Immunofluorescence Confocal Microscopy Research Articles

Exosomal HMGB1 educate PD-L1+-tumor associated macrophages via glycometabolic reprogramming to induce lung-tropic metastasis of triple negative breast cancer.

1090 Background: Lung metastasis occurs in one third of triple negative breast cancer (TNBC), the underlying molecular mechanism remains largely unclear. Mounting evidence suggests that PD-L1+-tumor associated macrophages (PD-L1+-TAMs) are closely associated with immune suppression, invasion, and metastasis. Tumor-derived exosomes have been reported to remodel tumor microenvironment and accelerate metastasis through packing and delivering a variety of biologically active molecules. The aim of this study was to investigate whether TNBC-derived exosomes were able to polarize lung macrophages towards PD-L1+-TAMs and promote a pre-metastatic immunosuppressive niche, therefore discovering the underlying mechanism of lung-tropic metastasis. Methods: Exosomes obtained from EO771 TNBC cell line (EO771/exo) and HC11 normal breast epithelial cell line (HC11/exo) were characterized. Absorption of exosomes by macrophages was observed by confocal microscopy. Mass spectrometry was used to identify the significantly highly expressed HMGB1 protein in EO771/exo, and ligands of HMGB1 on lung macrophages were detected by co-immunoprecipitation. Significance of EO771/exo and HMGB1 in inducing PD-L1+-TAMs and generating a pre-metastatic immunosuppressive niche were confirmed by both in vitro and in vivo studies. Functions of EO771/exo and HMGB1 on glycolysis and lactate production were evaluated by metabolism assays. Results: EO771/exo could polarize macrophages toward PD-L1+-TAMs and generate a pre-metastatic immunosuppressive niche. Mass spectrometry, clinical samples, and bioinformatics analysis showed that HMGB1 was significantly expressed in EO771/exo and was involved in cellular communication and metabolism processes. Co-immunoprecipitation and 3-dimensional modeling suggested a strong binding of HMGB1 with Tim-3 ligand on lung macrophages. Macrophages incubated with EO771/exo exhibited an enhanced glycolysis and lactate production. Flow cytometry demonstrated that green fluorescent protein (GFP)-EO771 tumor-bearing mice had more lung micro-metastases than brain and liver. Immunofluorescence and laser confocal microscopy demonstrated that EO771/exo were able to increase lung micro-metastases and escalate PD-L1 expression on lung macrophages. Moreover, elevated PD-L1+-TAMs and reduced CD3+CD8+ T lymphocytes were detected in TNBC positive axillary lymph nodes and lung metastasis specimens than TNBC negative axillary lymph nodes. Conclusions: Exosomal HMGB1 from TNBC could target Tim-3 ligand on lung macrophages and accelerate glycolysis and lactate production to induce PD-L1+-TAMs, thereby suppressing CD3+CD8+ T lymphocytes immunity and generating a pre-metastasis immune-suppressive niche. Our research would uncover a novel mechanism of lung-tropic metastasis.

Capillary contact points determine beta cell polarity, control secretion and are disrupted in the db/db mouse model of diabetes.

Almost all beta cells contact one capillary and insulin granule fusion is targeted to this region. However, there are reports of beta cells contacting more than one capillary. We therefore set out to determine the proportion of beta cells with multiple contacts and the impact of this on cell structure and function. We used pancreatic slices in mice and humans to better maintain cell and islet structure than in isolated islets. Cell structure was assayed using immunofluorescence and 3D confocal microscopy. Live-cell two-photon microscopy was used to map granule fusion events in response to glucose stimulation. We found that 36% and 22% of beta cells in islets from mice and humans, respectively, have separate contact with two capillaries. These contacts establish a distinct form of cell polarity with multiple basal regions. Both capillary contact points are enriched in presynaptic scaffold proteins, and both are a target for insulin granule fusion. Cells with two capillary contact points have a greater capillary contact area and secrete more, with analysis showing that, independent of the number of contact points, increased contact area is correlated with increased granule fusion. Using db/db mice as a model for type 2 diabetes, we observed changes in islet capillary organisation that significantly reduced total islet capillary surface area, and reduced area of capillary contact in single beta cells. Beta cells that contact two capillaries are a significant subpopulation of beta cells within the islet. They have a distinct form of cell polarity and both contact points are specialised for secretion. The larger capillary contact area of cells with two contact points is correlated with increased secretion. In the db/db mouse, changes in capillary structure impact beta cell capillary contact, implying that this is a new factor contributing to disease progression.

#1162 Paraneoplastic podocytopathy in patients with solid cancer: clinical characteristics and new insights into pathophysiological processes about 21 cases

