Experimental Murine Model Research Articles

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.

Abstract IA001: Cellular and molecular underpinnings of field cancerization and progression to invasive carcinoma in bladder cancer

Abstract Two major concerns in clinical management of bladder cancer are: (i) the high frequency of recurrence; and (ii) the morbidity and mortality associated with progression to invasive disease (invasive urothelial carcinoma, IUC). To gain a better understanding of these issues we have used the murine BBN (N-butyl-N-(4-hydroxylbutyl) nitrosamine) experimental model of bladder cancer, as well as tissue samples from normal human bladders and from bladders of patients at various stages of bladder cancer formation and progression. We have documented in the BBN model a CIS-like stage, prior to invasion, in which clonal expansion from a single cell occupies a significant fraction of the urothelium. With this experimental murine model alongside observations from patient samples and data from The Cancer Genome Atlas (TCGA), we have ascertained that a key event in progression of pre-invasive to invasive carcinoma is the silencing of the Sonic hedgehog (Shh) member of the Hedgehog gene family. Silencing-associated invasion appears due to the loss of SHH-induced differentiation signals, which when present slow growth and block invasion. Despite the consistent loss of SHH expression in invasive murine and human bladder cancers, however, TCGA data show few if any mutations in the SHH gene of IUC cells. Instead, our studies of patient samples and cancer-derived cell lines reveal that the loss of SHH expression is due to tighter chromosomal packaging and reduced accessibility of a bladder-specific enhancer located ~200 kb upstream of the SHH transcription start site. We have found that inhibitors of certain chromatin-modifying enzymes, including an FDA-approved drug, are capable of re-activating SHH expression, and are testing the possibility that these drugs may block or reverse invasion of urothelial carcinoma. The high frequency of bladder cancer recurrence is thought to result from field cancerization, that is, an increased probability of new cancer formation in apparent normal urothelium, even after successful resection of the primary tumor. To understand the basis for this field effect, we compared apparent normal urothelium from cancer patients to truly normal urothelium from bladders never affected by malignancy. We found that apparent normal samples display a dramatically increased basal character in transcriptomic assays, using as a metric of basalization an index constructed from single cell RNA-seq analysis of normal urothelium. We and others have demonstrated previously by lineage tracing and cell ablation studies in the BBN model that IUC arises from basal stem cells of the urothelium. The basalization of urothelium distant from the primary tumor that we observe thus may account for the increased probability of cancer formation in the apparent normal urothelium of bladder cancer patients. We have found that several FDA-approved antagonists of EGF signaling can reverse this basalization in the mouse BBN model as well as in organoid culture, and that these drugs dramatically reduce the formation of tumors following BBN exposure. Citation Format: Philip A. Beachy, Kris B. Prado, Aaron M. Kershner, Karim Mrouj, Bernard M. Kiss, Hua Dong, Ozgu Aydogdu, Ye Tian, Joseph C. Liao. Cellular and molecular underpinnings of field cancerization and progression to invasive carcinoma in bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr IA001.

Enhanced Therapeutic Effects of Human Mesenchymal stem Cells Transduced with Secreted Klotho in a Murine Experimental Autoimmune Encephalomyelitis Model.

