Quantitative Immunofluorescence Microscopy Research Articles

High-dose dexamethasone regulates microglial polarization via the GR/JAK1/STAT3 signaling pathway after traumatic brain injury.

Although microglial polarization and neuroinflammation are crucial cellular responses after traumatic brain injury, the fundamental regulatory and functional mechanisms remain insufficiently understood. As potent anti-inflammatory agents, the use of glucocorticoids in traumatic brain injury is still controversial, and their regulatory effects on microglial polarization are not yet known. In the present study, we sought to determine whether exacerbation of traumatic brain injury caused by high-dose dexamethasone is related to its regulatory effects on microglial polarization and its mechanisms of action. In vitro cultured BV2 cells and primary microglia and a controlled cortical impact mouse model were used to investigate the effects of dexamethasone on microglial polarization. Lipopolysaccharide, dexamethasone, RU486 (a glucocorticoid receptor antagonist), and ruxolitinib (a Janus kinase 1 antagonist) were administered. RNA-sequencing data obtained from a C57BL/6 mouse model of traumatic brain injury were used to identify potential targets of dexamethasone. The Morris water maze, quantitative reverse transcription-polymerase chain reaction, western blotting, immunofluorescence and confocal microscopy analysis, and TUNEL, Nissl, and Golgi staining were performed to investigate our hypothesis. High-throughput sequencing results showed that arginase 1, a marker of M2 microglia, was significantly downregulated in the dexamethasone group compared with the traumatic brain injury group at 3 days post-traumatic brain injury. Thus dexamethasone inhibited M1 and M2 microglia, with a more pronounced inhibitory effect on M2 microglia in vitro and in vivo. Glucocorticoid receptor plays an indispensable role in microglial polarization after dexamethasone treatment following traumatic brain injury. Additionally, glucocorticoid receptor activation increased the number of apoptotic cells and neuronal death, and also decreased the density of dendritic spines. A possible downstream receptor signaling mechanism is the GR/JAK1/STAT3 pathway. Overactivation of glucocorticoid receptor by high-dose dexamethasone reduced the expression of M2 microglia, which plays an anti-inflammatory role. In contrast, inhibiting the activation of glucocorticoid receptor reduced the number of apoptotic glia and neurons and decreased the loss of dendritic spines after traumatic brain injury. Dexamethasone may exert its neurotoxic effects by inhibiting M2 microglia through the GR/JAK1/STAT3 signaling pathway.

BNIP3 in hypoxia-induced mitophagy: Novel insights and promising target for non-alcoholic fatty liver disease

BNIP3 localizes to the outer mitochondrial membrane, has been demonstrated to be extensively involved in abnormalities to mitochondrial metabolic function and dynamicsand in non-alcoholic fatty liver disease (NAFLD). However, its role in NAFLD under hypoxia remains unclear. This study aimed to investigate the expression and the role of BNIP3 in NAFLD under hypoxia, and explore its involvement in regulating NAFLD mitophagy, fatty acid β-oxidation both in vivo and in vitro. BNIP3-mediated mitophagy level was analyzed using real-time quantitative polymerase chain reaction, Western blotting, immunofluorescence and electron microscopy. The role of BNIP3 in fatty acid β-oxidation was evaluated using lipid droplet staining, triglyceride content determination, and cellular energy metabolism. The results showed that compared with the HFD-2200 m, the body weight, inflammatory liver injury, and lipid deposition were significantly reduced in the HFD-4500 m group (P < 0.05), but autophagy and mitophagy were increased, and the expression of the mitophagy receptor BNIP3 was increased (P < 0.05). Compared to the control group, BNIP3 knockdown in the hypoxia group resulted in decreased levels of CPT1, ATGL, and p-HSL in lipid-accumulating hepatocytes, lipid droplet accumulation and triglyceride content increased (P < 0.05). Moreover, the ability of lipid-accumulating hepatocytes to oxidize fatty acids was reduced by BNIP3 knockdown in the hypoxia group (P < 0.05). Therefore, it can be concluded that, in NAFLD mice under hypoxia, BNIP3-mediated mitophagy promotes fatty acid β-oxidation. This study elucidated the role of BNIP3 in promoting fatty acid β-oxidation in NAFLD under hypoxia, and suggests BNIP3 may serve as a novel potential therapeutic target for NAFLD.

Extracellular Tau Oligomers Damage the Axon Initial Segment by an Endogenous Tau‐Dependent Mechanism

