Experimental Murine Model Research Articles

β3-Adrenergic receptor antagonism improves cardiac and vascular functions but did not modulate survival in a murine resuscitated septic shock model

BackgroundRecent findings suggest that β3-adrenergic receptors (β3-AR) could play a role in the hemodynamic regulation, but their function in septic shock remains unclear. This study investigates the modulation of β3-AR in an experimental murine model of resuscitated septic shock on in vivo hemodynamic, ex vivo vasoreactivity, inflammation and survival.MethodWild-type mice were used, undergoing cecal ligation and puncture (CLP) to induce septic shock, with SHAM as controls. Mice were treated with β3-AR agonist or antagonist three hours post-CLP, followed by resuscitation with fluids and antibiotics. Hemodynamic parameters were measured at 18 h following the surgery, and the expression of β-ARs in heart and aorta was assessed via immunostaining and western blot. Vascular reactivity was studied using myography, and inflammatory markers were analyzed through PCR and western blots. A 5-day survival study was conducted, documenting clinical severity scores and survival rates.Resultsβ3-AR was expressed in both endothelial and myocardial cells in healthy and septic mice. During septic shock model, β3-AR density on endothelial cells increased post-CLP, while β1- and β2-AR decreased or remained constant. β3-AR antagonist treatment improved hemodynamic parameters, increasing mean arterial pressure and cardiac index, unlike the agonist. Vascular reactivity to phenylephrine was enhanced in aortic rings from both β3-AR agonist and antagonist-treated mice. However, no significant differences in inducible NO synthase expression were observed among treated groups. Despite improved hemodynamic parameters with β3-AR antagonist treatment, survival rates in treated groups remained similar to CLP group.ConclusionsIn an experimental murine model of resuscitated septic shock, β3-AR is resistant to desensitization and its inhibition improves cardiac and vascular function without affecting the short-term survival.

Linsitinib inhibits IGF-1-induced cell proliferation and hyaluronic acid secretion by suppressing PI3K/Akt and ERK pathway in orbital fibroblasts from patients with thyroid-associated ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO), an autoimmune disorder of the retrobulbar tissue, is present in up to 50 percent of Graves's hyperthyroidism patients. Insulin-like growth factor 1 receptor (IGF-1R) has received attention as a target for the development of therapeutic agent for TAO. IGF-1R and TSHR (thyroid stimulating hormone receptor) interact with each other to form a physical or functional complex, further promoting the development of TAO. Linsitinib, OSI-906, is an inhibitor of IGF-1R and has been reported to inhibit cell proliferation of several tumor cells. Linsitinib has been receiving attention not only for its anticancer effect, but also for its anti-inflammatory effects. It has been reported that linsitinib reduces infiltration of inflammatory cells in orbital tissues, resulting in the reduction of muscle edema and adipose tissues in an experimental murine model for Graves' disease. In the current study, we investigated the issue of whether linsitinib inhibits the IGF-1-induced proliferation of orbital fibroblasts (OFs) via the suppression of phosphatidylinositol 3-kinase (PI3K) / Akt and extracellular signal-regulated kinase (ERK) pathway. Our results showed that pretreatment with linsitinib inhibited IGF-1-induced cell proliferation and hyaluronic acid secretion in the OFs of TAO patients. In addition, our results showed that pretreatment with linsitinib inhibited IGF-1-induced phosphorylation of IGF-1Rβ at Tyr1135, Akt at Ser473, and ERK in the OFs of patients with TAO. These results indicate that linsitinib inhibits IGF-1-induced cell proliferation and hyaluronic acid secretion in the OFs of TAO patients by suppressing the PI3K/Akt and ERK pathways, validating the use of linsitinib as a novel therapeutic agent for TAO.

Unveiling the Role of Resident Memory T Cells in Psoriasis.

