formation-related markers, namely IL-8, IL-18, CXCL1, and SDF-1, in infected cats. An increase in MPO-DNA complexes and cell-free dsDNA in infected cats compared to vehicle-inoculated was noted and supported by histopathologic severity in respiratory tissues. Immunofluorescence analyses further supported correlation of NET markers with tissue damage, especially 4 dpi. Differential gene expression analyses indicated an upregulation of genes associated with innate immune and neutrophil activation pathways. Transcripts involved in activation and NETosis pathways were upregulated by 4 dpi and downregulated by 12 dpi, suggesting peak activation of neutrophils and NET-associated markers in the early acute stages of infection. Correlation analyses conducted between NET-specific markers and clinical scores as well as histopathologic scores support association between neutrophil activation and disease severity during SARS-CoV-2 infection in this model. Overall, this study emphasizes the effect of neutrophil activation and NET release in SARS-CoV-2 infection in a feline model, prompting further investigation into therapeutic strategies aimed at mitigating excessive innate inflammatory responses in COVID-19.

Unraveling the Molecular Pathogenesis of Protein C Deficiency-Associated VTE: Insights from Protein C Mutations C238G and R189W in Thai Patients.

Protein C (PC) deficiency is a well-established risk factor for thromboembolism (TE), commonly manifesting in pediatric patients. This study aimed to elucidate the pathogenic mechanisms of two novel PC mutations, C238G and R189W, identified in Thai children with both venous and arterial TE. The effects of wild-type (WT), C238G, and R189W PC variants were investigated through transient transfection of HEK293T cells. PC secretion levels were measured, and immunofluorescence analysis was performed to assess intracellular localization. ER stress-related gene expression and UPR activation were evaluated. Structural analysis was conducted to explore the significance of the C238 and R189W residue in PC functionality. The C238G mutation led to a severe 95% reduction in PC secretion, while R189W showed a 30% decrease compared with WT. Immunofluorescence revealed that C238G-PC was predominantly retained in the ER, indicating protein misfolding. C238G-expressing cells exhibited significant upregulation of ER stress-related genes and UPR activation. In contrast, R189W resulted in only a modest increase in UPR gene expression, suggesting a less pronounced impact on protein folding and secretion. Structural analysis demonstrated the critical role of the C238 residue in maintaining PC's disulfide bond and overall conformation. This study reveals distinct molecular mechanisms by which the C238G and R189W mutations contribute to PC deficiency and increased thrombotic risk. The findings emphasize the essential role of the C238 residue in preserving PC structure and secretion, enhancing the understanding of PC deficiency-associated TE in pediatric patients.

Identification of antibody-resistant SARS-CoV-2 mutants via N4-Hydroxycytidine mutagenesis

Monoclonal antibodies targeting the Spike protein of SARS-CoV-2 are effective against COVID-19 and might mitigate future pandemics. However, their efficacy is challenged by the emergence of antibody-resistant virus variants. We developed a method to efficiently identify such resistant mutants based on selection from mutagenized virus pools. By inducing mutations with the active compound of Molnupiravir, N4-hydroxycytidine (NHC), and subsequently passaging the virus in the presence of antibodies, we identified specific Spike mutations linked to resistance. Validation of these mutations was conducted using pseudotypes and immunofluorescence analysis. From a Wuhan-like strain of SARS-CoV-2, we identified the following mutations conferring strong resistance towards the corresponding antibodies: Bamlanivimab – E484K, F490S and S494P; Sotrovimab – E340K; Cilgavimab – K444R/E and N450D. From the Omicron B.1.1.529 variant, the strongly selected mutations were: Bebtelovimab – V445A; Sotrovimab – E340K and K356M; Cilgavimab – K444R, V445A and N450D. We also identified escape mutations in the Wuhan-like Spike for the broadly neutralizing antibodies S2K146 – combined G485S and Q493R – and S2H97 – D428G, K462E and S514F. Structural analysis revealed that the selected mutations occurred at antibody-binding residues within the receptor-binding domains of the Spike protein. Most of the selected mutants largely maintained ACE2 binding and infectivity. Notably, many of the identified resistance-conferring mutations are prevalent in real-world SARS-CoV-2 variants, but some of them (G485S, D428G, and K462E) have not yet been observed in circulating strains. Our approach offers a strategy for predicting the therapeutic efficacy of antibodies against emerging virus variants.