AbstractBackgroundNeuronal polarity and synaptic connectivity are compromised in Alzheimer’s disease (AD) and other tauopathies. The axon initial segment (AIS) is a key structure for regulating polarity and functions of neurons. It occupies the first 20‐60 µm of the axon, comprises a diffusion barrier that segregates axon‐enriched from somatodendritic‐enriched molecules, and has a high concentration of voltage‐gated ion channels that generate action potentials. Extracellular amyloid‐β oligomers compromise AIS integrity. However, effects on the AIS of toxic tau species, including extracellular oligomers (xcTauOs) that spread tau pathology from neuron to neuron by a prion‐like process, whereas unknown. Therefore, we wanted to test the hypothesis that AIS structure is sensitive to xcTauOs.MethodPrimary cortical neurons derived from either wild type (WT), or tau knockout (KO) mice were exposed to xcTauOs or vehicle. Quantitative western blotting and immunofluorescence microscopy with an antibody against the AIS‐enriched protein TRIM46 was used to monitor effects on the AIS. The same methods were also used to compare TRIM46 and two other AIS proteins, ankyrin‐G and neurofascin‐186 in human hippocampal tissue obtained from AD and age‐matched non‐AD donors.ResultIn cultured WT, but not TKO neurons, xcTauOs cause a trend toward AIS shortening and reduce the concentration of the resident AIS protein, TRIM46, without affecting total TRIM46 levels. Lentiviral‐driven human tau expression in tau KO neurons rescues TRIM46 sensitivity to xcTauOs. In human AD hippocampus, AIS length and TRIM46 concentration within the AIS are reduced in neurons containing neurofibrillary tangles (NFTs), without affecting the overall protein levels of multiple resident AIS proteins.ConclusionThese collective findings demonstrate that in cultured neurons, xcTauOs cause partial AIS damage by a mechanism dependent on intracellular tau, thereby raising the possibility that AIS reduction in AD is caused by xcTauOs working in concert with endogenous neuronal tau.

Laminin β4 is a constituent of the cutaneous basement membrane zone and additional autoantigen of anti-p200 pemphigoid

Anti-p200 pemphigoid is a subepidermal autoimmune blistering disease (AIBD) characterized by autoantibodies against a 200kDa protein. Laminin γ1 has been described as target antigen in 70% to 90% of patients. No diagnostic assay is widely available for anti-p200 pemphigoid, which might be due to the unclear pathogenic relevance of anti-laminin γ1 autoantibodies. To identify a target antigen with higher clinical and diagnostic relevance. Immunoprecipitation, mass spectrometry, and immunoblotting were employed for analysis of skin extracts and sera of patients with anti-p200 pemphigoid (n=60), other AIBD (n=33), and healthy blood donors (n=29). To localize the new antigen in skin, cultured keratinocytes and fibroblasts, quantitative real-time polymerase chain reaction and immunofluorescence microscopy were performed. Laminin β4 was identified as target antigen of anti-p200 pemphigoid in all analyzed patients. It was located at the level of the basement membrane zone of the skin with predominant expression in keratinocytes. A higher number of sera needs to be tested to verify that laminin β4 is the diagnostically relevant antigen of anti-p200 pemphigoid. The identification of laminin β4 as an additional target antigen in anti-p200 pemphigoid will allow its differentiation from other AIBD and as such, improve the management of these rare disorders.

Continuous short-term acclimation to moderate cold elicits cardioprotection in rats, and alters β-adrenergic signaling and immune status

Moderate cold acclimation (MCA) is a non-invasive intervention mitigating effects of various pathological conditions including myocardial infarction. We aim to determine the shortest cardioprotective regimen of MCA and the response of β1/2/3-adrenoceptors (β-AR), its downstream signaling, and inflammatory status, which play a role in cell-survival during myocardial infarction. Adult male Wistar rats were acclimated (9 °C, 1–3-10 days). Infarct size, echocardiography, western blotting, ELISA, mitochondrial respirometry, receptor binding assay, and quantitative immunofluorescence microscopy were carried out on left ventricular myocardium and brown adipose tissue (BAT). MultiPlex analysis of cytokines and chemokines in serum was accomplished. We found that short-term MCA reduced myocardial infarction, improved resistance of mitochondria to Ca2+-overload, and downregulated β1-ARs. The β2-ARs/protein kinase B/Akt were attenuated while β3-ARs translocated on the T-tubular system suggesting its activation. Protein kinase G (PKG) translocated to sarcoplasmic reticulum and phosphorylation of AMPKThr172 increased after 10 days. Principal component analysis revealed a significant shift in cytokine/chemokine serum levels on day 10 of acclimation, which corresponds to maturation of BAT. In conclusion, short-term MCA increases heart resilience to ischemia without any negative side effects such as hypertension or hypertrophy. Cold-elicited cardioprotection is accompanied by β1/2-AR desensitization, activation of the β3-AR/PKG/AMPK pathways, and an immunomodulatory effect.