Abstract Background and Aims Minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) may occur in patients with solid cancer but this rare association has never been extensively investigated. Here, we report the first series of patients presenting this association, also named paraneoplastic podocytopathy with nephrotic syndrome (PPNS) . Previous study has demonstrated the specific induction in both podocytes and Reed-Sternberg cells of C-Maf Inducing Protein (CMIP) in patients with MCD and Hodgkin lymphoma. Here, we examined whether this molecular relationship was also relevant in patients developing PPNS in the setting of solid cancer. We have investigated the potential CMIP expression in kidney biopsies and in tumor tissues. Method We collected data from patients who presented with histologically proven cancer and concomitantly developed a nephrotic syndrome (± 3 months) related to MCD or FSGS Follow-up data were collected after 3, 6, 12 months and at last visit. We identified a control group of idiopathic nephrotic syndromes (INS) matched on sex, age and histological type, in order to compare the characteristics of these controls with patients with PPNS. Expression of CMIP was studied by immunohistochemistry and by immunofluorescence confocal microscopy on kidney biopsy and tumor samples. We used kidney biopsies from INS and tumors from patients without associated nephrotic syndrome as controls. Results Twenty-one PPNS patients and 21 controls with INS without cancer were identified. The median age of PPNS patients was higher (68 years) than usually observed in INS. Most of them (76%) had a MCD. A broad spectrum of cancers was found (bronchopulmonary, prostatic, colorectal…) . Baseline characteristics were similar between PPNS patients and INS controls, with a median urinary protein creatinine ratio of 865 and 900 mg/mmol, blood albumin t 15 and 15.3 g/L. Estimated glomerular filtration rate were 48 and 60 ml/min/1.73 m² respectively (no significant difference) . At presentation, PPNS patients were more likely to have hematuria (p = 0.031) , a lower median hemoglobin level than controls (p = 0.039) . Moreover, PPNS patients were less often treated with immunosuppressive drugs (p = 0.004) . After 12 months of follow-up, PPNS patients had a less favorable outcome than INS controls, with a mortality rate three times higher (30% vs 9.5%, p = 0.098) and a much lower rate of complete remission of nephrotic syndrome (25% vs 73.7%, p = 0.008, Table 1). CMIP labeling was performed on 13 tumor samples and 16 kidney biopsies in PPNS patients, and on 5 kidney biopsies with INS, and on 5 control tumor samples. Podocyte labeling of CMIP is observed on all but one kidney biopsy. CMIP labeling was positive on all case tumors, but only a scarce staining was detected in control patients with solid cancer without nephrotic syndrome (Fig. 1). In PPNS patients, confocal microscopy analysis on tumoral tissue showed high expression of CMIP in some epithelial cells and in immune infiltrating cells.. In addition, we found that CMIP was specifically express in CD4 T lymphocyte subset (Fig. 2), but not in CD20, FOXP3 or PD1 lymphocytes, defining a novel FOXP3−/PD1− CMIP high CD4+T lymphocyte subpopulation, within immune cell infiltrates. Conclusion We report the first series of patients with PPNS, who have a more severe renal and overall prognosis than controls. We show that patients with PPNS display higher induction of CMIP, both in the tumor and in podocytes. Strikingly, we identified within immune infiltrates a new subset of T-cell harboring a particular phenotype (CD4+, CMIPhigh, FOXP3−, PD1−) , which is not detected in patients with solid tumor without nephrotic syndrome and which could play a key role in the pathophysiology of PPNS.

The impact of von Willebrand factor on fibrin formation and structure unveiled with type 3 von Willebrand disease plasma

Normally, von Willebrand factor (VWF) remains inactive unless its A1A2 domains undergo a shear stress-triggered conformational change. We demonstrated the capacity of a recombinant A2 domain of VWF to bind and to affect fibrin formation, altering the fibrin clot structure. The data indicated that VWF contains an additional binding site for fibrin in the A2 domain that plays a role in the incorporation of VWF to the polymerizing fibrin. This study is to examine the hypothesis that active plasma VWF directly influence fibrin polymerization and the structure of fibrin clots. The study used healthy and type 3 von Willebrand disease (VWD) plasma, purified plasma VWF, fibrin polymerization assays, confocal microscopy and scanning electron microscopy. The exposed A2 domain in active VWF harbors additional binding sites for fibrinogen, and significantly potentiates fibrin formation (P < 0.02). Antibody against the A2 domain of VWF significantly decreased the initial rate of change of fibrin formation (P < 0.002). Clot analyses revealed a significant difference in porosity between normal and type 3 VWD plasma (P < 0.008), further supported by scanning electron microscopy, which demonstrated thicker fibrin fibers in the presence of plasma VWF (P < 0.0003). Confocal immunofluorescence microscopy showed punctate VWF staining along fibrin fibrils, providing visual evidence of the integration of plasma VWF into the fibrin matrix. The study with type 3 VWD plasma supports the hypothesis that plasma VWF directly influences fibrin polymerization and clot structure. In addition, a conformational change in the A1A2 domains exposes a hidden fibrin(ogen) binding site, indicating that plasma VWF determines the fibrin clot structure.

α1-Adrenergic Receptors Control the Activity of Sinoatrial Node by Modulating Transmembrane Transport of Chloride Anions

Norepinephrine (NE), which is released by sympathetic nerve endings, causes an increase in the frequency of spontaneous action potentials in the pacemaker cardiomyocytes of the sinoatrial node (SAN), also known as the “pacemaker” of the heart. This results in an increase in heart rate (HR). It is known that two types of postsynaptic adrenoreceptors (ARs), α1-AR and β-AR, can mediate the effects of NE. The role of α1-AR in the sympathetic control of heart rate and SAN automaticity, as well as the membrane mechanisms mediation the effects of α1-AR on the pacemaker, have not yet been elucidated. In this study, we utilized immunofluorescence confocal microscopy to examine the distribution of α1A-AR in the SAN of rats. Additionally, we assessed the expression of α1A-AR mRNA in the SAN tissue using RT-PCR. Furthermore, we investigated the impact of α1-AR stimulation on key functional parameters of the pacemaker, including the corrected sinus node recovery time (SNRT/cSNRT) and the SAN accommodation, using the Langendorff perfused heart technique. We also used optical mapping of the electrical activity of perfused, isolated tissue preparations to study the effect of α1-AR stimulation on the spatiotemporal characteristics of SAN excitation. We tested the effects of chloride transmembrane conductance blockade on alteration of functional parameters and pattern of SAN excitation caused by α1-AR. Fluorescent signals corresponding to α1A-AR have been identified in SAN cardiomyocytes, indicating the presence of α1A-AR at protein level. The expression of α1A-AR in SAN has been also confirmed at the mRNA level. The stimulation of α1-AR affects SAN functioning Phenylephrine (PHE) utilized as α1A-AR agonist causes a decrease in SNRT/cSNRT, as well as an acceleration of SAN accommodation. These effects were rate dependent and were observed at a high frequency of pacemaker tissue stimulation. PHE induces changes in the excitation pattern of the SAN. The effects of PHE on functional parameters and SAN excitation pattern are attenuated by Ca2+-dependent chloride channel blocker NPPB but remains unaffected by the protein kinase C inhibitor BIM. Our results suggest that cardiac α1-ARs are important for maintaining function of SAN pacemaker at high heart rates and that α1-AR signalling cascades in the SAN target Ca2+-dependent chloride channels are involved in the α1-adrenergic modulation of the electrophysiological properties of the heart pacemaker.

