Effect of Food and an Animal’s Sex on P-Glycoprotein Expression and Luminal Fluids in the Gastrointestinal Tract of Wistar Rats

The rat is one of the most commonly used animal models in pre-clinical studies. Limited information between the sexes and the effect of food consumption on the gastrointestinal (GI) physiology, however, is acknowledged or understood. This study aimed to investigate the potential sex differences and effect of food intake on the intestinal luminal fluid and the efflux membrane transporter P-glycoprotein (P-gp) along the intestinal tract of male and female Wistar rats. To characterise the intestinal luminal fluids, pH, surface tension, buffer capacity and osmolality were measured. Absolute P-gp expression along the intestinal tract was quantified via liquid chromatography-tandem mass spectrometry (LC-MS/MS). In general, the characteristics of the luminal fluids were similar in male and female rats along the GI tract. In fasted male rats, the absolute P-gp expression gradually increased from the duodenum to ileum but decreased in the colon. A significant sex difference (p < 0.05) was identified in the jejunum where P-gp expression in males was 83% higher than in females. Similarly, ileal P-gp expression in male rats was approximately 58% higher than that of their female counterparts. Conversely, following food intake, a significant sex difference (p < 0.05) in P-gp expression was found but in a contrasting trend. Fed female rats expressed much higher P-gp levels than male rats with an increase of 77% and 34% in the jejunum and ileum, respectively. A deeper understanding of the effects of sex and food intake on the absorption of P-gp substrates can lead to an improved translation from pre-clinical animal studies into human pharmacokinetic studies

A non-nutritive feeding intervention alters the expression of efflux transporters in the gastrointestinal tract

Intestinal interactions with nutrients, xenobiotics and endogenous hormones can influence the expression of clinically relevant membrane transporters. These changes in the gastrointestinal (GI) physiology can in turn affect the absorption of numerous drug substrates. Several studies have examined the effect of food on intestinal transporters in male and female humans and animal models. However, to our knowledge no studies have investigated the influence of a non-nutritive fibre meal on intestinal efflux transporters and key sex and GI hormones. Here, we show that a fibre meal increased the acute expression of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug-resistance-associated protein-2 (MRP2) in small intestinal segments in both male and female Wistar rats. Enzyme-linked immunosorbent assays were used for the protein quantification of efflux transporters and hormonal plasma concentration. In male rats, the fibre meal caused the plasma concentration of the GI hormone cholecystokinin (CCK) to increase by 75% and the sex hormone testosterone to decrease by 50%, whereas, in contrast, the housing food meal caused a decrease in CCK by 32% and testosterone saw an increase of 31%. No significant changes in the hormonal concentrations, however, were seen in female rats. A deeper understanding of the modulation of efflux transporters by sex, food intake and time can improve our understanding of inter- and intra-variability in the pharmacokinetics of drug substrates

Quantification of P-Glycoprotein in the Gastrointestinal Tract of Humans and Rodents: Methodology, Gut Region, Sex, and Species Matter

Intestinal efflux transporters affect the gastrointestinal processing of many drugs but further data on their intestinal expression levels are required. Relative mRNA expression and relative and absolute protein expression data of transporters are commonly measured by real-time polymerase chain reaction (RT-PCR), Western blot and mass spectrometry-based targeted proteomics techniques. All of these methods, however, have their own strengths and limitations, and therefore, validation for optimized quantification methods is needed. As such, the identification of the most appropriate technique is necessary to effectively translate preclinical findings to first-in-human trials. In this study, the mRNA expression and protein levels of the efflux transporter P-glycoprotein (P-gp) in jejunal and ileal epithelia of 30 male and female human subjects, and the duodenal, jejunal, ileal and colonic tissues in 48 Wistar rats were quantified using RT-PCR, Western blot and liquid chromatography-tandem mass spectrometry (LC-MS/MS). A similar sex difference was observed in the expression of small intestinal P-gp in humans and Wistar rats where P-gp was higher in males than females with an increasing trend from the proximal to the distal parts in both species. A strong positive linear correlation was determined between the Western blot data and LC-MS/MS data in the small intestine of humans (R^{2} = 0.85). Conflicting results, however, were shown in rat small intestinal and colonic P-gp expression between the techniques (R^{2} = 0.29 and 0.05, respectively). In RT-PCR and Western blot, an internal reference protein is experimentally required; here, beta-actin was used which is innately variable along the intestinal tract. Quantification via LC-MS/MS can provide data on P-gp expression without the need for an internal reference protein and consequently, can give higher confidence on the expression levels of P-gp along the intestinal tract. Overall, these findings highlight similar trends between the species and suggest that the Wistar rat is an appropriate preclinical animal model to predict the oral drug absorption of P-gp substrates in the human small intestine