Abstract P3200: SH3BGR Affects Energy Metabolism In Neonatal Rat Cardiomyocytes

Background: The SH3 domain-binding glutamic acid-rich (SH3BGR) gene maps to the genetic fragment associated with Down's syndrome (DS), one of the predominant causes of congenital heart diseases (CHD). We have previously shown that SH3BGR augments cardiomyocyte apoptosis, however, its possible role in cardiomyocyte metabolism is not known yet. Methods: Functional effects of SH3BGR knockdown or overexpression were studied in neonatal rat ventricular cardiomyocytes (NRVCMs) using seahorse metabolic assays, quantitative real-time PCR, immunoblotting, and immunofluorescence microscopy. Results: Since we found significantly altered cardiac levels of SH3BGR in human patients suffering from cardiac hypertrophy, which is reportedly associated with mitochondrial dysfunction, we determined if loss- or gain-of-function of SH3BGR impacts mitochondrial metabolism. We observed significant reduction in oxidative phosphorylation complexes and associated genes. Interestingly, seahorse metabolic activity assays revealed impairment in mitochondrial bioenergetics by both knockdown and overexpression of SH3BGR suggesting the importance of its stead-state levels in cardiomyocyte mitochondrial metabolism. NRVCMs showed inability to meet high energy demand on altered SH3BGR expression indicated by higher proton leak and impeded ATP production. These observations suggest altered mitochondrial respiration, which was further supported by alterations in the expression levels of respective metabolic genes. Combining these results with our previous findings suggests that SH3BGR plays an important role in cardiomyocyte oxidative phosphorylation, cell viability and apoptosis. Conclusion: Our data provide novel insights into potential role of SH3BGR in cardiomyocyte mitochondrial bioenergetics. These findings will help us understand how genetic deletion or SH3BGR mutations are associated with CHD e.g., using CRISPR/CAS mediated genetic manipulations using human patient derived induced pluripotent stem cells.

Effects of Yogurt with Carao (Cassia grandis) on Intestinal Barrier Dysfunction, α-glycosidase Activity, Lipase Activity, Hypoglycemic Effect, and Antioxidant Activity

Cell inflammation disrupts intestinal barrier functions and may cause disorders related to a leaky gut, possibly leading to diabetes. The objective of this study was to determine if carao (Cassia grandis) incorporated into yogurt enhances in vitro intestinal barrier function. To achieve this goal, Caco-2 cells were used as a model of intestinal barrier permeability. Caco-2 cells were treated with cytokines (interleukin-1β, tumor necrosis factor-α, interferon-γ, and lipopolysaccharide (LPS)) and yogurt with carao yogurt (CY) at different doses (1.3 g/L, 2.65 g/L, and 5.3 g/L). Real-time quantitative polymerase chain and immunofluorescence microscopy were applied to evaluate the expression and localization of tight junction proteins. Functional effects of the formulation of yogurt supplemented with carao were also evaluated in terms of the antioxidant activity, the α-glycosidase activity, and lipase inhibitory properties. In addition, the hypoglycemic potential was validated in vivo in a rat model. Compared to the control yogurt, Caco-2 TEER (transepithelial electrical resistance evaluation) by yogurt with 5.3 g/L of carao was significantly lower (p &lt; 0.05) after 48 h. Yogurt with 5.3 g/L of carao had a considerably lower permeability (p &lt; 0.05) than control yogurt in FD and LY flux. Yogurt with 5.3 g/L of carao enhanced the localization of ZO-1. Carao addition into yogurt increased the flavonoid content, apparent viscosity, lipase inhibition activity, and α-glycosidase activity. The rats fed with the yogurt with 5.3 g/L of carao demonstrated a higher blood glucose modulation.

Extracellular Tau Oligomers Damage the Axon Initial Segment.

In Alzheimer's disease (AD) brain, neuronal polarity and synaptic connectivity are compromised. A key structure for regulating polarity and functions of neurons is the axon initial segment (AIS), which segregates somatodendritic from axonal proteins and initiates action potentials. Toxic tau species, including extracellular oligomers (xcTauOs), spread tau pathology from neuron to neuron by a prion-like process, but few other cell biological effects of xcTauOs have been described. Test the hypothesis that AIS structure is sensitive to xcTauOs. Cultured wild type (WT) and tau knockout (KO) mouse cortical neurons were exposed to xcTauOs, and quantitative western blotting and immunofluorescence microscopy with anti-TRIM46 monitored effects on the AIS. The same methods were used to compare TRIM46 and two other resident AIS proteins in human hippocampal tissue obtained from AD and age-matched non-AD donors. Without affecting total TRIM46 levels, xcTauOs reduce the concentration of TRIM46 within the AIS and cause AIS shortening in cultured WT, but not TKO neurons. Lentiviral-driven tau expression in tau KO neurons rescues AIS length sensitivity to xcTauOs. In human AD hippocampus, the overall protein levels of multiple resident AIS proteins are unchanged compared to non-AD brain, but TRIM46 concentration within the AIS and AIS length are reduced in neurons containing neurofibrillary tangles. xcTauOs cause partial AIS damage in cultured neurons by a mechanism dependent on intracellular tau, thereby raising the possibility that the observed AIS reduction in AD neurons in vivo is caused by xcTauOs working in concert with endogenous neuronal tau.