Psoriasis is a chronic inflammatory skin disease characterized by periods of remission and relapse. In this pathology, keratinocytes, dendritic cells, and different subpopulations of T cells are critical to developing psoriatic lesions. Although current treatments can reduce symptoms, they reappear in previously injured areas months after stopping treatment. Evidence has pointed out that besides T helper 17 cells, other T cell subsets may be involved in relapses. This review focuses on the leading evidence linking resident memory T cells and P2X7 receptor to psoriasis' pathogenesis and their role in this pathology. Finally, we discuss some of the most widely used experimental murine models and novel strategies to investigate further the role of resident memory T cells in psoriasis.

Human liver stem cells and derived extracellular vesicles protect from sepsis-induced acute lung injury and restore bone marrow myelopoiesis in a murine model of sepsis

BackgroundSepsis is a condition with high mortality and morbidity, characterized by deregulation of the immune response against the pathogen. Current treatment strategies rely mainly on antibiotics and supportive care. However, there is growing interest in exploring cell-based therapies as complementary approaches. Human liver stem cells (HLSCs) are pluripotent cells of mesenchymal origin, showing some advantages compared to mesenchymal stem cells in terms of immunomodulatory properties. HSLC-derived extracellular vesicles (EVs) exhibited a superior efficacy profile compared to cells due to their potential to get through biological barriers and possibly to avoid tumorigenicity and showed to be effective in vivo and ex vivo models of liver and kidney disease. The potential of HLSCs and their EVs in recovering damage to distal organs due to sepsis other than the kidney remains unknown. This study aimed to investigate the therapeutic potential of the intravenous administration of HSLCs or HSLCs-derived EVs in a murine model of sepsis.ResultsSepsis was induced by caecal ligation and puncture (CLP) on C57/BL6 mice. After CLP, mice were assigned to receive either normal saline, HLSCs or their EVs and compared to a sham group which underwent only laparotomy. Survival, persistence of bacteraemia, lung function evaluation, histology and bone marrow analysis were performed. Administration of HLSCs or HLSC-EVs resulted in improved bacterial clearance and lung function in terms of lung elastance and oedema. Naïve murine hematopoietic progenitors in bone marrow were enhanced after treatment as well. Administration of HLSCs and HLSC-EVs after CLP to significantly improved survival.ConclusionsTreatment with HLSCs or HLSC-derived EVs was effective in improving acute lung injury, dysmyelopoiesis and ultimately survival in this experimental murine model of lethal sepsis.

Chondroprotective functions of neutrophil-derived extracellular vesicles by promoting the production of secreted frizzled-related protein 5 in cartilage

BackgroundOsteoarthritis (OA) is the most common degenerative joint disease characterized by cartilage degradation and various degrees of inflammation in the synovium. Growing evidence highlights that neutrophil extracellular vesicles (EVs) play a protective role in arthritic joints by promoting the resolution of inflammation and the synthesis of proteoglycans in cartilage. However, this homeostatic function is dependent on the activation state of neutrophils and the surrounding environment/tissues. Hence, we explored the chondroprotective functions of neutrophil-derived EVs under different stimulation conditions and the underlying molecular mechanism.MethodsHuman blood-derived neutrophils, murine bone marrow-derived neutrophils, C-28I2 cells and primary chondrocytes were used. Neutrophils were stimulated with different cytokines, and their EVs were isolated for chondrocyte stimulation and further subjected to RNA-sequencing analysis. Two experimental murine OA models were used, and the treatment was performed by intraarticular injections.ResultsConditioned medium from neutrophils stimulated with TGF-β (N-β) had the greatest inhibitory effect on the expression of catabolic factors in stimulated chondrocytes. These protective effects were not impaired when conditioned medium of N-β from AnxA1-deficient mice was used. Consistent with these results, EVs isolated from N-β significantly reduced the expression of catabolic factors in stimulated chondrocytes. Bulk RNA-seq analysis revealed that secreted frizzled-related protein 5 (SFRP5) is upregulated in N-β-EV-stimulated chondrocytes. Furthermore, recombinant SFRP5 treatment significantly reduced the expression of catabolic factors in vitro and catabolic process in experimental murine OA models.ConclusionsThe current study emphasizes the potential therapeutic application of neutrophils in OA and provides new knowledge on the molecular mechanisms underlying their function.