Treatment of multiple sclerosis (MS) remains a major challenge. The aim of this study was to evaluate the therapeutic potential of mesenchymal stem cells (MSCs) engineered with secreted Klotho (SKL) in an experimental autoimmune encephalomyelitis (EAE) mouse model of MS. EAE was induced in mice. MSCs or MSCs engineered with SKL (SKL-MSCs) were administered to EAE mice at the onset of disease. Hematoxylin-eosin and luxol fast blue staining were performed to evaluate histopathological changes. Expression of pro-inflammatory (TNF-α, IFN-γ, and IL-17) and anti-inflammatory (IL-10) cytokines was determined in the spinal cord using real-time PCR. Spinal cords were then processed for immunohistochemistry of the aforementioned cytokines. The frequencies of Th1, Th17, and regulatory T (Treg) cells were evaluated by flow cytometry of the spleen. The results showed that SKL-MSCs decreased clinical scores and reduced demyelination and inflammatory infiltration in the spinal cord more significantly than MSCs. Compared to MSCs, SKL-MSCs also exhibited a more profound capability of decreasing expression of TNF-α, IFN-γ, and IL-17 and increasing expression of IL-10 in the spinal cord with an enhanced homing to the inflamed tissue. Moreover, SKL-MSCs decreased the frequencies of Th1 and Th17 cells and increased the frequency of Treg cells in the spleen more potently than MSCs. Taken together, these findings demonstrate that SKL overexpression enhances the therapeutic potential of MSCs, as evidenced by significantly improved disease severity, decreased inflammation and tissue damage in the spinal cord, and a promoted shift in the Th17/Treg balance towards the anti-inflammatory Treg side in the EAE mice.

Novel 5-HT7 receptor antagonists modulate intestinal immune responses and reduce severity of colitis.

Inflammatory bowel disease (IBD) encompasses several debilitating chronic gastrointestinal (GI) inflammatory disorders, including Crohn's disease and ulcerative colitis. In both conditions, mucosal inflammation is a key clinical presentation associated with altered serotonin (5-hydroxytryptamine or 5-HT) signaling. This altered 5-HT signaling is also found across various animal models of colitis. Of the 14 known receptor subtypes, 5-HT receptor type 7 (5-HT7) is one of the most recently discovered. We previously reported that blocking 5-HT signaling with either a selective 5-HT7 receptor antagonist (SB-269970) or genetic ablation alleviated intestinal inflammation in murine experimental models of colitis. Here, we developed novel antagonists, namely, MC-170073 and MC-230078, which target 5-HT7 receptors with high selectivity. We also investigated the in vivo efficacy of these antagonists in experimental colitis by using dextran sulfate sodium (DSS) and the transfer of CD4+CD45RBhigh T cells to induce intestinal inflammation. Inhibition of 5-HT7 receptor signaling with the antagonists, MC-170073 and MC-230078, ameliorated intestinal inflammation in both acute and chronic colitis models, which was accompanied by lower histopathological damage and diminished levels of proinflammatory cytokines compared with vehicle-treated controls. Together, the data reveal that the pharmacological inhibition of 5-HT7 receptors by these selective antagonists ameliorates the severity of colitis across various experimental models and may, in the future, serve as a potential treatment option for patients with IBD. In addition, these findings support that 5-HT7 is a viable therapeutic target for IBD.NEW & NOTEWORTHY This study demonstrates that the novel highly selective 5-HT7 receptor antagonists, MC-170073 and MC-230078, significantly alleviated the severity of colitis across models of experimental colitis. These findings suggest that inhibition of 5-HT7 receptor signaling by these new antagonists may serve as an alternative mode of treatment to diminish symptomology in those with inflammatory bowel disease.

The Immunomodulatory Effects and Mechanism of Wogonin for the Treatment of Multiple Sclerosis in a Murine Experimental Autoimmune Encephalomyelitis Model

Abstract Multiple sclerosis (MS) stands out as the most prevalent inflammatory autoimmune disease affecting the central nervous system (CNS). The primary driver of MS involves myelin-specific autoreactive T cells that infiltrate the CNS, initiating attacks on myelinated axons and triggering neuroinflammatory responses. Wogonin, a flavonoid, exhibits pharmacologic properties, including neuroprotective, anticancer, antiviral, anti-inflammatory, and anti-oxyradical effects. Our study demonstrates that wogonin significantly suppresses TLR-dependent DC activation, migration, and the expression of adhesion molecules in LPS-stimulated bone marrow-derived dendritic cells (BMDCs). In the experimental autoimmune encephalomyelitis (EAE) model, preventive treatment with wogonin led to reduced lymphocyte infiltration, prevented demyelination in the CNS, and consequently mitigated disease symptoms. Moreover, wogonin suppressed the release of neuroinflammatory cytokines by activated microglial cells, Th1 cells, Th2 cells, and Th17 cells in vitro and exerted therapeutic effects on attenuating the disease severity in the early disease onset stage and late disease progression stage. Our study suggests that wogonin treatment may not only prevent the onset of MS by inhibiting DC activation and migration but also potentially ameliorate the progression of MS by reducing neuroinflammation. This opens avenues for the development of new approaches in the therapy of autoimmune diseases.