Abstract 4935: Cannabidiol and cannabis extract protect cells against platinum-based chemotherapy via cellular uptake

Abstract Cannabinoids including cannabidiol belong between constituents of cannabis sp. (marijuana plant). Purified or as a part of cannabis products, the compounds are frequently used by cancer patients to attenuate chemotherapy-induced side effects such as pain, nausea, and vomiting. The presence of cannabis products on the public market expands each year while the illegality status weakens in an increasing number of countries. U.S. Food and Drug Administration has approved several cannabis-based drugs for seizures induced by Lennox-Gastaut syndrome, Dravet syndrome, or tuberous sclerosis complex, also for the lost appetite of AIDS patients, and chemotherapy-induced nausea and vomiting. Oncological patients are further and strongly motivated to take the drugs in combination with chemotherapy by numerous reports demonstrating the anticancer effects of cannabinoids. Nevertheless, here we present unsettling data that cannabis extract together with CBD negatively interferes with worldwide approved platinum-based drugs cisplatin, carboplatin, and oxaliplatin. The phenomenon is proven across three cellular models including primary cells. Even low concentrations of cannabidiol or cannabis extract reduced the toxicity of platinum-based drugs using long-term colony forming assay and/or short-term crystal violet assay. Increased resistance of cells was also accompanied by decreased cellular stress as evaluated by quantitative immunofluorescence microscopy and immunoblotting. Most importantly, concomitant cannabidiol and cisplatin exposure results in the decreased presence of cisplatin-modified DNA. Following the information, we determined platinum content in cells by ICP-MS. The experiment uncovered significantly decreased intracellular platinum content indicating an effect on cellular transport. Furthermore, the mechanism is translational independent as shown by the experiment with cycloheximide. This study offers crucial information that patients and physicians should take into account regarding possible interactions between platinum-based drugs and cannabis-based products. Furthermore, the study on the contradictory effects of cannabis products in cancer treatment may be motivated by this project. Citation Format: Tereza Buchtová, Lucie Beresova, Katarina Chroma, Martin Mistrik. Cannabidiol and cannabis extract protect cells against platinum-based chemotherapy via cellular uptake. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4935.

Paclitaxel, but Not Cisplatin, Affects Satellite Glial Cells in Dorsal Root Ganglia of Rats with Chemotherapy-Induced Peripheral Neurotoxicity

Chemotherapy-induced peripheral neurotoxicity is one of the most common dose-limiting toxicities of several widely used anticancer drugs such as platinum derivatives (cisplatin) and taxanes (paclitaxel). Several molecular mechanisms related to the onset of neurotoxicity have already been proposed, most of them having the sensory neurons of the dorsal root ganglia (DRG) and the peripheral nerve fibers as principal targets. In this study we explore chemotherapy-induced peripheral neurotoxicity beyond the neuronocentric view, investigating the changes induced by paclitaxel (PTX) and cisplatin (CDDP) on satellite glial cells (SGC) in the DRG and their crosstalk. Rats were chronically treated with PTX (10 mg/Kg, 1qwx4) or CDDP (2 mg/Kg 2qwx4) or respective vehicles. Morpho-functional analyses were performed to verify the features of drug-induced peripheral neurotoxicity. Qualitative and quantitative immunohistochemistry, 3D immunofluorescence, immunoblotting, and transmission electron microscopy analyses were also performed to detect alterations in SGCs and their interconnections. We demonstrated that PTX, but not CDDP, produces a strong activation of SGCs in the DRG, by altering their interconnections and their physical contact with sensory neurons. SGCs may act as principal actors in PTX-induced peripheral neurotoxicity, paving the way for the identification of new druggable targets for the treatment and prevention of chemotherapy-induced peripheral neurotoxicity.

Heat treatment-induced autophagy promotes breast cancer cell invasion and metastasis via TGF-β2-mediated epithelial-mesenchymal transitions.

Insufficient thermal ablation can accelerate malignant behaviors and metastases in some solid tumors, and epithelial-mesenchymal transition (EMT) and autophagy are involved in tumor metastasis. It has been found that TGF-β2 which belongs to the family of transforming growth factors often associated with cancer cell invasiveness and EMT. However, whether the interactions between autophagy and TGF-β2 induce EMT in breast cancer (BC) cells following insufficient microwave ablation (MWA) remains unclear. BC cells were treated with sublethal heat treatment to simulate insufficient MWA, and the effects of heat treatment on the BC cell phenotypes were explored. CCK-8, colony formation, flow cytometry, Transwell, and wound healing assays were performed to evaluate the influence of sublethal heat treatment on the proliferation, apoptosis, invasion, and migration of BC cells. Western blotting, real-time quantitative PCR, immunofluorescence, and transmission electron microscopy were carried out to determine the changes in markers associated with autophagy and EMT following sublethal heat treatment. Results showed that heat treatment promoted the proliferation of surviving BC cells, which was accompanied by autophagy induction. Heat treatment-induced autophagy up-regulated TGF-β2/Smad2 signaling and promoted EMT phenotype, thereby enhancing BC cells' migration and invasion abilities. An increase or decrease of TGF-β2 expression resulted in the potentiation and suppression of autophagy, as well as the enhancement and abatement of EMT. Autophagy inhibitors facilitated apoptosis and repressed proliferation of BC cells in vitro, and thwarted BC cell tumor growth and pulmonary metastasis in vivo. Heat treatment-induced autophagy promoted invasion and metastasis via TGF-β2/Smad2-mediated EMTs. Suppressing autophagy may be a suitable strategy for overcoming the progression and metastasis of residual BC cells following insufficient MWA.