Experimental murine models of interstitial cystitis/bladder pain syndrome: A review.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic enigmatic disease of the urinary bladder characterized by persistent bladder/pelvic pain in conjunction with lower urinary tract symptoms. IC/BPS is categorized as either Hunner-type IC (HIC) or BPS based on the presence/absence of the Hunner lesion, a reddish mucosal lesion in the bladder. HIC and BPS present with similar symptoms, however, the etiologies are completely different. Recent evidence suggests that HIC is an immune-mediated inflammatory disease of the urinary bladder. In contrast, BPS, other forms of HIC lacking Hunner lesions, is a minimally inflamed condition comprising various clinical phenotypes. Based on this evidence, basic research into IC/BPS has shifted to target each subtype of IC/BPS. Today, experimental murine models of autoimmune cystitis are used for HIC research, whereas models related to neurophysiological and psychosocial dysfunctions have been developed for BPS research. This emerging concept of a subtype-tailored approach may contribute to a better understanding of the full picture of IC/BPS, thereby improving current clinical management strategies and the development of novel therapies.

Sucupira oleoresin-based doxorubicin pH-sensitive nanoemulsions: A potential nanomedicine approach to enhance the safety profile and anticancer activity

Plant derivatives-based nanocarriers as a matrix for delivery of chemotherapeutic drugs are capable of enhancing conventional antitumor drugs' cytotoxicity against cancer cells. Bearing in mind that the use of doxorubicin-loaded sucupira oleoresin nanoemulsions can play a significant role in increasing specificity, and efficacy, whilst improvement the chemotherapy safety profile we hypothesize that this nanocarrier may be a powerful alternative strategy for cancer therapy. Thus, the aimed of this research was to prepare and characterize this nanoemulsion, as well as assess the antitumor activity and toxicity in an experimental murine breast cancer model. In the first place, the phytocompounds present in sucupira oleoresin were identified by Liquid chromatography docked Orbitrap-mass spectrometer. Afterward, the doxorubicin-loaded sucupira oleoresin nanoemulsion was prepared using hot homogenization followed by ultrasonic calibration method. Then, nanoemulsions were characterized in terms of mean diameter, polydispersity, zeta potential, entrapment efficiency, and kinetic release. Toxicity and in vivo antitumor activity were also evaluated. The bioactive compounds found in sucupira oleoresin were terpenes, especially sesqui- and vouacapan diterpenes, which have a wide range of biological activities. The sucupira oleoresin nanoemulsions with or without doxorubicin were stable under refrigeration, monodisperse featuring an average size of around 60 nm, and negative zeta potential. High encapsulation doxorubicin content (98 %) was related to the ion-pair formed between doxorubicin and oleic acid. Stability studies in different biological media suggest that the proposed formulation can remain stable after intravenous administration. Meanwhile, the release outcomes indicate a pH-responsive profile. In vivo studies show that nanoemulsions with a combination of doxorubicin and oleoresin exhibit better efficacy and reduced effects on organs of potential toxicity such as the liver, heart, and intestine as well as less body weight loss likened to the free drug. Owing to promising results, we highlight that the doxorubicin-loaded sucupira oleoresin nanoemulsion could supply gain better insights for the combined breast cancer treatment.

In situ assessment of neuroinflammatory cytokines in different stages of ovine natural prion disease.

