Leukocyte Infiltration Research Articles

Metabolomic analysis reveals an important role of sphingosine 1-phosphate in the development of HFMD due to EV-A71 infection.

Hand, foot, and mouth disease (HFMD) is a serious pediatric infectious disease that causes immeasurable physical and mental health burdens. Currently, there is a lack of information on the mechanisms of HFMD severity and early diagnosis. We performed metabolomic profiling of sera from 84 Enterovirus A71 (EV-A71) infections and 45 control individuals. Targeted metabolomics assays were employed to further validate some of the differential metabolic molecules. We identified significant molecular changes in the sera of HFMD patients compared to healthy controls (HCs). A total of 54, 60, 35, and 62 differential metabolites were screened between mild cases and HCs, severe cases and HCs, severe cases and mild cases, and among the three groups, respectively. These differential metabolites implicated dysregulation of the tricarboxylic acid cycle, alanine, aspartate, and glutamate metabolism, and valine, leucine, and isoleucine biosynthesis. The diagnostic panel based on some overlapped differential metabolites could effectively discriminate severe cases from mild cases with an AUC of 0.912 (95% CI: 0.85-0.97) using the logistic regression model. Next, we found the elevation of serum sphingosine 1-phosphate (S1P) level in EV-A71 infection mice, which was similar to clinical observation. Importantly, after blocking the release of S1P by MK571, the clinical symptoms and survival of mice were significantly improved, involving the reduction of leukocyte infiltration in infected brain tissues. Collectively, our data provided a landscape view of metabolic alterations in EV-A71 infected children and revealed regulating S1P metabolism was an exploitable therapeutic target against EV-A71 infection.

CD3+CD4-CD8- Double-Negative Lymphocytes Are Increased in the Aqueous Humor of Patients with Retinitis Pigmentosa: Their Possible Role in Mediating Inflammation

Recently, evidence has supported a significant role for immune and oxidative-mediated damage underlying the pathogenesis of different types of retinal diseases, including retinitis pigmentosa (RP). Our study aimed to evaluate the presence of immune cells and mediators in patients with RP using flow cytometric analysis of peripheral blood (PB) and aqueous humor (AH) samples. We recruited 12 patients with RP and nine controls undergoing cataract surgery. Flow cytometric analysis of PB and AH samples provided a membrane staining that targeted surface molecules (CD14, CD16, CD19, CD3, CD4, CD8, and CD161) identifying monocytes, natural killer (NK) cells, B cells, T cells, and T subpopulations, respectively. Moreover, lymphocytes were polyclonally stimulated to evaluate cytokine (CK) production at single-cell level. The circulating immune cell distribution was comparable between patients with RP and controls. Conversely, in the AH of controls we could detect no cells, while in the RP AH samples we found infiltrating leukocytes, consisting of T (CD3+), B (CD19+), NK (CD16+CD3-) cells, and monocytes (CD14+). In patients with RP, the frequency of most infiltrating immune cell populations was similar between the AH and PB. However, among T cell subpopulations, the frequency of CD3+CD4+ T cells was significantly lower in the RP AH compared to RP PB, whereas CD3+CD4-CD8- double-negative (DN) T cells were significantly higher in the RP AH compared to RP PB. Cytokine production analysis revealed a trend toward an increased frequency of CD3+CD8-CD161+IFN-ɣ-producing cells and a decreased frequency of CD3+CD8+IL-4-producing cells in the RP AH compared to RP PB. The detection of immune cells, particularly DN T cells, and a Th1-skewed phenotype in RP AH suggests immune-mediated and inflammatory mechanisms in the disease.

Tissue-matched analysis of MRI evaluating the tumor infiltrating lymphocytes in hepatocellular carcinoma.