Intestinal Epithelial Digestive, Transport, and Barrier Protein Expression Is Increased in Environmental Enteric Dysfunction

Environmental enteric dysfunction (EED) is characterized by malabsorption and diarrhea that result in irreversible deficits in physical and intellectual growth. We sought to define the expression of transport and tight junction proteins by quantitative analysis of duodenal biopsies from patients with EED. Biopsies from Pakistani children with confirmed EED diagnoses were compared to those from age-matched North American healthy controls, patients with celiac disease, and patients with nonceliac disease with villous atrophy or intraepithelial lymphocytosis. Expression of brush border digestive and transport proteins and paracellular (tight junction) proteins was assessed by quantitative multiplex immunofluorescence microscopy. EED was characterized by partial villous atrophy and marked intraepithelial lymphocytosis. Epithelial proliferation and enteroendocrine, tuft, and Paneth cell numbers were unchanged, but there was significant goblet cell expansion in EED biopsies. Expression of proteins involved in nutrient and water absorption and that of the basolateral Cl− transport protein NKCC1 were also increased in EED. Finally, the barrier–forming tight junction protein claudin-4 (CLDN4) was significantly upregulated in EED, particularly within villous enterocytes. In contrast, expression of CFTR, CLDN2, CLDN15, JAM-A, occludin, ZO-1, and E-cadherin was unchanged. Upregulation of a barrier–forming tight junction protein and brush border and basolateral membrane proteins that support nutrient and water transport in EED is paradoxical, as their increased expression would be expected to be correlated with increased intestinal barrier function and enhanced absorption, respectively. These data suggest that EED activates adaptive intestinal epithelial responses to enhance nutrient absorption but that these changes are insufficient to restore health.

Optimization of extracellular matrix for primary human hepatocyte cultures using mixed collagen-Matrigel matrices.

Loss of differentiation of primary human hepatocytes (PHHs) ex vivo is a known problem of in vitro liver models. Culture optimizations using collagen type I and Matrigel reduce the dedifferentiation process but are not able to prevent it. While neither of these extracellular matrices (ECMs) on their own correspond to the authentic hepatic ECM, a combination of them could more closely resemble the in vivo situation. Our study aimed to systematically analyze the influence of mixed matrices composed of collagen type I and Matrigel on the maintenance and reestablishment of hepatic functions. Therefore, PHHs were cultured on mixed collagen-Matrigel matrices in monolayer and sandwich cultures and viability, metabolic capacity, differentiation markers, cellular arrangement and the cells' ability to repolarize and form functional bile canaliculi were assessed by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), functional assays and immunofluorescence microscopy. Our results show that mixed matrices were superior to pure matrices in maintaining metabolic capacity and hepatic differentiation. In contrast, Matrigel supplementation can impair the development of a proper hepatocytic polarization. Our systematic study helps to compose an optimized ECM to maintain and reestablish hepatic differentiation on cellular and multicellular levels in human liver models.

Translational potential of synaptic alterations in Alzheimer's disease patients and amyloid precursor protein knock-in mice.

Synaptic dysfunction is an early event in Alzheimer's disease. Post-mortem studies suggest that alterations in synaptic proteins are associated with cognitive decline in Alzheimer's disease. We measured the concentration of three synaptic proteins, zinc transporter protein 3, dynamin1 and AMPA glutamate receptor 3 in cerebrospinal fluid of subjects with mild cognitive impairment (n = 18) and Alzheimer's disease (n = 18) and compared the levels to cognitively and neurologically healthy controls (n = 18) by using ELISA assay. In addition, we aimed to assess the translational potential of these synaptic proteins in two established amyloid precursor protein knock-in Alzheimer's disease mouse models by assessing the cerebrospinal fluid, hippocampal and cortical synaptic protein concentrations. Using ELISA, we measured in parallel these three proteins in cerebrospinal fluid and/or brain of 12- and 24-month-old AppNL-F and AppNL-G-F knock-in mice and AppWt control mice. The regional distribution and expression of these proteins were explored upon aging of the App knock-in models by quantitative immunofluorescence microscopy. Notably, we found a significant increase in concentrations of zinc transporter protein 3 and AMPA glutamate receptor 3 in cerebrospinal fluid of both patient groups compared with cognitively healthy controls. Dynamin1 concentration was significantly higher in Alzheimer's disease patients. Remarkably, patients with mild cognitive impairment who converted to Alzheimer's disease (n = 7) within 2 years exhibited elevated baseline cerebrospinal fluid zinc transporter protein 3 concentrations compared with mild cognitive impairment patients who did not convert (n = 11). Interestingly, similar to the alterations in Alzheimer's disease subjects, cerebrospinal fluid AMPA glutamate receptor 3 concentration was significantly higher in AppNL-G-F knock-in mice when compared with wild-type controls. Furthermore, we have detected age and brain regional specific changes of the three synaptic proteins in the hippocampus and prefrontal cortex of both AppNL-F and AppNL-G-F knock-in mice. Notably, all the three cerebrospinal fluid synaptic protein concentrations correlated negatively with concentrations in hippocampal lysates. The elevated zinc transporter protein 3 concentrations in the cerebrospinal fluid of converter versus non-converter mild cognitive impairment patients suggests a prospective role of zinc transporter 3 in differentiating dementia patients of the biological continuum of Alzheimer's disease. The increased cerebrospinal fluid concentrations of synaptic proteins in both patient groups, potentially reflecting synaptic alterations in the brain, were similarly observed in the amyloid precursor protein knock-in mouse models highlighting the translational potential of these proteins as markers for synaptic alterations. These synaptic markers could potentially help reduce the current disparities between human and animal model-based studies aiding the translation of preclinical discoveries of pathophysiological changes into clinical research.