Histological differences related to autophagy in the minor salivary gland between primary and secondary types of Sjögren’s syndrome

Some forms of Sjögren’s syndrome (SS) follow a clinical course accompanied by systemic symptoms caused by lymphocyte infiltration and proliferation in the liver, kidneys, and other organs. To better understand the clinical outcomes of SS, here we used minor salivary gland tissues from patients and examine their molecular, biological, and pathological characteristics. A retrospective study was performed, combining clinical data and formalin-fixed paraffin-embedded (FFPE) samples from female patients over 60 years of age who underwent biopsies at Okayama University Hospital. We employed direct digital RNA counting with nCounter® and multiplex immunofluorescence analysis with a PhenoCycler™ on the labial gland biopsies. We compared FFPE samples from SS patients who presented with other connective tissue diseases (secondary SS) with those from stable SS patients with symptoms restricted to the exocrine glands (primary SS). Secondary SS tissues showed enhanced epithelial damage and lymphocytic infiltration accompanied by elevated expression of autophagy marker genes in the immune cells of the labial glands. The close intercellular distance between helper T cells and B cells positive for autophagy-associated molecules suggests accelerated autophagy in these lymphocytes and potential B cell activation by helper T cells. These findings indicate that examination of FFPE samples from labial gland biopsies can be an effective tool for evaluating molecular histological differences between secondary and primary SS through multiplexed analysis of gene expression and tissue imaging.

First-in-Class Mitogen-Activated Protein Kinase p38α: MAPK-Activated Protein Kinase-2 (MK2) Dual Signal Modulator with Anti-inflammatory and Endothelial-stabilizing Properties.

We previously identified a small molecule, UM101, predicted to bind to the substrate-binding groove of p38aMitogen-activated Protein Kinase (MAPK) near the binding site of its proinflammatory substrate, MAPK-activated protein kinase (MK2). UM101 exhibited anti-inflammatory, endothelial-stabilizing, and lung-protective effects. To overcome its limited aqueous solubility and p38a binding affinity, we designed an analog of UM101, GEn-1124, with improved aqueous solubility, stability, and p38a binding affinity. Compared with UM101, GEn-1124 has 18-fold greater p38a-binding affinity as measured by Surface Plasmon Resonance (SPR), 11-fold greater aqueous solubility, enhanced barrier-stabilizing activity in thrombin-stimulated human pulmonary artery endothelial cells (hPAEC) in vitro, and greater lung protection in vivo GEn-1124 improved survival from 10% to 40% in murine acute lung injury (ALI) induced by combined exposure to intratracheal bacterial endotoxin lipopolysaccharide (LPS) instillation and febrile-range hyperthermia (FRH) and from 0% to 50% in a mouse influenza pneumonia model. Gene expression analysis by RNASeq in TNFa-treated hPAEC showed that the gene-modifying effects of GEn-1124 were much more restricted to TNFa-inducible genes than the catalytic site p38 inhibitor, SB203580. Gene expression pathway analysis, confocal immunofluorescence analysis of p38aand MK2 subcellular trafficking, and SPR analysis of phosphorylated p38a:MK2 binding affinity supports a novel mechanism of action. GEn-1124 destabilizes the activated p38a:MK2 complex, dissociates nuclear export of MK2 and p38a, thereby promoting intranuclear retention and enhanced intranuclear signaling by phosphorylated p38a retention, and accelerated inactivation of p38-free cytosolic MK2 by unopposed phosphatases. Significance Statement We describe an analog of our first-in-class small molecule modulator of p38a/MK2 signaling targeted to a pocket near the ED substrate binding domain of p38a, which destabilizes the p38a:MK2 complex without blocking p38 catalytic activity or ablating downstream signaling. The result is a rebalancing of downstream pro- and anti-inflammatory signaling, yielding anti-inflammatory, endothelial-stabilizing, and lung-protective effects with therapeutic potential in ARDS.

Abstract C061: Loss of CaMK2B accelerates tumor formation and enhances metastatic competency in genetically engineered mouse models of PDAC

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer- related deaths in the United States and is often not diagnosed until it has already metastasized. Our prior work established that calcium signaling pathways facilitate a pro-metastatic phenotype through the induction of an epithelial-to-mesenchymal transition (EMT) program in PDAC tumor cells, but the downstream signaling molecules mediating this phenotype have remained elusive. MethodsUsing a novel genetically engineered model of pancreatic cancer, we demonstrate that loss of Calcium/calmodulin-dependent kinase-II B (CaMK2B), a downstream effector kinase activated by calcium, enhances cellular plasticity, and increases metastasis. To this end, we generated a genetically engineered mouse model where Camk2b was knocked out in tumor cells in a lineage-labeled Kras- and p53-driven PDAC mouse model (herein KPCY-Camk2bcKO). Results Genetic deletion of Camk2b in the KPCY genetically engineered model accelerated tumor formation and dramatically reduced the overall median survival of KPCY- Camk2b cKO mice (78 days) relative to KPCY controls (136 days). Pathological and immunofluorescence analysis of KPCY-Camk2b cKO animals showed an increased number of poorly differentiated tumor regions and a higher metastatic burden, with >85% of KPCY- Camk2b cKO animals presenting with multi-focal metastases. Further analysis of tumor cells in KPCY-Camk2b cKO mice showed enhanced expression of markers of the highly aggressive squamous PDAC subtypes. Strikingly, Camk2b deletion combined with oncogenic Kras G12D (in the absence of p53 alterations), was sufficient to form lethal metastatic PDAC tumors, while age-matched control Kras G12D tumors with intact Camk2b only progressed to the acinar-to-ductal metaplasia (ADM) or Pancreatic Intraepithelial Neoplasia (PanIN) stage. Collectively, these results establish Camk2b loss as a new oncogenic and pro-metastatic mechanism in PDAC Conclusion This work demonstrates that Camk2b loss enhances tumor cell evolution and plasticity within PDAC to promote metastasis. In this context, we speculate that loss of Camk2b rewires calcium signaling to drive an aggressive subtype of PDAC with increased metastasis. Citation Format: Jessica Peura, Nikita Bhalerao, Calvin Johnson, Yamini Ogoti, Faith Keller, Emma Watson, Zhen Zhao, Jason Pitarresi. Loss of CaMK2B accelerates tumor formation and enhances metastatic competency in genetically engineered mouse models of PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C061.