According to the neuroinflammatory hypothesis, a cytokine-mediated host innate immune response may be involved in the mechanisms that contribute to the process of neurodegeneration. Specifically, regarding prion diseases, some experimental murine models have evidenced an altered profile of inflammatory intermediaries. However, the local inflammatory response has rarely been assessed, and never in tissues from different natural models throughout the progression of neurodegeneration. The aim of this study was to use immunohistochemistry (IHC) to in situ assess the temporal protein expression of several cytokines in the cerebellum of sheep suffering from various clinical stages of scrapie. Clear changes in the expression of most of the assessed markers were observed in the affected sheep compared to the healthy control sheep, and from different stages. In summary, this preliminary IHC study focusing in the Purkinje cell layer changes demonstrate that all cytokines or respective receptors studied (IL-1, IL-1R, IL-2R, IL-6, IL-10R, and TNFαR) except for IFNγR are disease-associated signaling proteins showing an increase or decrease in relation to the progression of clinical disease. In the future, this study will be extended to other inflammatory mediators and brain regions, focusing in particular on the release of these inflammatory mediators by astroglial and microglial populations.

A murine experimental model of the pulmonary thrombotic effect induced by the venom of the snake Bothrops lanceolatus.

The venom of Bothrops lanceolatus, a viperid species endemic to the Lesser Antillean Island of Martinique, induces thrombosis in a number of patients. Previous clinical observations indicate that thrombotic events are more common in patients bitten by juvenile specimens. There is a need to develop an experimental model of this effect in order to study the mechanisms involved. The venoms of juvenile and adult specimens of B. lanceolatus were compared by (a) describing their proteome, (b) assessing their ability to induce thrombosis in a mouse model, and (c) evaluating their in vitro procoagulant activity and in vivo hemostasis alterations. Venom proteomes of juvenile and adult specimens were highly similar, albeit showing some differences. When injected by the intraperitoneal (i.p.) route, the venom of juvenile specimens induced the formation of abundant thrombi in the pulmonary vasculature, whereas this effect was less frequent in the case of adult venom. Thrombosis was not abrogated by the metalloproteinase inhibitor Batimastat. Both venoms showed a weak in vitro procoagulant effect on citrated mouse plasma and bovine fibrinogen. When administered intravenously (i.v.) venoms did not affect classical clotting tests (prothrombin time and activated partial thromboplastin time) but caused a partial drop in fibrinogen concentration. The venom of juvenile specimens induced partial alterations in some rotational thromboelastometry parameters after i.v. injection. When venoms were administered i.p., only minor alterations in classical clotting tests were observed with juvenile venom, and no changes occurred for either venom in rotational thromboelastometry parameters. Both juvenile and adult venoms induced a marked thrombocytopenia after i.p. injection. An experimental model of the thrombotic effect induced by B. lanceolatus venom was developed. This effect is more pronounced in the case of venom of juvenile specimens, despite the observation that juvenile and adult venom proteomes are similar. Adult and juvenile venoms do not induce a consumption coagulopathy characteristic of other Bothrops sp venoms. Both venoms induce a conspicuous thrombocytopenia. This experimental model reproduces the main clinical findings described in these envenomings and should be useful to understand the mechanisms of the thrombotic effect.

Could the Adoptive Transfer of Memory Lymphocytes be an Alternative Treatment for Acinetobacter baumannii Infections?

We evaluated the efficacy of the adoptive transfer of memory B, CD4+, and CD8+ T lymphocytes compared with sulbactam and tigecycline in an experimental murine pneumonia model by two multidrug-resistant Acinetobacter baumannii strains, colistin-susceptible AbCS01 and colistin-resistant AbCR17. Pharmacodynamically optimized antimicrobial dosages were administered for 72 h, and intravenous administration of 2 × 106 of each of the memory cells in a single dose 30 min post-infection. Bacterial lung and blood counts and mortality rates were analyzed. Results showed that a single dose of memory B or CD4+ T cells was as effective as sulbactam in terms of bacterial clearance from the lungs and blood compared with the untreated mice or the tigecycline-treated mice inoculated with the AbCS01 strain. In the pneumonia model by AbCR17, a single dose of memory B or CD4+ T cells also reduced the bacterial load in the lungs compared with both antibiotic groups and was more efficacious than tigecycline in terms of blood clearance. Regarding survival, the adoptive transfer of memory B or CD4+ T cells was as effective as three days of sulbactam treatment for both strains. These data suggest that adoptive memory cell transfer could be a new effective treatment of multidrug-resistant A. baumannii infections.