Spatiotemporal profile of neutrophil extracellular trap formation in a mouse model of ischemic stroke

BackgroundThromboinflammatory processes modulate the complex pathophysiology of cerebral ischemia-reperfusion (I/R) injury in ischemic stroke, but the exact underlying mechanisms remain poorly understood. Emerging evidence indicates that neutrophil extracellular traps (NETs) might play an important role in the thromboinflammatory cascade. In addition, the link between von Willebrand factor (VWF) and neutrophil recruitment in the ischemic brain might promote thromboinflammation, possibly by the formation of NETs. ObjectivesTo study NET formation in a murine model of cerebral I/R injury in ischemic stroke. MethodsThe filament–induced transient middle cerebral artery occlusion model was used to induce 60 minutes of focal cerebral ischemia after which reperfusion was allowed. At different time points postischemia, NETs were identified in the ischemic mouse brain using quantitative immunofluorescence microscopy. ResultsNETs could be identified in the ipsilateral brain hemisphere. Interestingly, NETs could already be detected at 6 hours poststroke. Their presence increased at 12 hours, was highest at 24 hours, and decreased again 48 hours postischemia. Remarkably, NETs were predominantly localized within the brain vasculature postischemia, suggesting that NETs play a role in secondary microthrombosis. Strikingly, NET formation was significantly decreased in VWF–deficient mice compared to littermate wild-type mice 24 hours postischemia, indicating a possible role for VWF in promoting NETosis in the ischemic brain. ConclusionThis study identified the spatiotemporal profile of NET formation in a mouse model of cerebral I/R injury in ischemic stroke. NETs, potentially in combination with VWF, might be attractive targets for the development of novel therapeutic strategies in ischemic stroke treatment.

Resolvin D1 modulates periodontal ligament fibroblast function.

The resolution of inflammation is an active process mediated by specialized lipid mediators called lipoxins and resolvins. Periodontal ligament fibroblasts (PDLFs) play a significant role in periodontal regeneration. The purpose of the current study was to determine the impact of resolvin D1 (RvD1) on human PDLF cell wound healing and proliferation, receptor expression (G-protein-coupled receptor 32 [GPR32] and formyl peptidereceptor2 [ALX/FPR2]), and cytokine expression and release. PDLFs were stimulated with interleukin-1β (IL-1β) (500pg/ml) with and without RvD1 (100nM). RvD1 receptor expression was determined by quantitative real-time polymerase chain reaction (qPCR), immunofluorescence microscopy, and fluorescence-activated cell sorting. Wound closure was measured by a scratch assay, and proliferation was determined by bromodeoxyuridine incorporation. Interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemoattractant protein-1, cyclooxygenase-2, matrix metalloproteinases-1, -2, and -3 (MMP-1, -2, and -3), tissue inhibitors of metalloproteinases-1 and -2 (TIMP-1 and -2), prostaglandin E2, and osteoprotegerin (OPG) gene expression and production were measured using qPCR and Western blotting, multiplex immunoassay, and enzyme-linked immunosorbent assay. PDLF expressed GPR32 and ALX/FPR2. RvD1 reversed IL-1β-induced inhibition of wound healing and proliferation of PDLF. IL-1β also induced the production of proinflammatory cytokines and MMPs. This effect was reversed by RvD1. RvD1 reversed IL-1β-induced inhibition of TIMP-1, TIMP-2, and OPG. The data suggested that RvD1 has a pro-wound healing, proliferative, and anti-inflammatory impact on the PDLF that favors periodontal regeneration.

The Matricellular Protein SPARC Decreases in the Lacrimal Gland At Adulthood and During Inflammation.

Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein abundantly expressed in basement membranes and capsules surrounding a variety of organs and tissues. It mediates extracellular matrix organization and has been implicated in cell contraction. Here, we evaluated the expression of SPARC in the murine lacrimal gland at adulthood and during inflammation. Lacrimal glands of young mice (4-6 weeks old) and adult mice (32-40 weeks old) were used for extraction of DNA, RNA, and protein. The presence of SPARC was assessed by quantitative PCR, ELISA, and immunofluorescence microscopy. 5-Methylcytosine and DNA methylation were evaluated using ELISA and bisulfite genomic sequencing, respectively. The effects of cytokines and inflammation in Sparc expression were evaluated in vitro and in the non-obese diabetic (NOD) mouse model of Sjögren's syndrome. The mRNA and protein levels of SPARC were downregulated in lacrimal glands of mature adult mice presenting age-related histological alterations such as increased deposition of lipofuscin and lipids. Epigenetic analyses indicated that glands in adult mice contain higher levels of global DNA methylation and show increased hypermethylation of specific CpG sites within the Sparc gene promoter. Analysis of smooth muscle actin (SMA)-green fluorescent protein (GFP) transgenic mice revealed that SPARC localizes primarily to myoepithelial cells within the gland. Treatment of myoepithelial cells with IL-1β or TNF-α and the development of inflammation in the NOD mice led to decreased transcription of Sparc. SPARC is a novel matricellular glycoprotein expressed by myoepithelial cells in the lacrimal gland. Loss of SPARC during adulthood and chronic inflammation might have detrimental consequences on myoepithelial cell contraction and the secretion of tear fluid.

P392: PHARMACOLOGICAL INHIBITION OF SYK CONFERS ANTI-PROLIFERATIVE AND NOVEL ANTI-TUMOR IMMUNE RESPONSES IN AML

Background: Spleen tyrosine kinase (SYK) acts as a key integrator of signals from cell surface receptors containing an immunoreceptor tyrosine-based activation motif to boost cellular proliferation. In acute myeloid leukemia (AML), SYK serves as a relay to an oncogenic transcriptional regulatory network linked to NPM1, HOXA9 and MEIS1. The selective, orally bioavailable SYK inhibitor entospletinib (ENTO) has demonstrated clinical activity and tolerability in HOXA9/MEIS1-driven AML. ENTO is currently being investigated in a global Phase 3 trial, AGILITY (NCT05020665), in combination with intensive induction/consolidation chemotherapy in patients (pts) with treatment-naive NPM1-mutated (NPM1m) AML. Lanraplenib (LANRA) is a next-generation SYK inhibitor with similar potency and selectivity to ENTO but with more favorable pharmacologic properties that is currently being evaluated in combination with gilteritinib in pts with relapsed or refractory FLT3-mutated AML (NCT05028751). Aims: To investigate the effects of ENTO and LANRA in T-cell responses in AML. Methods: All pt-derived bone marrow (BM) and peripheral blood samples were obtained in accordance with IRB (OHSU#004422) approval. Results from gene expression profiling studies (RNA sequence [RNA-seq]) in bone marrow/peripheral blood mononuclear cells (MNCs) derived from 152 pts with AML, for which matching NPM1 status and ex vivo sensitivity to ENTO was available, previously published by Tyner et al (2018), were reanalyzed using trimmed mean of M values normalization and differential expression using edgeR. ENTO/LANRA sensitivity was assessed ex vivo after 72 hours in culture at 37°C by MTS assay. T-cell functional assays were performed by culturing MNCs with anti-CD3 and treating with ENTO or LANRA, singly and in combination with anti-PD1. CD3+ T-cell proliferation as measured by Ki67 expression and pSYK activation were assessed by quantitative single-cell immunofluorescence microscopy. Six formalin-fixed, paraffin-embedded BM biopsies from newly diagnosed AML pts were assessed for spatial expression of mRNA using a digital spatial profiling method. Results: In AML, mutations in NPM1 with co-expression of HOXA9/MEIS1 at baseline predicted anti-proliferative activity to ENTO in ex vivo drug sensitivity studies. Accordingly, treatment of leukemic cells with either ENTO or LANRA inhibited SYK auto-phosphorylation in a dose-dependent manner. Pathway analysis of archival RNA-seq data from pts enrolled in the Leukemia and Lymphoma Society BEAT AML Master Protocol (NCT03013998 [BAML-16-001-S6]) revealed gene expression signatures at baseline associated with the observed sensitivity to ENTO, including significant enrichment in the expression of genes associated with leukemogenesis, myeloid differentiation and immune regulation. This was supported by T-cell functional studies, which demonstrated that ENTO and LANRA, singly as well as in combination with anti-PD1, could restore T-cell proliferation in primary AML pt samples that exhibited suppression. Lastly, Cancer Transcriptome Atlas analysis on archival BM biopsies obtained from these AML pts showed a strong correlation between immune checkpoint response and gene expression associated with immune pathway signaling. Summary/Conclusion: Inhibition of SYK is a promising therapeutic approach in NPM1m AML. Our studies illustrate that ENTO and LANRA may restore T-cell proliferation in a subset of AML pts with dysfunctional T-cell responses, suggesting a novel mechanism of action. Additional studies are required to fully understand the mechanism of SYK-mediated antitumor immune responses in AML.