Abstract PO1-06-11: Exosomal HMGB1 induce PD-L1+-tumor associated macrophages through glycometabolic reprogramming to promote lung-tropic metastasis of triple negative breast cancer

Abstract Background: Lung metastasis occurs in 30% of triple negative breast cancer (TNBC), the detailed molecular mechanism remains largely unexplored. Accumulating evidence suggests that PD-L1+-tumor associated macrophages (PD-L1+-TAMs) are closely related to immune suppression, invasion, and metastasis. Tumor-derived exosomes have been reported to remodel tumor microenvironment and accelerate metastasis via packing and delivering a variety of biologically active molecules. Our previous studies found that exosomes from breast cancer cells polarized macrophages toward TAMs within the primary tumor nest. The aim of this study was to reveal whether TNBC-derived exosomes were able to polarize lung macrophages towards PD-L1+-TAMs and generate a pre-metastatic immunosuppressive niche, therefore discovering the underlying mechanism of lung-tropic metastasis. Methods: Exosomes isolated from EO771 TNBC cell line (EO771/exo) and HC11 normal breast epithelial cell line (HC11/exo) were characterized. Uptake of exosomes by macrophages was visualized by confocal microscopy. Mass spectrometry was applied to identify the significantly highly expressed HMGB1 protein in EO771/exo, and ligands of HMGB1 on lung macrophages were detected by co-immunoprecipitation. Functions of EO771/exo and HMGB1 in inducing PD-L1+-TAMs and generating a pre-metastatic immunosuppressive niche were confirmed by both in vitro and in vivo studies. Roles of EO771/exo and HMGB1 on glycolysis and lactate production were evaluated by metabolism assays. Associations between PD-L1+-TAMs and CD3+CD8+ T lymphocytes within TNBC axillary lymph nodes and lung metastasis specimens were analyzed by immunofluorescence and immunohistochemistry. Results: Confocal microscopy showed constant internalization and absorption of EO771/exo by macrophages. EO771/exo could polarize macrophages toward PD-L1+-TAMs and generate a pre-metastatic immunosuppressive niche, with respect to HC11/exo. Mass spectrometry, clinical samples, and bioinformatics analysis revealed that HMGB1 was highly expressed in EO771/exo and was involved in cellular communication and metabolism processes. Co-immunoprecipitation and 3-dimensional modeling indicated a strong binding of HMGB1 with Tim-3 ligand on lung macrophages. Macrophages treated with EO771/exo displayed an enhanced glycolysis and lactate production. Flow cytometry demonstrated that green fluorescent protein (GFP)-EO771 tumor-bearing mice had more lung micro-metastases than brain and liver. Immunofluorescence and laser confocal microscopy confirmed that EO771/exo, with respect to HC11/exo, were able to increase lung micro-metastases and escalate PD-L1 expression on lung macrophages. Moreover, elevated PD-L1+-TAMs and reduced CD3+CD8+ T lymphocytes were found in TNBC positive axillary lymph nodes and lung metastasis specimens than TNBC negative axillary lymph nodes. Conclusions: Exosomal HMGB1 from TNBC could target Tim-3 ligand on lung macrophages and accelerate glycolysis and lactate production to induce PD-L1+-TAMs, thereby suppressing CD3+CD8+ T lymphocytes immunity and generating a pre-metastasis immune-suppressive niche. Our research would uncover a novel mechanism of lung-tropic metastasis and provide a new therapeutic approach for TNBC treatment. Keywords: breast cancer; lung metastasis; exosomes; tumor-associated macrophages; metabolic reprogramming Citation Format: Wei-Xian Chen, Jia-Hao Wu, Tian-Cheng Cheng, Sujin Yang. Exosomal HMGB1 induce PD-L1+-tumor associated macrophages through glycometabolic reprogramming to promote lung-tropic metastasis of triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-06-11.