Tenascin-C induces transdifferentiation of retinal pigment epithelial cells in proliferative vitreoretinopathy

Proliferation and transdifferentiation of the retinal pigment epithelium (RPE) are hallmarks of proliferative vitreoretinopathy (PVR); however, the critical regulators of this process remain to be elucidated. Here, we investigated the role of tenascin-C in PVR development. In vitro, exposure of human ARPE-19 (hRPE) cells to TGF-β2 increased tenascin-C expression. Tenascin-C was shown to be involved in TGF-β2-induced transdifferentiation of hRPE cells, which was inhibited by pretreatment with tenascin-C siRNA. In PVR mouse models, a marked increase in the expression of tenascin-C mRNA and protein was observed. Additionally, immunofluorescence analysis demonstrated a dramatic increase in the colocalization of tenascin-C with RPE65 or α-smooth muscle actin(α-SMA) in the epiretinal membranes of patients with PVR. There was also abundant expression of integrin αV and β-catenin in the PVR membranes. ICG-001, a β-catenin inhibitor, efficiently attenuated PVR progression in a PVR animal model. These findings suggest that tenascin-C is secreted by transdifferentiated RPE cells and promotes the development of PVR via the integrin αV and β-catenin pathways. Therefore, tenascin-C could be a potential therapeutic target for the inhibition of epiretinal membrane development associated with PVR.

CYTOSKELETON DYNAMICS AND SPATIAL ORGANIZATION DURING EPITHELIAL-TO-MESENCHYMAL TRANSITION

Epithelial-to-mesenchymal transition (EMT) is a crucial process that occurs during normal development, such as embryogenesis and organ formation. If it is inappropriately activated, it can lead to pathological processes like metastasis. Although EMT is well studied at the morphological and transcriptome level, cytoskeleton changes during this process are less understood. In our work we aimed to describe the morphological changes that occurred in MCF-7, A-549 and HaCaT cells after EMT, analyze microtubule dynamics, spatial distribution and its contribution to cell motility, identify changes in actin filament organization and study focal adhesion turnover in the cells after EMT. We hypothesized that the dynamics of microtubules in cells undergoing EMT might change. Cells undergoing EMT were expected to have more dynamic microtubules. Also, cells undergoing EMT are expected to adhere more efficiently to diverse substrates, and therefore spread and move more easily. Focal contacts in cells undergoing EMT were expected to be more pronounced and dynamic than in cells that not undergoing EMT. The research methods used in the study included inducing EMT in modified MCF-7 cells through an inserted inducible Tet-on system and stimulation of EMT in A-549 and HaCaT cells using TGF-β. The cells were observed using bright field microscopy, and immunofluorescence analysis was conducted to visualize microtubules and actin filaments. Transfection with EB-3–RFP protein was done to describe and measure microtubule dynamics, while transduction with Talin-RFP and transient transfection with Ptag-RFP-vinculin was done to visualize focal adhesions. Time-lapse fluorescent microscopy was used to record films, and the Fiji Image J program was used to analyze the data. All statistical analysis was performed using GraphPad Prism (Dotmatics, USA), and a nonparametric Mann-Whitney U test or parametric t-test with Welch correction. The actin filament measurements were completed using Matlab scripts. The major research findings: In all three-cell models after EMT, cell morphology changed. Cells increased in size. MCF-7 and HaCaT became spread out, while A-549 became elongated. Some of the cells lost cell-cell contacts. All three cell models after EMT had alterations in microtubule organization and dynamics. MCF-7 and HaCaT cells showed more frequently individual MTs at cell edges, while A-549 had less covered nucleus by MTs after EMT. Microtubule dynamics (growth velocity) increased, and the length of microtubule growth tracks became longer. The average angle of MT growth trajectories to cell radius decreased. Actin fibers rearranged into more pronounced stress fibers after EMT. Alterations in the focal adhesion behaviors were different in three models used. These results indicate that cytoskeletal changes during EMT mainly include increased microtubule dynamics.