The effect of PARP inhibition on androgen receptor localization and activity in castration resistant prostate cancer.

e17037 Background: The poly(ADP-ribose) polymerase inhibitors (PARPi) olaparib and rucaparib have been approved for the treatment of metastatic castration resistant prostate cancer (mCRPC) in the setting of homologous recombination deficiency (HRD). Additionally, PARPi have been shown to modulate androgen receptor (AR) signaling, with a recent report demonstrating mono-ADP-ribosylation of cysteine residues in AR by Parp7. Here, we further evaluated the effect of PARPi on AR activity and localization. Methods: The effect of PARPi on cell growth and survival of CRPC cell lines was evaluated in vitro and in vivo. AR ribosylation was assessed in CRPC cell lines by immunoprecipitation (IP) assays and proximity ligation assays (PLA). The subcellular localization of AR was determined by quantitative immunofluorescence microscopy in CRPC cell lines and xenograft models. Changes in AR activity with PARPi treatment were evaluated by a luciferase reporter assay and AR target gene expression in a PDX model. Finally, PARPi mediated alteration in the AR protein interactome was evaluated by liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics. Results: The PARPi olaparib and talazoparib, and to a lesser extent veliparib, inhibited CRPC cell growth. Evidence of AR ribosylation was seen by IP and PLA. PARPi treatment of multiple in vitro and in vivo prostate cancer models resulted in a shift of AR subcellular localization, from predominantly nuclear to cytoplasmic compartments. In luciferase reporter assays, AR transactivation activity was decreased after PARPi treatment in a dose dependent manner. In vivo, in prostate cancer xenograft models, decreased AR target gene expression was seen upon PARPi treatment. LC-MS/MS proteomic studies revealed that PARP inhibition resulted in significant changes in the composition of AR interaction partners, in particular of proteins related to intracellular trafficking and nuclear transport. This suggests a potential link between altered AR complex assembly and the observed changes in AR subcellular localization in the context of PARPi treatment. Conclusions: We describe a novel sequela of PARPi therapy to alter AR localization and activity in CRPC. Single agent PARP inhibition can alter prostate cancer cell growth in vitro and in vivo. With PARPi treatment, AR localization is shifted to the cytoplasm, the AR interactome is altered, and AR transcriptional activity decreased. These findings implicate a collateral mechanism of PARPi in preventing prostate cancer cell growth/survival that may augment previously described mechanisms related to HDR and synthetic lethality. It is essential to understand the role of PARPi beyond synthetic lethality in the context of HRD in order to better define the spectrum of response to PARPi across patients, and for development of biology-informed combination therapies.

Safety, pharmacodynamic, and clinical response evaluation of nilotinib and paclitaxel in adults with refractory solid tumors.

3026 Background: The combination of the BCR-Abl kinase inhibitor nilotinib and the anti-tubulin agent paclitaxel was identified in the NCI-ALMANAC study to have greater-than-additive activity in the NCI-60 cell line panel and greater-than-single-agent antitumor activity in xenograft models, in which this combination induces tumor epithelial-mesenchymal transition (EMT). A phase 1 study was initiated to establish the safety, tolerability, and recommended phase 2 dose (RP2D) of this combination in patients (pts) with advanced solid tumors and to examine the pharmacokinetic (PK) and pharmacodynamic (PD) effects of the combination to understand the mechanism of action (NCT02379416). The dose escalation phase established the RP2D as 300 mg oral nilotinib twice daily and 80 mg/m2 intravenous paclitaxel on days (D) 1, 8, and 15 of each 28-day cycle. Here, we report the safety, preliminary PD, and efficacy data for this combination. Methods: Nilotinib and paclitaxel were administered as noted above, with a 1-day (escalation cohort) or 2-day (expansion cohort) paclitaxel-only run-in during the first cycle to enable comparison of the PK and PD effects of the combination vs. single-agent paclitaxel. Paired biopsies to assess tumor molecular response were collected from expansion cohort pts at baseline, cycle (C) 1 D2, and C1D28, with an optional biopsy at progression; accrual continued until ≥ 12 sufficient-quality paired biopsies were obtained. Blood specimens to assess molecular responses in circulating tumor cells (CTCs) were obtained at several timepoints during C1 and longitudinally every cycle thereafter. EMT biomarkers were measured in tumor and CTC specimens using quantitative immunofluorescence microscopy assays. Results: A total of 44 pts were enrolled. Three pts had partial responses (PR), and 1 had an unconfirmed PR (9%); 23 pts (52%) had a best response of stable disease (SD), including 7 pts on study for ≥ 10 cycles. The most common grade (Gr) 3-4 treatment-related adverse events were hematologic and hypophosphatemia. No pts experienced Gr ≥ 3 peripheral neuropathy. The median time on treatment was 67 days. Two pts with granulosa cell ovarian carcinoma had durable responses, completing 74+ and 64 cycles. Multiple patient biopsies and corresponding CTC specimens exhibited treatment-induced EMT. Longitudinal analysis of CTC EMT phenotypes in the 2 pts with extended PR revealed a substantial increase in mesenchymal-like CTCs prior to progression for the pt on study for 64 cycles; such increases were not observed in the pt still on study after 74+ cycles. Further PD analyses are ongoing. Conclusions: The combination of nilotinib and paclitaxel demonstrates promising disease control with durable response in select patients. Tumor PD analyses to discover the underlying pharmacology of this active regimen are ongoing. Funded by NCI Contract No. HHSN261201500003I. Clinical trial information: NCT02379416.