Intestinal anti-inflammatory effect of a plant sterol food supplement in experimental murine colitis and cell co-culture models

Intestinal anti-inflammatory effect of a plant sterol food supplement in experimental murine colitis and cell co-culture models

Oral administration of Lactobacillus delbrueckii UFV-H2b20 protects mice against Aspergillus fumigatus lung infection.

Probiotics provide therapeutic benefits not only in the gut but also other mucosal organs, including the lungs. To evaluate the effects of the probiotic strain L. delbrueckii UFV-H2b20 oral administration in an experimental murine model of A. fumigatus pulmonary infection. BALB/c mice were associated with L. delbrueckii and infected with Aspergillus fumigatus and compared with non-associated group. We investigated survival, respiratory mechanics, histopathology, colony forming units, cytokines in bronchoalveolar lavage, IgA in feces, efferocytosis, production of reactive oxygen species and the cell population in the mesenteric lymph nodes. L. delbrueckii induces tolerogenic dendritic cells, IL-10+macrophages and FoxP3+regulatory T cells in mesenteric lymph nodes and increased IgA levels in feces; after infection with A. fumigatus, increased survival and decreased fungal burden. There was decreased lung vascular permeability without changes in the leukocyte profile. There was enhanced neutrophilic response and increased macrophage efferocytosis. L. delbrueckii-treated mice displayed more of FoxP3+Treg cells, TGF-β and IL-10 levels in lungs, and concomitant decreased IL-1β, IL-17A, and CXCL1 production. Uur results indicate that L. delbrueckii UFV H2b20 ingestion improves immune responses, controlling pulmonary A. fumigatus infection. L. delbrueckii seems to play a role in pathogenesis control by promoting immune regulation.

SIRPα engagement regulates ILC2 effector function and alleviates airway hyperreactivity via modulating energy metabolism

Group-2 innate lymphoid cells (ILC2) are part of a growing family of innate lymphocytes known for their crucial role in both the development and exacerbation of allergic asthma. The activation and function of ILC2s are regulated by various activating and inhibitory molecules, with their balance determining the severity of allergic responses. In this study, we aim to elucidate the critical role of the suppressor molecule signal regulatory protein alpha (SIRPα), which interacts with CD47, in controlling ILC2-mediated airway hyperreactivity (AHR). Our data indicate that activated ILC2s upregulate the expression of SIRPα, and the interaction between SIRPα and CD47 effectively suppresses both ILC2 proliferation and effector function. To evaluate the function of SIRPα in ILC2-mediated AHR, we combined multiple approaches including genetically modified mouse models and adoptive transfer experiments in murine models of allergen-induced AHR. Our findings suggest that the absence of SIRPα leads to the overactivation of ILC2s. Conversely, engagement of SIRPα with CD47 reduces ILC2 cytokine production and effectively regulates ILC2-dependent AHR. Furthermore, the SIRPα-CD47 axis modulates mitochondrial metabolism through the JAK/STAT and ERK/MAPK signaling pathways, thereby regulating NF-κB activity and the production of type 2 cytokines. Additionally, our studies have revealed that SIRPα is inducible and expressed on human ILC2s, and administration of human CD47-Fc effectively suppresses the effector function and cytokine production. Moreover, administering human CD47-Fc to humanized ILC2 mice effectively alleviates AHR and lung inflammation. These findings highlight the promising therapeutic potential of targeting the SIRPα-CD47 axis in the treatment of ILC2-dependent allergic asthma.