Metallothionein I as a direct link between therapeutic hematopoietic stem/progenitor cells and cerebral protection in stroke

National Institutes of Health/National Heart, Lung, and blood Institute (NIH/NHLBI (HL125572-01A1)); Malcolm Feist Cardiovascular Fellowship program; National Institute of General Medical Sciences COBRE Grant (P30- GM110703) and Japan Society for the Promotion of Science (Grants-in-Aid for Scientific Research- KAKENHI, 16H07356)

Exploring the Role of Platelets in Virus-Induced Inflammatory Demyelinating Disease and Myocarditis

Data Availability Statement: The data generated for this study can be found in the GEO at the NCBI (accession no. GSE253385, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE253385 (accessed on 15 March 2024)).Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms25063460/s1 .Theiler’s murine encephalomyelitis virus (TMEV) infection has been used as a mouse model for two virus-induced organ-specific immune-mediated diseases. TMEV-induced demyelinating disease (TMEV-IDD) in the central nervous system (CNS) is a chronic inflammatory disease with viral persistence and an animal model of multiple sclerosis (MS) in humans. TMEV infection can also cause acute myocarditis with viral replication and immune cell infiltration in the heart, leading to cardiac fibrosis. Since platelets have been reported to modulate immune responses, we aimed to determine the role of platelets in TMEV infection. In transcriptome analyses of platelets, distinct sets of immune-related genes, including major histocompatibility complex (MHC) class I, were up- or downregulated in TMEV-infected mice at different time points. We depleted platelets from TMEV-infected mice by injecting them with platelet-specific antibodies. The platelet-depleted mice had significantly fewer viral antigen-positive cells in the CNS. Platelet depletion reduced the severities of TMEV-IDD and myocarditis, although the pathology scores did not reach statistical significance. Immunologically, the platelet-depleted mice had an increase in interferon (IFN)-γ production with a higher anti-TMEV IgG2a/IgG1 ratio. Thus, platelets may play roles in TMEV infection, such as gene expression, viral clearance, and anti-viral antibody isotype responses.This work is supported by the Ministry of Education, Culture, Sports, Science and Technology, Japan, through the Monbukagakusho (MEXT, 2021-2025) Scholarship (I.A.), Grant-in Aid for Scientific Research KAKENHI from the Japan Society for the Promotion of Science (JSPS) (JP22K07527 (S.O.), JP23K06493 (F.S.), JP21K07167 (H.T.), and JP22K18378 and JP23K08901 (I.T.)), Novartis Pharma Research Grants (S.O. and I.T.), the Royal Society Wolfson Foundation (RSWF\R3\183001, (F.N.E.G.)), the Hokkaido Lawyer Association for Hepatitis B (Orange funding) (H.T.), and Hokkaido Organization for Translational Research (A seeds) (H.T.)

Formyl Peptide Receptor as a Novel Therapeutic Target for Anxiety-Related Disorders

Formyl peptide receptors (FPR) belong to a family of sensors of the immune system that detect microbe-associated molecules and inform various cellular and sensorial mechanisms to the presence of pathogens in the host. Here we demonstrate that Fpr2/3-deficient mice show a distinct profile of behaviour characterised by reduced anxiety in the marble burying and light-dark box paradigms, increased exploratory behaviour in an open-field, together with superior performance on a novel object recognition test. Pharmacological blockade with a formyl peptide receptor antagonist, Boc2, in wild type mice reproduced most of the behavioural changes observed in the Fpr2/3(-/-) mice, including a significant improvement in novel object discrimination and reduced anxiety in a light/dark shuttle test. These effects were associated with reduced FPR signalling in the gut as shown by the significant reduction in the levels of p-p38. Collectively, these findings suggest that homeostatic FPR signalling exerts a modulatory effect on anxiety-like behaviours. These findings thus suggest that therapies targeting FPRs may be a novel approach to ameliorate behavioural abnormalities present in neuropsychiatric disorders at the cognitive-emotional interface