L. plantarum and L. casei synergistically mitigate Gastro-Intestinal Acute Radiation Syndrome (GI-ARS)

Introduction: Incidental or accidental acute exposure to ionizing radiation causes radiation toxicity, for which there are no known medical countermeasures (MCMs). Ionizing radiation alters host gut epithelium as well as the gut microbiome. We sought to investigate if radiation induced gut dysbiosis occurred as a result of depletion of gut-protective bacteria, and if probiotic supplementation of such bacteria could help mitigate GI-ARS post ionizing radiation exposure. Methods: Adult C57BL/6 mice were subjected to total body irradiation, and a defined concentration of probiotics (either individually or combined) were given 24 h after radiation exposure. Mice were sacrificed 24 hours post treatment (i.e., 48 h post irradiation). Vascular-to-luminal flux of FITC-inulin was measured to evaluate intestinal mucosal permeability and endotoxemia by measuring plasma lipopolysaccharide. Gut microbiota was analyzed by 16S rRNA sequencing. Intestinal epithelial junctions were analyzed by immunofluorescence confocal microscopy and mucosal inflammatory response by RT-PCR for cytokine expression. Systemic inflammation was evaluated by ELISA for plasma cytokines. Results and conclusion: Our data show that radiation altered the taxonomic composition of the microbiota compared to age-matched sham treated mice. Shannon index analysis showed a significant decrease in α-diversity in irradiated mice. Bray-Curtis analysis indicated significant differences in microbiota composition between Sham and IR groups. E. coli abundance was increased whereas Lactobacillus abundance was decreased, particularly L. plantarum and L. casei. Probiotic supplementation with individual Lactobacillus species each mitigated radiation induced intestinal barrier dysfunction, gut specific as well as systemic inflammatory cytokine expression and endotoxemia. Our preliminary data further indicates that L. plantarum affects Epidermal Growth Factor Receptor (EGFR)-mediated signaling whereas L. casei was EGFR-independent. Interestingly, combined supplementation in a 1:1 ratio further enhanced recovery compared to adding the individual effects. Individual supplementation of L. plantarum and L. casei to irradiated mice reduced intestinal (ileal) permeability by 2-fold and 3-fold respectively, whereas combined supplementation reduced ileal permeability almost 8-fold compared to irradiated but untreated mice. This data indicates that L. plantarum and L. casei mitigate GI-ARS by distinct cellular mechanisms. Furthermore, data show that a mixture of these probiotic species is more effective in radio-mitigation than individually. Further mechanistic analysis will be required to identify cross-talking targets between mechanisms of these probiotics and synergistic radio-mitigation. In conclusion, Lactobacillus species are potential MCMs for the treatment of GI-ARS. Funding acknowledgements: NIH/NIAID, UO1-AI170019, UO1-AI172991. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The interplay between calcineurin inhibitors (CNIs) and WNK bodies

KS-WNK1 is a kidney-specific scaffolding isoform of the With-No-Lysine (WNK) kinase signaling pathway. It is the predominant WNK1 isoform in the kidney, lacks a functional kinase domain, and is highly expressed in the distal convoluted tubule (DCT) where it drives the formation of biomolecular condensates called WNK bodies. During hypokalemia, WNK bodies bring together KS-WNK1, L-WNK1, WNK4, and SPAK, facilitating activation of the kinase cascade. This leads to increased downstream phosphorylation of the SLC12 cotransporter NCC. Mechanisms governing the reversibility and size of WNK body condensate scaffolds are poorly understood. We propose that phosphatases regulate KS-WNK1 and WNK body formation--a likely candidate being the phosphatase calcineurin (PP3). Prior work suggests that calcineurin dephosphorylates and inhibits the WNK-SPAK-NCC pathway, whereas calcineurin inhibitors (CNIs) such as tacrolimus increase phosphorylation and activation of the pathway. Here, we tested the hypothesis that CNIs also increase the abundance of KS-WNK1, causing increased WNK body size and abundance, and activation of the WNK-SPAK-NCC pathway. To study the effect of endogenous WNK kinases on KS-WNK1 function, WT HEK293 cells or HEK293 L-WNK1 KO cells were transiently transfected with KS-WNK1-HA-mRuby, treated with tacrolimus and studied by immunofluorescence confocal microscopy and immunoblotting of supernatant and detergent-insoluble pellet fractions. In both WT and KO cells, tacrolimus significantly increased WNK body abundance and size. Consistent with its localization within WNK bodies, KS-WNK1 protein was enriched in the pellet fraction by 10-fold in both WT and KO cells. KS-WNK1 protein abundance was increased by tacrolimus in a dose- (0-400ng/ml) and time-dependent manner (0-72h), an effect that was independent of endogenous L-WNK1 expression. Consistent with previous studies, SPAK abundance and phosphorylation was increased with tacrolimus treatment in a dose and time-dependent manner. Interestingly, SPAK localization was shifted into the pellet fraction in WT cells expressing KS-WNK1, whereas it was localized in the supernatant in L-WNK1 KO cells. The tacrolimus-induced increase in KS-WNK1 and pSPAK correlated with increased SLC12 cotransporter phosphorylation in WT cells. These preliminary results suggest that tacrolimus promotes WNK body formation and activity via KS-WNK1, and that SPAK localization in HEK cell-reconstituted WNK bodies requires full length WNK1. There is growing evidence that biomolecular condensates concentrate biomolecules to optimize signaling pathways and enhance kinase specificity through spatiotemporal control. Here we propose a novel concept that WNK bodies are biomolecular condensates that are regulated by calcineurin inhibitors. NIH K08DK118211, U54DK137329, ASN Gottschalk Award, P30DK081943. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Curative effects and mechanisms of AG1296 and LY294002 co-therapy in Angiostrongylus cantonensis-induced neurovascular unit dysfunction and eosinophilic meningoencephalitis