Upregulation of HCN2 in ventral tegmental area is involved in morphine-induced conditioned place preference in rats.

Morphine is an opioid commonly used to treat pain in clinic, but it also has the potential to be highly addictive, which can lead to abuse. Despite these known risks, the cellular and molecular mechanism of morphine conditioned place preference (CPP) is still unclear. In this study, using a rat model of chronic morphine administration, we found that compared with the control group, the mRNA and protein expression of HCN2 channel in the ventral tegmental area (VTA) were upregulated. Further immunofluorescence analysis showed that the fluorescence intensity of HCN2 channel of VTA dopaminergic neurons in morphine group was significantly enhanced, while the patch clamp recording of brain slices showed that both the magnitude and the density of Ih (HCN channel current) of VTA neurons were significantly increased. Moreover, intra-VTA infusion of ZD7288, a selective inhibitor of HCN channel, into rats of the morphine group decreased morphine CPP. Taken together, our results show that chronic morphine administration induces an upregulation of HCN2 in VTA dopamine neurons, while HCN inhibition reduces morphine CPP, suggesting that HCN channel may be a potential target for the treatment of morphine addiction.

Pyroptosis inhibition alleviates acute lung injury via E-twenty-six variant gene 5-mediated downregulation of gasdermin D

BackgroundAcute lung injury (ALI) is a life-threatening condition characterized by excessive pulmonary inflammation, yet its precise pathophysiology remains elusive. Pyroptosis, a programmed cell death mechanism controlled by gasdermin D (GSDMD), has been linked to the etiology of ALI. This study investigated the regulatory functions of the transcription factor E-twenty-six variant gene 5 (ETV5) and GSDMD in ALI. MethodsLipopolysaccharide (LPS) was used to treat BEAS-2B cells (50 mmol/mL) and establish an LPS-induced mouse model of ALI (by intratracheal administration, 3 mg/kg). Protein-protein docking, immunofluorescence analysis, western blotting, real-time quantitative polymerase chain reaction, and dual-luciferase reporter gene assay were used to examine ETV5-mediated negative feedback regulation of GSDMD and its effects on pyroptosis and ALI. ResultsOur results showed that the physiological function of ETV5 was reduced by its downregulated expression, which impeded its nuclear translocation in ALI mice. Increased pyroptosis and enhanced production of inflammatory cytokines were associated with LPS-induced ALI. ETV5 overexpression in LPS-treated BEAS-2B cells decreased the expression of total and membrane-bound GSDMD, negatively regulated GSDMD, and prevented pyroptosis. The expression of inflammatory cytokines was subsequently reduced due to this inhibition, which, in turn, reduced ALI. Molecular docking analysis and dual-luciferase reporter gene assay results indicated a direct interaction between ETV5 and GSDMD, which inhibited GSDMD production. ConclusionOur results indicate that ETV5 inhibits pyroptosis, decreases the expression of inflammatory cytokines, and negatively regulates GSDMD expression to ameliorate ALI symptoms.

Association of novel DNAH11 variants with asthenoteratozoospermia lead to male infertility

BackgroundBi-allelic variants in DNAH11 have been identified as causative factors in Primary Ciliary Dyskinesia, leading to abnormal respiratory cilia. Nonetheless, the specific impact of these variants on human sperm flagellar and their involvement in male infertility remain largely unknown.MethodsA collaborative effort involving two Chinese reproductive centers conducted a study with 975 unrelated infertile men. Whole-exome sequencing was employed for variant screening, and Sanger sequencing confirmed the identified variants. Morphological and ultrastructural analyses of sperm were conducted using Scanning Electron Microscopy and Transmission Electron Microscopy. Western Blot Analysis and Immunofluorescence Analysis were utilized to assess protein levels and localization. ICSI was performed to evaluate its efficacy in achieving favorable pregnancy outcomes for individuals with DNAH11 variants.ResultsIn this study, we identified seven novel variants in the DNAH11 gene in four asthenoteratozoospermia subjects. These variants led the absence of DNAH11 proteins and ultrastructure defects in sperm flagella, particularly affecting the outer dynein arms (ODAs) and adjacent structures. The levels of ODA protein DNAI2 and axoneme related proteins were down regulated, instead of inner dynein arms (IDA) proteins DNAH1 and DNAH6. Two out of four individuals with DNAH11 variants achieved clinical pregnancies through ICSI. The findings confirm the association between male infertility and bi-allelic deleterious variants in DNAH11, resulting in the aberrant assembly of sperm flagella and contributing to asthenoteratozoospermia. Importantly, ICSI emerges as an effective intervention for overcoming reproductive challenges caused by DNAH11 gene variants.