Targeting of Formyl Peptide Receptor 2 for in vivo imaging of acute vascular inflammation

© The author(s). Inflammatory conditions are associated with a variety of diseases and can significantly contribute to their pathophysiology. Neutrophils are recognised as key players in driving vascular inflammation and promoting inflammation resolution. As a result, neutrophils, and specifically their surface formyl peptide receptors (FPRs), are attractive targets for non-invasive visualization of inflammatory disease states and studying mechanistic details of the process. Methods: A small-molecule Formyl Peptide Receptor 2 (FPR2/ALX)-targeted compound was combined with two rhodamine-derived fluorescent tags to form firstly, a targeted probe (Rho-pip-C1) and secondly a targeted, pH-responsive probe (Rho-NH-C1) for in vivo applications. We tested internalization, toxicity and functional interactions with neutrophils in vitro for both compounds, as well as the fluorescence switching response of Rho-NH-C1 to neutrophil activation. Finally, in vivo imaging (fluorescent intravital microscopy [IVM]) and therapeutic efficacy studies were performed in an inflammatory mouse model. Results: In vitro studies showed that the compounds bound to human neutrophils via FPR2/ALX without causing internalisation at relevant concentrations. Additionally, the compounds did not cause toxicity or affect neutrophil functional responses (e.g. chemotaxis or transmigration). In vivo studies using IVM showed Rho-pip-C1 bound to activated neutrophils in a model of vascular inflammation. The pH-sensitive (“switchable”) version termed Rho-NH-C1 validated these findings, showing fluorescent activity only in inflammatory conditions. Conclusions: These results indicate a viable design of fluorescent probes that have the ability to detect inflammatory events by targeting activated neutrophils.British Pharmacological Society; Wilkinson Trust; EPSRC; German Research Foundation

Targeting AnxA1/Fpr2/ALX Regulates Neutrophil Function Promoting Thrombo-Inflammation Resolution in Sickle Cell Disease

NIH/NHLBI (HL134959-01A1 [FNEG]; HL098435, HL133497, HL141155, and GM12130 [AWO]; HL142604 [RP]), the AHA (19PRE34380751 [ZAY]) and the Royal Society Wolfson Foundation (RSWF\R3\183001 [FNEG])

Anti-Allergic Cromones Inhibit Histamine and Eicosanoid Release from Activated Human and Murine Mast Cells by Releasing Annexin A1

PMCID: PMC3601088This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Bryostatin-1 Attenuates Ischemia-Elicited Neutrophil Transmigration and Ameliorates Graft Injury after Kidney Transplantation

Data Availability Statement: Not applicable.Copyright: © 2022 by the authors. Ischemia reperfusion injury (IRI) is a form of sterile inflammation whose severity determines short- and long-term graft fates in kidney transplantation. Neutrophils are now recognized as a key cell type mediating early graft injury, which activates further innate immune responses and intensifies acquired immunity and alloimmunity. Since the macrolide Bryostatin-1 has been shown to block neutrophil transmigration, we aimed to determine whether these findings could be translated to the field of kidney transplantation. To study the effects of Bryostatin-1 on ischemia-elicited neutrophil transmigration, an in vitro model of hypoxia and normoxia was equipped with human endothelial cells and neutrophils. To translate these findings, a porcine renal autotransplantation model with eight hours of reperfusion was used to study neutrophil infiltration in vivo. Graft-specific treatment using Bryostatin-1 (100 nM) was applied during static cold storage. Bryostatin-1 dose-dependently blocked neutrophil activation and transmigration over ischemically challenged endothelial cell monolayers. When applied to porcine renal autografts, Bryostatin-1 reduced neutrophil graft infiltration, attenuated histological and ultrastructural damage, and improved renal function. Our novel findings demonstrate that Bryostatin-1 is a promising pharmacological candidate for graft-specific treatment in kidney transplantation, as it provides protection by blocking neutrophil infiltration and attenuating functional graft injury.Royal Society Wolfson Foundation (RSWF\R3\18300 to F.N.E.G); Eastern Star New Idea Award, LSUHSC-S (to F.N.E.G. and J.S.A.)