BackgroundCo-therapy with albendazole and steroid is commonly used in patients with eosinophilic meningoencephalitis caused by Angiostrongylus cantonensis infections. However, anthelminthics often worsen symptoms, possibly due to the inflammatory reaction to antigens released by dying worms. Therefore, the present study was to investigate the curative effects and probable mechanisms of the platelet-derived growth factor receptor-beta (PDGFR-β) inhibitor AG1296 (AG) and the phosphoinositide 3-kinase inhibitor (PI3K) LY294002 (LY) in A. cantonensis-induced neurovascular unit dysfunction and eosinophilic meningoencephalitis. MethodsWestern blots were used to detect matrix protein degradation and the expressions of PDGFR-β/PI3K signaling pathway. The co-localization of PDGFR-β and vascular smooth muscle cells (VSMCs), and metalloproteinase-9 (MMP-9) and VSMCs on the blood vessels were measured by confocal laser scanning immunofluorescence microscopy. Sandwich enzyme-linked immunosorbent assays were used to test S100B, interleukin (IL)-6, and transforming growth factor beta in the cerebrospinal fluid to determine their possible roles in mouse resistance to A. cantonensis. ResultsThe results showed that AG and LY cotherapy decreased the MMP-9 activity and inflammatory reaction. Furthermore, S100B, IL-6 and eosinophil counts were reduced by inhibitor treatment. The localization of PDGFR-β and MMP-9 was observed in VSMCs. Furthermore, we showed that the degradation of the neurovascular matrix and blood-brain barrier permeability were reduced in the mouse brain. ConclusionsThese findings demonstrate the potential of PDGFR-β inhibitor AG and PI3K inhibitor LY co-therapy as anti-A. cantonensis drug candidates through improved neurovascular unit dysfunction and reduced inflammatory response.

Macrophage Targeted Dendrimer-Tesaglitazar Therapy for Atherosclerosis

Background: Atherosclerosis is a leading contributor to cardiovascular disease, the number one cause of death globally. Although atherosclerosis is clearly a multicellular disease that also affects endothelial cells as well as smooth muscle cells of arteries, it is increasingly recognized as an inflammatory disease. Our goal is to determine if targeting immune cells implicated in atherosclerosis can halt plaque accumulation and resulting derangement of vascular cells in the aorta. Peroxisome proliferator activated receptors (PPARs) within macrophages present a promising therapeutic target as PPAR-α and PPAR-γ agonists have been shown to improve lipid metabolism and inhibit macrophage foam cell formation. However, due to high doses required and associated side effects, clinical translation was halted. We target anti-atherogenic pathways in immune cells using the novel treatment D-Tesa, a dendrimer conjugate of a dual PPAR-α/γ agonist, Tesaglitazar. Methods: 25-week-old, male, apolipoprotein E knockout (ApoE−/−) mice were fed high fat diet (HFD) for 16 weeks to induce atherosclerosis and randomized into two groups: 1) treatment group: D-Tesa for 6 weeks (20μg/kg twice weekly by oral gavage and 2) untreated group. HFD was continued for the duration of the study. Pulse wave velocity (PWV: Doppler), blood pressure (BP: tail cuff on trained, conscious mice), and body weight were monitored. Dendrimer mediated delivery of drug payload to plaque macrophages was studied using D-Cy5, a fluorescently labeled dendrimer, in an atherosclerotic mouse. Aortas were isolated and used for vasoreactivity studies and western blot analysis. THP-1 cell derived foam cells were used to study D-Tesa’s mechanism of action in vitro. Results: Immunofluorescence staining and confocal microscopy revealed that D-Cy5 localized in macrophages within the atherosclerotic plaque in ApoE−/− mice on HFD, indicating that the dendrimer can deliver drugs specifically into the plaque. D-Tesa slowed plaque progression, preserved endothelial function, and decreased arterial stiffness. BP was similar and unchanged between groups. D-Tesa surprisingly decreased body weight, which was the opposite effect observed from the free drug in clinical trials. Western blotting of aorta revealed that D-Tesa treated mice experienced an upregulation of PPAR-α, PPAR-γ, and ABCA1. The in vitro studies with THP-1 derived foam cells confirmed the upregulation of the cholesterol efflux pathway and anti-inflammatory benefit of D-Tesa. Conclusion: D-Tesa offers a novel therapy to ameliorate multiple damaging consequences of atherosclerosis on the cardiovascular system including improvements in plaque burden, endothelial function, arterial stiffness, and body weight in an ApoE−/− model of atherosclerosis. Excitingly, these effects were seen at 2-15 times lower doses despite continued HFD than those reported in literature with a combination of free Tesa and low-fat diet. Thus, D-Tesa offers great translational relevance because of potential reduction in negative side effects and higher patient benefit due to noted lack of compliance with dietary changes in the clinic. Further research is ongoing to evaluate sex differences, dose response, and long-term effects of this highly promising therapy. This work was supported by a NHLBI grant R01HL148112 01 (L.S.) and the Traystman Endowment (S.K.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Immunohistochemistry of the nasal cavity-associated lymphoid tissue in the dolphin (Stenella coeruleoalba, Meyen 1833).

The striped dolphin (Stenella coeruleoalba) is a medium-sized pelagic dolphin with a single external nasal opening (blowhole) located in the rostral and dorsal regions of the skull. The nasal cavity is divided into three sections: the olfactory, respiratory, and vestibular areas. The surface epithelium lining the regio vestibularis is the first tissue in the nose to be directly affected by environmental antigens. Cetaceans have a significant amount of mucosa-associated lymphoid tissue (MALT) located throughout their bodies. The lymphoid tissue found in the nasal mucosa is known as nose- or nasopharynx-associated lymphoid tissue (NALT). NALT has not yet been studied in dolphins, but it has been identified and documented in humans and laboratory rodents. This study utilized toll-like receptor 2 (TLR2), CD4, Langerin/CD207, and inducible nitric oxide synthase to characterize, for the first time, immune cells in the mucosal regio vestibularis of the S. coeruleoalba nasal cavity using confocal microscopy immunofluorescence techniques. The findings revealed scattered immune cells immunoreactive to the tested antibodies, present in both the epithelial tissue lining the nasal cavity vestibulum and the underlying connective tissue. This study enhances our comprehension of the immune system of cetaceans. RESEARCH HIGHLIGHTS: This study provides new insights into NALT in S. coeruleoalba. This research deepens the knowledge of the skin of cetaceans.