IL-10 deficiency aggravates cell senescence and accelerates BLM-induced pulmonary fibrosis in aged mice via PTEN/AKT/ERK pathway

BackgroundPulmonary fibrosis (PF) is an aging-related progressive lung disorder. The aged lung undergoes functional and structural changes termed immunosenescence and inflammaging, which facilitate the occurrence of fibrosis. Interleukin-10 (IL-10) is a potent anti-inflammatory and immunoregulatory cytokine, yet it remains unclear how IL-10 deficiency-induced immunosenescence participates in the development of PF.MethodsFirstly we evaluated the susceptibility to fibrosis and IL-10 expression in aged mice. Then 13-month-old wild-type (WT) and IL-10 knockout (KO) mice were subjected to bleomycin(BLM) and analyzed senescence-related markers by PCR, western blot and immunohistochemistry staining of p16, p21, p53, as well as DHE and SA-β-gal staining. We further compared 18-month-old WT mice with 13-month-old IL-10KO mice to assess aging-associated cell senescence and inflamation infiltration in both lung and BALF. Moreover, proliferation and apoptosis of alveolar type 2 cells(AT2) were evaluated by FCM, immunofluorescence, TUNEL staining, and TEM analysis. Recombinant IL-10 (rIL-10) was also administered intratracheally to evaluate its therapeutic potential and related mechanism. For the in vitro experiments, 10-week-old naïve pramily lung fibroblasts(PLFs) were treated with the culture medium of 13-month PLFs derived from WT, IL-10KO, or IL-10KO + rIL-10 respectively, and examined the secretion of senescence-associated secretory phenotype (SASP) factors and related pathways.ResultsThe aged mice displayed increased susceptibility to fibrosis and decreased IL-10 expression. The 13-month-old IL-10KO mice exhibited significant exacerbation of cell senescence compared to their contemporary WT mice, and even more severe epithelial-mesenchymal transition (EMT) than that of 18 month WT mice. These IL-10 deficient mice showed heightened inflammatory responses and accelerated PF progression. Intratracheal administration of rIL-10 reduced lung CD45 + cell infiltration by 15%, including a 6% reduction in granulocytes and a 10% reduction in macrophages, and increased the proportion of AT2 cells by approximately 8%. Additionally, rIL-10 significantly decreased α-SMA and collagen deposition, and reduced the expression of senescence proteins p16 and p21 by 50% in these mice. In vitro analysis revealed that conditioned media from IL-10 deficient mice promoted SASP secretion and upregulated senescence genes in naïve lung fibroblasts, which was mitigated by rIL-10 treatment. Mechanistically, rIL-10 inhibited TGF-β-Smad2/3 and PTEN/PI3K/AKT/ERK pathways, thereby suppressing senescence and fibrosis-related proteins.ConclusionsIL-10 deficiency in aged mice leads to accelerated cell senescence and exacerbated fibrosis, with IL-10KO-PLFs displaying increased SASP secretion. Recombinant IL-10 treatment effectively mitigates these effects, suggesting its potential as a therapeutic target for PF.Graphical

Unveiling the impact of SUMOylation at K298 site of heat shock factor 1 on glioblastoma malignant progression

BackgroundGlioblastoma (GBM) poses a significant medical challenge due to its aggressive nature and poor prognosis. Mitochondrial unfolded protein response (UPRmt) and the heat shock factor 1 (HSF1) pathway play crucial roles in GBM pathogenesis. Post-translational modifications, such as SUMOylation, regulate the mechanism of action of HSF1 and may influence the progression of GBM. Understanding the interplay between SUMOylation-modified HSF1 and GBM pathophysiology is essential for developing targeted therapies. MethodsWe conducted a comprehensive investigation using cellular, molecular, and in vivo techniques. Cell culture experiments involved establishing stable cell lines, protein extraction, Western blotting, co-immunoprecipitation, and immunofluorescence analysis. Mass spectrometry was utilized for protein interaction studies. Computational modeling techniques were employed for protein structure analysis. Plasmid construction and lentiviral transfection facilitated the manipulation of HSF1 SUMOylation. In vivo studies employed xenograft models for tumor growth assessment. ResultsOur research findings indicate that HSF1 primarily undergoes SUMOylation at the lysine residue K298, enhancing its nuclear translocation, stability, and downstream heat shock protein expression, while having no effect on its trimer conformation. SUMOylated HSF1 promoted the UPRmt pathway, leading to increased GBM cell proliferation, migration, invasion, and reduced apoptosis. In vivo studies have confirmed that SUMOylation of HSF1 enhances its oncogenic effect in promoting tumor growth in GBM xenograft models. ConclusionThis study elucidates the significance of SUMOylation modification of HSF1 in driving GBM progression. Targeting SUMOylated HSF1 may offer a novel therapeutic approach for GBM treatment. Further investigation into the specific molecular mechanisms influenced by SUMOylated HSF1 is warranted for the development of effective targeted therapies to improve outcomes for GBM patients.