Murine kidney transplant outcome is best measured by transdermal glomerular filtration rate

Mouse kidney transplantation provides a powerful preclinical model for the study of kidney transplant alloimmunity. However, accurate measurement of graft function is difficult because of the inaccuracy of traditional surrogate markers serum creatinine and urea. We report the use of transdermal glomerular filtration rate measurement under the experimental conditions of unilateral nephrectomy and allogeneic kidney transplantation. Our findings demonstrate that transdermal glomerular filtration rate measurement is easy to perform, reproducible, and has more interexperimental consistency than serum creatinine or urea measurements. Most importantly, it significantly reduces the numbers of experimental animals required to detect subtle and yet clinically relevant differences in kidney function as often is the case in experimental murine kidney transplantation models.

An Experimental Murine Model to Study Lipoatrophia Semicircularis.

Lipoatrophia semicircularis is a benign pathology characterized by subcutaneous tissue atrophy that affects the skin and related structures. Its etiology remains unclear; however, in the recent few years, it has been proposed that electrostatic charges could be a potential factor. Based on this hypothesis, the aim of this work is to study the cause-effect relation between electrostatic energy and LS, providing insights into the molecular mechanisms. For this purpose, an experimental murine model was created using obese mice. One group served as a control and the other groups involved charging clothes with varying connections to the ground: through the skin, through the clothes or not connected to the ground). Skin biopsies showed that the most significant lesions, including lipophagic granulomas with inflammatory infiltrate, were found in the first group (connected to the ground through the skin). Lipophagic reactions without an inflammatory infiltrate were observed in the other groups subjected to electrical discharges. In the control mice, no histological changes were observed. Oxidative processes were also measured in lower limbs tissue. Malondialdehyde levels significantly increased in the lower limbs after electrostatic discharges. However, the presence of ground through a wire attached to highly conductive clothes around the thigh significantly reduced the effect of electrostatic charges on lipid peroxidation. To our knowledge, this is the first study in which an experimental model has been used to reproduce LS induced by electrostatic energy, suggesting a cause-effect relationship between electrostatic charge and discharge with fat tissue lesion.

Ascorbyl palmitate (ASC16) as a potential inhibitor of toxicity induced by Crotalus durissus terrificus venom

It is well known that C. d. terrificus venom causes pathophysiological effects such as neuropathies, coagulopathies, and even death. Previous studies have reported that ASC16 can interact with monomeric phospholipases A2 from the venom of various snake species (e.g., Vipera russelli and Echis carinatus). As a result, ASC16 has been proposed as an inhibitor of the toxic effects induced by the heterodimeric complex (crotoxin) and other components of the venom of C. d. terrificus. To investigate this further, in silico studies were designed using the crotoxin (CTX) protein complex as a model, and experimental assays were conducted to evaluate the inhibitory effect of ASC16 on CTX, as well as on other venom enzymes such as thrombin-like enzyme (TLE), phosphodiesterase (PDE) and l-aminoxidase (LAAO). For in vitro assays, specific substrates were used, and lethal activity was measured over 48 h using an in vivo murine experimental model (CF01). In silico studies have indicated that the hydrophilic portion of ASC16 adopts a stable conformation while interacting with the catalytic site of crotoxin. At the highest concentrations, ASC16 significantly inhibited the activities of PLA2 (40.89 ± 0.09 %), TLE (11.03 ± 0.69 %), PDE (51.33 ± 2.83 %), and LAAO (56.79 ± 2.91 %). Furthermore, ASC16 neutralized the 2 LD50 lethality of crotalic venom. These findings lay the groundwork for designing promising adjuvants that can facilitate the incorporation of a larger quantity of proteins in immunization schemes. Consequently, this approach aims to achieve higher antibody titers, reduce the number of required immunizations, and minimize local damage in the producer animal.

Postoperative Ileus Murine Model.