ORF virus causes tumor-promoting inflammation in sheep and goats.

ORF virus (ORFV) causes contagious ecthyma ("ORF"), a disease of sheep and goats characterized by lesions ranging from vesicles and pustules to atypical papilloma-like and angiomatous lesions in the skin and mucosae. The authors investigated the molecular factors leading to the ORF-associated atypical tumor-like changes. Fifteen lambs, 15 kids, and an adult ram clinically affected by natural ORFV infection were enrolled in the study and examined by several methods. ORFV was detected by viral culture or real-time polymerase chain reaction (RT-PCR) in the lesioned tissues and in the blood of the clinically affected sheep and goats. Surprisingly, ORFV was also detected in the blood of healthy goats from an affected herd. Microscopically, they found a pseudo-papillomatous proliferation of the epithelium, while the dermis and lamina propria were expanded by a proliferating neovascular component that highly expressed the viral vascular endothelial growth factor (vVEGF) and its host receptor vascular endothelial growth factor receptor 2 (VEGFR2). Immunohistochemistry, immunofluorescence, and in situ hybridization for mRNA showed that epidermal growth factor receptor (EGFR) was expressed in the fibrovascular component, in the infiltrating CD163+ macrophages, and in the basal stratum of the epidermis. Confocal immunofluorescence microscopy demonstrated that CD163+ macrophages were associated with VEGF and VEGFR2. Finally, they found by quantitative RT-PCR the overexpression of the interleukin-6 and VEGFR2 genes in the lesioned tissues. These findings suggest that ORFV activates an inflammatory reaction characterized by CD163+ macrophages expressing EGFR and VEGFR2, which might play an oncogenic role through synergistic action with vVEGF signaling.

Intimomedial tears of the aorta heal by smooth muscle cell-mediated fibrosis without atherosclerosis.

BACKGROUNDDisease of the aorta varies from atherosclerosis to aneurysms, with complications including rupture, dissection, and poorly characterized limited tears. We studied limited tears without any mural hematoma, termed intimomedial tears, to gain insight into aortic vulnerability to excessive wall stresses. Our premise is that minimal injuries in aortas with sufficient medial resilience to prevent tear progression correspond to initial mechanisms leading to complete structural failure in aortas with significantly compromised medial resilience.METHODSIntimomedial tears were macroscopically identified in 9 of 108 ascending aortas after surgery and analyzed by histology and immunofluorescence confocal microscopy.RESULTSNonhemorrhagic, nonatheromatous tears correlated with advanced aneurysmal disease and most lacked distinctive symptoms or radiological signs. Tears traversed the intima and part of the subjacent media, while the resultant defects were partially or completely filled with neointima characterized by differentiated smooth muscle cells, scattered leukocytes, dense fibrosis, and absent elastic laminae despite tropoelastin synthesis. Healed lesions contained organized fibrin at tear edges without evidence of plasma and erythrocyte extravasation or lipid accumulation.CONCLUSIONThese findings suggest a multiphasic model of aortic wall failure in which primary lesions of intimomedial tears either heal if the media is sufficiently resilient or progress as dissection or rupture by medial delamination and tear completion, respectively. Moreover, mural incorporation of thrombus and cellular responses to injury, two historically important concepts in atheroma pathogenesis, contribute to vessel wall repair with adequate conduit function, but even together are not sufficient to induce atherosclerosis.FUNDINGNIH (R01-HL146723, R01-HL168473) and Yale Department of Surgery.

Characterization of the First Secreted Sorting Nexin Identified in the Leishmania Protists.

Proteins of the sorting nexin (SNX) family present a modular structural architecture with a phox homology (PX) phosphoinositide (PI)-binding domain and additional PX structural domains, conferring to them a wide variety of vital eukaryotic cell's functions, from signal transduction to membrane deformation and cargo binding. Although SNXs are well studied in human and yeasts, they are poorly investigated in protists. Herein, is presented the characterization of the first SNX identified in Leishmania protozoan parasites encoded by the LdBPK_352470 gene. In silico secondary and tertiary structure prediction revealed a PX domain on the N-terminal half and a Bin/amphiphysin/Rvs (BAR) domain on the C-terminal half of this protein, with these features classifying it in the SNX-BAR subfamily of SNXs. We named the LdBPK_352470.1 gene product LdSNXi, as it is the first SNX identified in Leishmania (L.) donovani. Its expression was confirmed in L. donovani promastigotes under different cell cycle phases, and it was shown to be secreted in the extracellular medium. Using an in vitro lipid binding assay, it was demonstrated that recombinant (r) LdSNXi (rGST-LdSNXi) tagged with glutathione-S-transferase (GST) binds to the PtdIns3P and PtdIns4P PIs. Using a specific a-LdSNXi antibody and immunofluorescence confocal microscopy, the intracellular localization of endogenous LdSNXi was analyzed in L. donovani promastigotes and axenic amastigotes. Additionally, rLdSNXi tagged with enhanced green fluorescent protein (rLdSNXi-EGFP) was heterologously expressed in transfected HeLa cells and its localization was examined. All observed localizations suggest functions compatible with the postulated SNX identity of LdSNXi. Sequence, structure, and evolutionary analysis revealed high homology between LdSNXi and the human SNX2, while the investigation of protein-protein interactions based on STRING (v.11.5) predicted putative molecular partners of LdSNXi in Leishmania.

Stress-Induced Changes in Nucleocytoplasmic Localization of Crucial Factors in Gene Expression Regulation.