Brain Ischemic Tolerance Triggered by Preconditioning Involves Modulation of Tumor Necrosis Factor-α-Stimulated Gene 6 (TSG-6) in Mice Subjected to Transient Middle Cerebral Artery Occlusion

Ischemic preconditioning (PC) induced by a sub-lethal cerebral insult triggers brain tolerance against a subsequent severe injury through diverse mechanisms, including the modulation of the immune system. Tumor necrosis factor (TNF)-α-stimulated gene 6 (TSG-6), a hyaluronate (HA)-binding protein, has recently been involved in the regulation of the neuroimmune response following ischemic stroke. Thus, we aimed at assessing whether the neuroprotective effects of ischemic PC involve the modulation of TSG-6 in a murine model of transient middle cerebral artery occlusion (MCAo). The expression of TSG-6 was significantly elevated in the ischemic cortex of mice subjected to 1 h MCAo followed by 24 h reperfusion, while this effect was further potentiated (p < 0.05 vs. MCAo) by pre-exposure to ischemic PC (i.e., 15 min MCAo) 72 h before. By immunofluorescence analysis, we detected TSG-6 expression mainly in astrocytes and myeloid cells populating the lesioned cerebral cortex, with a more intense signal in tissue from mice pre-exposed to ischemic PC. By contrast, levels of TSG-6 were reduced after 24 h of reperfusion in plasma (p < 0.05 vs. SHAM), but were dramatically elevated when severe ischemia (1 h MCAo) was preceded by ischemic PC (p < 0.001 vs. MCAo) that also resulted in significant neuroprotection. In conclusion, our data demonstrate that neuroprotection exerted by ischemic PC is associated with the elevation of TSG-6 protein levels both in the brain and in plasma, further underscoring the beneficial effects of this endogenous modulator of the immune system.

Fractionated irradiation promotes radioresistance and decreases oxidative stress by increasing Nrf2 of ALDH-positive nasopharyngeal cancer stem cells

Radiotherapy is widely regarded as the primary therapeutic modality for nasopharyngeal cancer (NPC). Studies have shown that cancer cells with high resistance to radiation, known as radioresistant cancer cells, may cause residual illness, which in turn might contribute to the occurrence of cancer recurrence and metastasis. It has been shown that cancer stem-like cells (CSCs) exhibit resistance to radiation therapy. In the present study, fractionated doses of radiation induced epithelial-mesenchymal transition (EMT) and ALDH+ CSCs phenotype of NPC tumor spheroids. Furthermore, it has been shown that cells with elevated ALDH activity have increased resistance to the effects of fractionated irradiation. Nuclear factor erythroid-2-related factor 2 (Nrf2) plays a pivotal role in regulating cellular antioxidant systems. A large body of evidence suggests that Nrf2 plays a significant role in the development of radioresistance in cancer. Our research revealed that the application of fractionated irradiation resulted in a decline in Nrf2-dependent reactive oxygen species (ROS) levels, thereby mitigating DNA damage in ALDH+ stem-like NPC cells. In addition, immunofluorescence analysis revealed that subsequent to the process of fractionated irradiation of ALDH+ cells, activated Nrf2 was predominantly localized inside the nucleus. Immunofluorescent analysis also revealed that the presence of the nuclear Nrf2+/NQO1+/ALDH1+ axis might potentially serve as an indicator of poor prognosis and resistance to radiotherapy in patients with NPC. Thus, our findings strongly suggest that the radioresistance of ALDH-positive NPC CSCs to fractionated irradiation is regulated by nuclear Nrf2 accumulation. Nrf2 exerts its effects through the downstream effector NQO1/ALDH1, which depends on ROS attenuation.

Unique Pathology in the Locus Coeruleus of Individuals with Down Syndrome.

Down syndrome (DS) is one of the most commonly occurring chromosomal conditions. Most individuals with DS develop Alzheimer's disease (AD) by 50 years of age. Recent evidence suggests that AD pathology in the locus coeruleus (LC) is an early event in sporadic AD. It is likely that the widespread axonal network of LC neurons contributes to the spread of tau pathology in the AD brain, although this has not been investigated in DS-AD. The main purpose of this study was to profile AD pathology and neuroinflammation in the LC, comparing AD and DS-AD in postmortem human tissues. We utilized immunofluorescence and semi-quantitative analyses of pTau (4 different forms), amyloid-β (Aβ), glial, and neuronal markers in the LC across 36 cases (control, DS-AD, and AD) to compare the different pathological profiles. Oligomeric tau was highly elevated in DS-AD cases compared to LOAD or EOAD cases. The distribution of staining for pT231 was elevated in DS-AD and EOAD compared to the LOAD group. The DS-AD group exhibited increased Aβ immunostaining compared to AD cases. The number of tau-bearing neurons was also significantly different between the EOAD and DS-AD cases compared to the LOAD cases. While inflammation, pTau, and Aβ are all involved in AD pathology, their contribution to disease progression may differ depending on the diagnosis. Our results suggest that DS-AD and EOAD may be more similar in pathology than LOAD. Our study highlights unique avenues to further our understanding of the mechanisms governing AD neuropathology.