Most patients experience postoperative ileus (POI) after surgery, which is associated with increased morbidity, mortality, and hospitalization time. POI is a consequence of mechanical damage during surgery, resulting in disruption of motility in the gastrointestinal tract. The mechanisms of POI are related to aberrant neuronal sensitivity, impaired epithelial barrier function, and increased local inflammation. However, the details remain enigmatic. Therefore, experimental murine models are crucial for elucidating the pathophysiology and mechanism of POI injury and for the development of novel therapies. Here, we introduce a murine model of POI generated via intestinal manipulation (IM) that is similar to clinical surgery; this is achieved by mechanical damage to the small intestine by massaging the abdomen 1-3 times with a cotton swab. IM delayed gastrointestinal transit 24 h after surgery, as assessed by FITC-dextran gavage and fluorescence detection of the segmental digestive tract. Moreover, tissue swelling of the submucosa and immune cell infiltration were investigated by hematoxylin and eosin staining and flow cytometry. Proper pressure of the IM and a hyperemic effect on the intestine are critical for the procedure. This murine model of POI can be utilized to study the mechanisms of intestinal damage and recovery after abdominal surgery.

Soluble Fibrinogen–Like Protein 2 Downregulation and Th17/Treg Imbalance in a Taurocholate‐Induced Murine Experimental Model of Severe Acute Pancreatitis

ABSTRACTBackgroundSevere acute pancreatitis (SAP) is associated with tremendous systemic inflammation, T‐helper 17 (Th17) cells, and regulatory T (Treg) cells play an essential role in the inflammatory responses. Meanwhile, soluble fibrinogen–like protein 2 (Sfgl2) is a critical immunosuppressive effector cytokine of Treg cells and modulates immune responses. However, the impact of SAP induction on Sfgl2 expression and the role of Sfgl2 in immunomodulation under SAP conditions are largely unknown.MethodsA taurocholate‐induced mouse SAP model was established. The ratios of CD4+CD25+Foxp3+ Treg cells or CD4+IL‐17+ Th17 cells in blood and pancreatic tissues as well as surface expression of CD80, CD86, and major histocompatibility complex class II (MHC‐II) were determined by flow cytometry. Gene mRNA expression was determined by qPCR. Serum amylase and soluble factors were quantitated by commercial kits. Bone marrow–derived dendritic cells (DCs) were generated, and NF‐κB/p65 translocation was measured by immunofluorescence staining.ResultsSAP induction in mice decreased the Th17/Treg ratio in the pancreatic tissue and increased the Th17/Treg ratio in the peripheral blood. In addition, SAP was associated with a reduced level of Sfgl2 in the pancreatic tissue and blood: higher levels of serum IL‐17, IL‐2, IFN‐α, and TNF‐α, and lower levels of serum IL‐4 and IL‐10. Furthermore, the SAP‐induced reduction in Sfgl2 expression was accompanied by dysregulated maturation of bone marrow–derived DCs.ConclusionsSAP causes reduced Sfgl2 expression and Th17/Treg imbalance, thus providing critical insights for the development of Sfgl2‐ and Th17/Treg balance‐targeted immunotherapies for patients with SAP.

FPR1, as a Potential Biomarker of Diagnosis and Infliximab Therapy Responses for Crohn's Disease, is Related to Disease Activity, Inflammation and Macrophage Polarization.

Crohn's disease (CD) represents a multifaceted inflammatory gastrointestinal condition, with a profound significance placed on unraveling its molecular pathways to enhance both diagnostic capabilities and therapeutic interventions. This study focused on identifying a robust macrophage-related signatures (MacroSig) for diagnosing CD, emphasizing the role of FPR1 in macrophage polarization and its implications in CD. Expression profiles from intestinal biopsies and macrophages of 1804 CD patients were retrieved from the Gene Expression Omnibus (GEO). Utilizing CIBERSORTx, differential expression analysis, and weighted correlation network analysis to to identify macrophage-related genes (MRGs). By unsupervised clustering, distinct clusters of CD were identified. Potential biomarkers were identified via using four machine learning algorithms, leading to the establishment of MacroSig which combines insights from 12 machine learning algorithms. Furthermore, the expression of FPR1 was verified in intestinal biopsies of CD patients and two murine experimental colitis models. Finally, we further explored the role of FPR1 in macrophage polarization through single-cell analysis as well as through the study of RAW264.7 cells and peritoneal macrophages. Two distinct clusters with differential levels of macrophage infiltration and inflammation were identified. The MacroSig, which included FPR1 and LILRB2, exhibited high diagnostic accuracy and outperformed existing biomarkers and signatures. Clinical analysis demonstrated a strong correlation of FPR1 with disease activity, endoscopic inflammation status, and response to infliximab treatment. The expression levels of FPR1 were validated in our CD cohort by immunohistochemistry and confirmed in two colitis mouse models. Single-cell analysis indicated that FPR1 is predominantly expressed in macrophages and monocytes. In vitro studies demonstrated that FPR1 was upregulated in M1 macrophages, and its activation promoted M1 polarization. We developed a promising diagnostic signature for CD, and targeting FPR1 to modulate macrophage polarization may represent a novel therapeutic strategy.