This study investigates the toxic effect of harmful materials, unfiltered by the placenta, on neonatal umbilical cord (UC) vessels, focusing on stress-induced adaptations in transcriptional and translational processes. It aims to analyze changes in pathways related to mRNA condensate formation, transcriptional regulation, and DNA damage response under maternal smoking-induced stress. UC vessels from neonates born to smoking (Sm) and nonsmoking mothers (Ctr) were examined. Immunofluorescence staining and confocal microscopy assessed the localization of key markers, including Transcription Complex Subunit 1 (CNOT1) and the largest subunit of RNA polymerase II enzyme (RPB1). Additionally, markers of DNA damage response, such as Poly(ADP-ribose) polymerase-1, were evaluated. In Sm samples, dissolution of CNOT1 granules in UC vessels was observed, potentially aiding stalled translation and enhancing transcription via RPB1 assembly and translocation. Control vessels showed predominant cytoplasmic RPB1 localization. Despite adaptive responses, Sm endothelial cells exhibited significant damage, indicated by markers like Poly(ADP-ribose) polymerase-1. Ex vivo metal treatment on control vessels mirrored Sm sample alterations, emphasizing marker roles in cell survival under toxic exposure. Maternal smoking induces specific molecular adaptations in UC vessels, affecting mRNA condensate formation, transcriptional regulation, and DNA damage response pathways. Understanding these intricate molecular mechanisms could inform interventions to improve neonatal health outcomes and mitigate adverse effects of toxic exposure during pregnancy.

Acute Rift Valley fever virus infection induces inflammatory cytokines and cell death in ex vivo rat brain slice culture.

Rift Valley fever virus (RVFV) is an emerging arboviral disease with pandemic potential. While infection is often self-limiting, a subset of individuals may develop late-onset encephalitis, accounting for up to 20 % of severe cases. Importantly, individuals displaying neurologic disease have up to a 53 % case fatality rate, yet the neuropathogenesis of RVFV infection remains understudied. In this study, we evaluated whether ex vivo postnatal rat brain slice cultures (BSCs) could be used to evaluate RVFV infection in the central nervous system. BSCs mounted an inflammatory response after slicing, which resolved over time, and they were viable in culture for at least 12 days. Infection of rat BSCs with pathogenic RVFV strain ZH501 induced tissue damage and apoptosis over 48 h. Viral replication in BSCs reached up to 1×107 p.f.u. equivalents/ml, depending on inoculation dose. Confocal immunofluorescent microscopy of cleared slices confirmed direct infection of neurons as well as activation of microglia and astrocytes. Further, RVFV-infected rat BSCs produced antiviral cytokines and chemokines, including MCP-1 and GRO/KC. This study demonstrates that rat BSCs support replication of RVFV for ex vivo studies of neuropathogenesis. This allows for continued and complementary investigation into RVFV infection in an ex vivo postnatal brain slice culture format.

Abstract 2711: A novel bispecific antibody to reprogram the immune suppressive GBM TME

Abstract GBM (glioblastoma) is the most common and aggressive primary brain tumor in adults. Immunotherapy is an emerging and promising treatment modality for GBM. Unfortunately, most immunotherapy trials have provided only modest benefit to GBM patients thus far. A significant barrier that limits efficacy of immunotherapy in GBM is the paucity of tumor infiltrating lymphocytes and abundance of immune suppressive myeloid cells. A critical step in enhancing the efficacy of immunotherapy for GBM requires approaches that enhance T cell infiltration and reprogram the myeloid cells to a more pro-inflammatory state. S100A4 is a major regulator of stemness, epithelial-mesenchymal transition, and immunosuppression in glioma stem cells and a critical regulator of immune suppressive myeloid and T cell phenotypes in GBM. Targeting S100A4 in either glioma cells or stromal cells is sufficient to reprogram the tumor immune landscape and extend survival of glioma bearing mice, indicating that S100A4 is a promising immunotherapy target. Since S100A4 has both intracellular and extracellular function as well as cytokine-like function, we developed a S100A4 blocking antibody. While the anti-S100A4 antibody blocks breast cancer metastasis efficiently, it did not cross the BBB. Hence, we generated a bispecific S100A4-TfR antibody (BsA) that allows robust BBB penetration. Using ELISA, confocal immunofluorescent microscopy, and multiplexed flow cytometry, our preliminary results suggest that systemic administration of the S100A4-TfR BsA results in accumulation of BsA in the brain and glioma tissue, increased T cell infiltration and immune remodeling. Furthermore, the BsA is taken up by glioma cells and accumulates intracellularly, which is correlated with decreased nuclear SOX2 expression. These results suggest that S100A4-TfR BsA is a promising agent to treat GBM to reprogram the GBM immune landscape and reduce stemness. We are currently performing single cell RNA-sequencing to elucidate molecular and cellular changes induced by the bispecific antibody treatment in different cell types and performing long term survival analyses to evaluate its therapeutic potential. Citation Format: Thomas Wong, Jia-Shiun Leu, Xuejun Fan, Reece Kang, Nourhan Abdelfattah, Fransisca Leonard, Ningyan Zhang, Kyuson Yun. A novel bispecific antibody to reprogram the immune suppressive GBM TME [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2711.