Multiple cell death modalities and immune response in pulpitis.

To investigate the level and distribution of apoptosis, pyroptosis, necroptosis, and NETosis in pulpitis with or without necrosis on a basis of histological classification. Additionally, to examine the effect of pulpitis with necrosis (PWN) on the number and activation of peripheral and bone marrow (BM) neutrophils, as well as spleen lymphocytes, in a mouse model of pulpitis. The material comprised 20 permanent teeth, with or without caries, which were classified into three histological categories based on the distribution of inflammatory cells and the presence or absence of necrosis: (i) healthy pulp (HP), (ii) pulpitis without necrosis (PWON), and (iii) PWN. The levels of the four regulated cell death (RCD) pathways were detected by immunohistochemical and immunofluorescent staining with specific markers: apoptosis (caspase-8, cleaved caspase-3), pyroptosis (cleaved caspase-1, membrane-binding gasdermin D), necroptosis (receptor-interacting kinase 3, phosphorylated MLKL), and NETosis (myeloperoxidase, citrullinated histone H3). Acute pulpitis was induced in C57BL/6J mice via pulp exposure, and the mice were divided into four groups: (i) control (no tooth preparation, n = 6), (ii) Day 1 (sacrificed at 1 day after pulp exposure, n = 3), (iii) Day 3 (n = 3), and (iv) Day 5 (n = 7). The control and Day 5 groups were used for further immunofluorescent analysis to assess the levels of RCD and flow cytometry to monitor the changes in peripheral and BM neutrophils, as well as spleen lymphocytes. Human dental pulp stem cells (hDPSCs) were isolated and cultured from extracted healthy third molars. Apoptosis and necroptosis in hDPSCs were induced by staurosporine, whilst pyroptosis was induced by lipopolysaccharide and nigericin. One-way analysis of variance (ANOVA) with Tukey's test, Welch's ANOVA with Tamhane's test, and Student's t-tests were used to compare immunohistochemical labelling and flow cytometric data amongst groups (p < .05). The pulpal tissue of PWN can be divided into the abscess core (PWN-AC) and fibrous tissue (PWN-FT). The ratio of total necrotic cells (TUNEL-positive) in PWN-AC was significantly higher than in PWN-FT and PWON (both p < .01). Compared with HP, the expression levels of markers for apoptosis and pyroptosis were increased in PWON, whilst the expression levels of markers for apoptosis, pyroptosis, and NETosis were elevated in PWN, primarily detected in PWN-AC. Interestingly, myeloperoxidase (MPO) was exclusively observed in PWN-AC, with minimal detection in PWN-FT and PWON. Additionally, the frequency of MPO+ cells was significantly higher than that of MB-GSDMD+ cells and Cl-cas3+ cells in PWN-AC (both p < .01). Histological observation and TUNEL staining showed abundant necrotic cells in mouse pulpal tissue after pulp exposure, indicating a simulation of human PWN. In mouse pulpitis tissue, markers of apoptosis, pyroptosis, and NETosis were detected. Invitro, various cell deaths including apoptosis, pyroptosis, and necroptosis were also triggered in hDPSCs under various cell death treatments. Furthermore, in terms of systemic changes, pulp exposure-induced pulpitis could increase the number (p < .05) and cellular activity (p < .01) of neutrophils from BM in a mouse model. No significant changes in peripheral blood neutrophils, spleen T cells, B cells, or the CD4/CD8 ratio were detected between the control and pulpitis mice. Our findings uncover distinct patterns of mixed cell death at different histological stages of human pulpitis and the impact of pulpitis on the number and activity of BM neutrophils. Notably, NETosis occurs specifically and predominates in the abscess area of pulpitis, suggesting a potential effect of neutrophil extracellular traps (NETs) on pulpitis progression and NETs-targeted diagnostic strategy may play a role in decision making for vital pulp therapy.

IGF-I concentration determines cell fate by converting signaling dynamics as a bifurcation parameter in L6 myoblasts

Insulin-like growth factor (IGF)-I mediates long-term activities that determine cell fate, including cell proliferation and differentiation. This study aimed to characterize the mechanisms by which IGF-I determines cell fate from the aspect of IGF-I signaling dynamics. In L6 myoblasts, myogenic differentiation proceeded under low IGF-I levels, whereas proliferation was enhanced under high levels. Mathematical and experimental analyses revealed that IGF-I signaling oscillated at low IGF-I levels but remained constant at high levels, suggesting that differences in IGF-I signaling dynamics determine cell fate. We previously reported that differential insulin receptor substrate (IRS)-1 levels generate a driving force for cell competition. Computational simulations and immunofluorescence analyses revealed that asynchronous IRS-1 protein oscillations were synchronized during myogenic processes through cell competition. Disturbances of cell competition impaired signaling synchronization and cell fusion, indicating that synchronization of IGF-I signaling oscillation is critical for myoblast cell fusion to form multinucleate myotubes.