Efficacy of Tamoxifen Metabolites in Combination with Colistin and Tigecycline in Experimental Murine Models of Escherichia coli and Acinetobacter baumannii.

This study aimed to evaluate the potential of tamoxifen and N-desmethyltamoxifen metabolites as therapeutic agents against multidrug-resistant Escherichia coli and Acinetobacter baumannii, using a repurposing approach to shorten the time required to obtain a new effective treatment against multidrug-resistant bacterial infections. Characterisation and virulence studies were conducted on E. coli (colistin-susceptible C1-7-LE and colistin-resistant MCR-1+) and A. baumannii (tigecycline-susceptible Ab#9 and tigecycline-resistant Ab#186) strains. The efficacy of the metabolite mix (33.3% each) and N-desmethyltamoxifen in combination with colistimethate sodium (CMS) or tigecycline was evaluated in experimental models in mice. In the pneumonia model, N-desmethyltamoxifen exhibited significant efficacy against Ab#9 and both E. coli strains, especially E. coli MCR-1+ (-2.86 log10 CFU/g lungs, -5.88 log10 CFU/mL blood, and -50% mortality), and against the Ab#186 strain when combined with CMS (-2.27 log10 CFU/g lungs, -2.73 log10 CFU/mL blood, and -40% mortality) or tigecycline (-3.27 log10 CFU/g lungs, -4.95 log10 CFU/mL blood, and -50% mortality). Moreover, the metabolite mix in combination with both antibiotics decreased the bacterial concentrations in the lungs and blood for both A. baumannii strains. In the sepsis model, the significant efficacy of the metabolite mix was restricted to the colistin-susceptible E. coli C1-7-LE strain (-3.32 log10 CFU/g lung, -6.06 log10 CFU/mL blood, and -79% mortality). N-desmethyltamoxifen could be a new therapeutic option in combination with CMS or tigecycline for combating multidrug-resistant GNB, specifically A. baumannii.

Live-attenuated CHIKV vaccine with rearranged genome replicates in vitro and induces immune response in mice.

Chikungunya fever virus (CHIKV) is a mosquito-borne alphavirus that causes wide-spread human infections and epidemics in Asia, Africa and recently, in the Americas. CHIKV is considered a priority pathogen by CEPI and WHO. Despite recent approval of a live-attenuated CHIKV vaccine, development of additional vaccines is warranted due to the worldwide outbreaks of CHIKV. Previously, we developed immunization DNA (iDNA) plasmid capable of launching live-attenuated CHIKV vaccine in vivo. Here we report the use of CHIKV iDNA plasmid to prepare a novel, live-attenuated CHIKV vaccine V5040 with rearranged RNA genome. In V5040, genomic RNA was rearranged to encode capsid gene downstream from the glycoprotein genes. Attenuated mutations derived from experimental CHIKV 181/25 vaccine were also engineered into E2 gene of V5040. The DNA copy of rearranged CHIKV genomic RNA with attenuated mutations was cloned into iDNA plasmid pMG5040 downstream from the CMV promoter. After transfection in vitro, pMG5040 launched replication of V5040 virus with rearranged genome and attenuating E2 mutations. Furthermore, V5040 virus was evaluated in experimental murine models for general safety and immunogenicity. Vaccination with V5040 virus subcutaneously resulted in elicitation of CHIKV-specific, virus-neutralizing antibodies. The results warrant further evaluation of V5040 virus with rearranged genome as a novel live-attenuated vaccine for CHIKV.