Abstract 6225: Tumor evolution through selection by ECM stiffness

Abstract In this study, we used experimental evolution to investigate the extent to which the mechanical stiffness of the tumor extracellular matrix (ECM) exerts selection pressure on a genetically diverse population of breast cancer cells. Polyacrylamide hydrogels were fabricated and conjugated with collagen type I to create the ECM with different stiffness. MDA-MB-231 cells were continuously cultured on the hydrogels of the same corresponding stiffness. Growth rate was determined at certain time points by harvesting and counting the cells. Abundance of clonal MDA-MB-231 cells labeled with heritable DNA barcodes underwent the same selection was identified with sequencing of DNA barcode tags. Cellular phenotypes were characterized through immunofluorescence staining and confocal microscopy. 3D migration of the cancer cells was examined using Boyden chamber assay. Cellular contractility was measured through traction force microscopy. Whole genome transcriptome sequencing (RNA-seq), assay for transposase-accessible chromatin with sequencing, and reduced representation bisulfite sequencing were performed for transcriptomic and epigenetic analysis. Sustained culture of MDA-MB-231 breast cancer cells overall three months on soft ECM resulted in an increased proliferation rate and beta1 integrin-dependent spreading area of the cells. Such changes did not occur in clonal cells with low genetic variation and did not occur in ancestral cells on stiff substrates. Barcode analysis revealed two distinct surviving populations post-genetic selection, with one demonstrating robust proliferation on both soft and stiff ECM, and the other exhibiting preferential adaptation to the soft matrix. The soft-selected populations exhibited nuclear localization of the transcriptional regulator yes-associated protein and less wrinkled nuclei compared with ancestral populations when both populations were grown on soft ECM. Aggressive migration was observed in the soft-selected populations, indicating a more malignant selected phenotype. The soft-selected cells showed increased RhoA activity and contractile tension on a soft substrate compared to the ancestral cells. RNA-seq analysis revealed systematic differences between ancestral, stiff-selected, and soft-selected populations corresponding to enriched biological processes related to cell adhesion and ECM organization. Significant differences in DNA methylation and chromatin accessibility were observed in soft selected populations which were correlated with changes in RNA expression. ECM stiffness exerts selection pressure on genetically variable cancer cell populations, selecting specific genetic variants in a cell population optimally adapted for that stiffness. Selected variants on soft ECM tend to be highly migratory, proliferative and generate high traction even on soft ECM. Our results highlight the importance of ECM stiffness-driven evolution in cancer development. Citation Format: Ting-Ching Wang, Suchitaa Sawhney, Daylin Morgan, Richa Rashmi, Marcos R. Estecio, Amy Brock, Irtisha Singh, Richard L. Bennett, Charles F. Baer, Jonathan D. Licht, Tanmay P. Lele. Tumor evolution through selection by ECM stiffness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6225.

Elevations in the Mitochondrial Matrix Protein Cyclophilin D Correlate With Reduced Parvalbumin Expression in the Prefrontal Cortex of Patients With Schizophrenia.

Cognitive deficits in schizophrenia are linked to dysfunctions of the dorsolateral prefrontal cortex (DLPFC), including alterations in parvalbumin (PV)-expressing interneurons (PVIs). Redox dysregulation and oxidative stress may represent convergence points in the pathology of schizophrenia, causing dysfunction of GABAergic interneurons and loss of PV. Here, we show that the mitochondrial matrix protein cyclophilin D (CypD), a critical initiator of the mitochondrial permeability transition pore (mPTP) and modulator of the intracellular redox state, is altered in PVIs in schizophrenia. Western blotting was used to measure CypD protein levels in postmortem DLPFC specimens of schizophrenic patients (n = 27) and matched comparison subjects with no known history of psychiatric or neurological disorders (n = 26). In a subset of this cohort, multilabel immunofluorescent confocal microscopy with unbiased stereological sampling methods were used to quantify (1) numbers of PVI across the cortical mantle (20 unaffected comparison, 14 schizophrenia) and (2) PV and CypD protein levels from PVIs in the cortical layers 2-4 (23 unaffected comparison, 18 schizophrenia). In schizophrenic patients, the overall number of PVIs in the DLPFC was not significantly altered, but in individual PVIs of layers 2-4 PV protein levels decreased along a superficial-to-deep gradient when compared to unaffected comparison subjects. These laminar-specific PVI alterations were reciprocally linked to significant CypD elevations both in PVIs and total DLPFC gray matter. Our findings support previously reported PVI anomalies in schizophrenia and suggest that CypD-mediated mPTP formation could be a potential contributor to PVI dysfunction in schizophrenia.

Human Adenovirus Entry and Early Events during Infection of Primary Murine Neurons: Immunofluorescence Studies In Vitro.

Human adenovirus (HAdV) is a common pathogen, which can lead to various clinical symptoms and-in some cases-central nervous system (CNS) dysfunctions, such as encephalitis and meningitis. Although the initial events of virus entry have already been identified in various cell types, the mechanism of neuronal uptake of adenoviruses is relatively little understood. The aim of this study was to investigate early events during adenoviral infection, in particular to determine the connection between cellular coxsackievirus and adenovirus receptor (CAR), clathrin, caveolin, and early endosomal proteins (EEA1 and Rab5) with the entry of HAdVs into primary murine neurons in vitro. An immunofluorescence assay and confocal microscopy analysis were carried out to determine HAdV4, 5, and 7 correlation with CAR, clathrin, caveolin, and early endosomal proteins in neurons. The quantification of Pearson's coefficient between CAR and HAdVs indicated that the HAdV4 and HAdV5 types correlated with CAR and that the correlation was more substantial for HAdV5. Inhibition of clathrin-mediated endocytosis using chlorpromazine limited the infection with HAdV, whereas inhibition of caveolin-mediated endocytosis did not affect virus entry. Thus, the entry of tested HAdV types into neurons was most likely associated with clathrin but not caveolin. It was also demonstrated that HAdVs correlate with the Rab proteins (EEA1, Rab5) present in early vesicles, and the observed differences in the manner of correlation depended on the serotype of the virus. With our research, we strove to expand knowledge regarding the mechanism of HAdV entry into neurons, which may be beneficial for developing potential therapeutics in the future.