CD25+FOXP3+CD45RA− regulatory T-cell infiltration as a prognostic biomarker for endometrial carcinoma

BackgroundRegulatory T (Treg) cells reportedly play crucial roles in tumor angiogenesis as well as antitumor immunity. In order to explore their therapeutic potential, we investigated the precise prognostic impact of Treg markers in endometrial carcinoma.MethodsWe performed multiplexed immunofluorescence and quantitative image analyses of CD25, FOXP3, CTLA4, and CD45RA in tumor specimens from 176 consecutive patients treated at our institution for primary endometrial carcinomas. Bioinformatics analyses were further conducted to corroborate the findings.ResultsHigh CD25+, FOXP3+, and CD25+FOXP3+CD45RA− stromal cell counts correlated with better overall survival (OS) (p = 0.00019, 0.028 and 0.0012) and MSI-high (p = 0.015, 0.016 and 0.047). High CD45RA+ stromal cell count was associated with superficial myometrial invasion (p = 0.0038). Bioinformatics survival analysis by Kaplan-Meier plotter showed that high CD25, FOXP3, CTLA4, and CD45RA mRNA expressions correlated with better OS (p = 0.046, 0.00042, 0.000044, and 0.0022). Univariate and multivariate analyses with various clinicopathologic prognostic factors indicated that high CD25+ or CD25+FOXP3+CD45RA− stromal cell count was significant and independent for favorable OS (p = 0.0053 and 0.0015). We subsequently analyzed the correlations between the multiplexed immunofluorescence results and treatment-free interval (TFI) after primary chemotherapy in recurrent cases, finding no significant associations. Further analysis revealed that high ratio of CD25+ : CD8+ cell count or CD25+FOXP3+CD45RA− : CD8+ cell count correlated with longer TFI (p = 0.021 and 0.021).ConclusionThe current observations suggest that the balance between CD25+ or CD25+FOXP3+CD45RA− cells and CD8+ cells, corresponding to promoting or inhibiting effect on tumor angiogenesis, affect tumor chemosensitivity leading to prognostic significance. CD25+FOXP3+CD45RA− effector Treg tumor infiltration may serve as a useful prognostic biomarker and a potential target for immunotherapeutic manipulation of tumor chemosensitivity by novel management for advanced/recurrent endometrial carcinomas.

Enhanced lipid metabolism reprogramming in CHF rats through IL-6-mediated cardiac glial cell modulation by digilanid C and electroacupuncture stimulation combination.

Cardiac lipid metabolism reprogramming is recognized as a critical pathological factor in the progression of chronic heart failure (CHF). The therapeutic potential of digilanid C and electroacupuncture stimulation (ES) in enhancing lipid metabolism and cardiac function has been established. However, the optimal synergistic regulatory strategies of these interventions on cardiac lipid metabolism have yet to be elucidated. This study aimed to comprehensively evaluate the impact of a digilanid C-ES combination on cardiac steatosis remodeling in CHF. Assessments were conducted across various dimensions, including myocardial oxygen consumption, mitochondrial function, and lipid metabolism. Additionally, we sought to uncover the underlying neuromolecular mechanisms. Our findings, at both molecular and morphological levels, indicated that the synergistic application of digilanid C and ES significantly inhibited myocardial fibrosis and steatosis. This combination therapy facilitated the repair of cardiac neuro-vascular uncoupling and induced a reprogramming of lipid metabolism. Notably, the digilanid C-ES combination ameliorated cardiomyocyte apoptosis and enhanced mitochondrial biogenesis in CHF, leading to a restructured energy supply pattern. Cardiac immunofluorescence analyses revealed the aggregation of cardiac glial cells (CGCs) at sites of abnormal neurovascular coupling, a response to cardiac lipid degeneration. This was accompanied by a marked reduction in the abnormally elevated expression of interleukin 6 (IL-6) and glutamatergic signaling, which correlated with the severity of cardiac steatosis and the aberrant activation of CGCs. The combined therapy was found to activate the Janus kinase 1 (JAK1)/signal transducer and activator of transcription 3 (STAT3) pathway, effectively attenuated lipid accumulation and over-recruitment of CGCs and deprivation of glutamatergic nerves. These findings underscore the potential of digilanid C and ES combination therapy as a novel approach to modulate the complex interplay between neurovascular dynamics and metabolic dysregulation in CHF.