The effect of ageing in an experimental model of myocardial infarction in senescent mice

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Funded by the Spanish Ministry of Science and Innovation (ISCIII) PI19/01714 and PI22/1083 co-financed by the European Regional Development Fund (ERDF), and by the Generalitat Valenciana. Introduction The murine experimental model of myocardial infarction allows the study of underlying molecular and cellular mechanisms in cardiac function. Specifically, intercellular communication mediated by small non-coding RNAs, microRNAs (miRNAs) which can modulate gene expression post-transcriptionally, is one of the fundamental processes in the investigation of this pathology that needs to be explored in animal models. The Senescence-Accelerated Mouse Prone (SAMP8) and its control strain SAM Resistant (SAMR1) have been established as an experimental model to study ageing-associated vascular dysfunction but scarcely used in epigenetic studies. Purpose To assess the suitability of the SAMR1/SAMP8 ageing model for the study of miRNAs expression following an acute myocardial infarction (AMI), and to determine the optimal time after AMI to measure circulating miRNA levels. Methods Six-month old male and female SAMR1 and SAMP8 (n = 6 per group) underwent AMI by ligation of the left anterior descending coronary artery and sham surgery. All interventions were performed under full anesthesia and analgesia. Animals were euthanized at different times (1h, 4h and 24h) and blood were collected. AMI was confirmed by immediate discoloration of the left ventricle, by ST segment elevation in the electrocardiogram, and by triphenyltetrazolium chloride staining of the non-ischemic areas of heart sections. RNA was obtained from non-hemolyzed serum. Circulating miRNA previously considered as possible AMI biomarkers, miR-1-3p, miR-133-3p, miR-208-3p and miR-499-5p, were measured by qRT-PCR. Results are shown as mean ± SEM of the relative expression (2-ΔΔCt), using U6 snRNA as endogenous control. p-values were calculated using ANOVA and statistical significance was considered when p<0.05. The investigation complies with ethical standards and was approved by the Ethics Committee for Animal Welfare of University of Valencia (2020/VSC/PEA/0128). Results miR-1-3p, miR-133-3p, miR-208-3p and miR-499-5p were significantly increased 4 hours after AMI when compared with sham group in both SAMR1 and SAMP8. It was noteworthy that only with the sham surgery all biomarkers exhibited a time-dependent decrease (p < 0.05). While miR-1-3p rose at 4 hours after AMI (6.46 ± 1.32, p<0.001), miR-133-3p, miR-208-3p and miR-499-5p showed a time-dependent upregulation, being maximal at 24 hours after AMI (p < 0.001 vs. sham group). When comparing samples from SAMR1 and SAMP8, the expression of the analyzed miRNAs was higher in SAMP8 4 hours after AMI (p < 0.05), to a greater extent than in SAMR1. Conclusion Through the use of the SAMR1/SAMP8 mice model, the impact of ageing on miRNA expression following an AMI was notably confirmed, particularly at 4 hours after AMI. Consequently, this study proposes the SAMR1/SAMP8 mouse model as suitable for studying the effects of both AMI and ageing on circulating miRNA expression.