Tumor-infiltrating lymphocytes (TILs) play critical roles in the tumor microenvironment and immunotherapy response. This study aims to explore the feasibility of multi-parametric magnetic resonance imaging (MRI) in evaluating TILs and to develop an evaluation model that considers spatial heterogeneity. Multi-parametric MRI was performed on hepatocellular carcinoma (HCC) mice (N = 28). Three-dimensional (3D) printing was employed for tissue sampling, to match the multi-parametric MRI data with tumor tissues, followed by flow cytometry analysis and next-generation RNA-sequencing. Pearson's correlation, multivariate logistic regression, and receiver operating characteristic (ROC) curve analyses were utilized to model TIL-related MRI parameters. MRI quantitative parameters, including T1 relaxation times and perfusion, were correlated with the infiltration of leukocytes, T-cells, CD4+ T-cells, CD8+ T-cells, PD1 + CD8+ T-cells, B-cells, macrophages, and regulatory T-cells (correlation coefficients ranged from -0.656 to 0.482, p <.05) in tumor tissues. TILs were clustered into inflamed and non-inflamed subclasses, with the proportion of T-cells, CD8+ T-cells, and PD1 + CD8+ T-cells significantly higher in the inflamed group compared to the non-inflamed group (43.37% vs. 25.45%, 50.83% vs. 34.90%, 40.45% vs. 29.47%, respectively; p <.001). The TIL evaluation model, based on the Z-score combining Kep and T1post, was able to distinguish between these subgroups, yielding an area under the curve of 0.816 (95% confidence interval 0.721-0.910) and a cut-off value of -0.03 (sensitivity 68.4%, specificity 91.3%). Additionally, the Z-score was related to the gene expression of T-cell activation, chemokine production, and cell adhesion. The tissue-matched analysis of multi-parametric MRI offers a feasible method of regional evaluation and can distinguish between TIL subclasses.

Protective Effect of Pentoxifylline on the Development of Acute Gastric Mucosal Injury in a Model of LPS-Induced Sepsis

Alterations in the gastric mucosal barrier, one of whose fundamental components is phosphatidylcholine (PC), may play an important role in the pathophysiology of erosive gastritis secondary to sepsis. Pentoxifylline (PTX) has been shown to reduce tissue damage in various experimental models of sepsis. The aim of this study was to investigate the effect of PTX on gastric mucosa PC synthesis, leukocyte infiltration, arachidonic acid-related metabolites, inflammation, oxidative stress, NO, CO, and somatostatin in a rat model of LPS-induced sepsis. Rats were administered LPS (10 mg/kg b.w.) intraperitoneally. After 30 min (early treatment group) or 120 min (late treatment group) of LPS administration, they were randomly divided into two groups that were intraperitoneally administered saline (5 mL/kg; LPS + Saline group) or PTX (45 mg/kg; 5 mL/kg; LPS + PTX group). Control rats received only saline instead of LPS and/or PTX. Two hours after saline or PTX administration (total of 150 or 240 min of procedure), animals were anesthetized, and then gastric lavage, gastric mucosa and plasma samples were obtained and kept frozen until determination. LPS-induced sepsis changed the gastric mucosal barrier by reducing its phospholipid content, PGI2, and somatostatin levels, as well as increasing MPO, TXB2, LTB4, PLA2, and MDA. Alterations may be mediated, at least in part, by modifications in the production of NO, CO, and cGMP content. PTX had a beneficial effect on gastric lesions secondary to sepsis by restoring PC production.

Comparative transcriptomic analysis revealed potential mechanisms regulating the hypertrophy of goose pectoral muscles

Pectoral muscle development is an important economic trait. According to the different essence, muscle development can be divided into 2 processes: embryonic muscle fiber generation and postnatal muscle fiber hypertrophy, and postnatal muscle fiber hypertrophy has a greater impact on muscle development than the number of muscle fibers formed during the embryonic phase in poultry. However, the underlying mechanisms regulating the hypertrophy of goose pectoral muscles have not been elucidated. Therefore, the purpose of the present study was to conduct transcriptome sequencing in pectoral muscles of both Landes (LD) and Sichuan White (SW) geese at 6, 10, and 30 weeks of age to reveal the molecular mechanisms regulating pectoral muscle hypertrophy through intra-breed and inter-breed bioinformatics analyses. Phenotypically, the pectoral muscle weight/index of LD and SW geese increased from 6 to 30 weeks of age, and except for the pectoral muscle index at 10 weeks of age (P = 0.962), at the same age, the pectoral muscle weight/index of LD geese were significantly higher than that of SW geese (P < 0.05). In transcriptional regulation, intra-breed bioinformatics analysis identified 3331 genes whose expression levels were opposite to the trend of pectoral muscle hypertrophy both in LD and SW geese, and the 3331 genes were mainly enriched into abundant KEGG pathways related to lipid metabolism, proliferation/apoptosis, and immune response. Moreover, 23 genes (including SLC2A10, TNFRSF1A, PRKAA1, SLC27A4, ITGB2, THY1, RHOA, MYL10, ACTB, PRKCB, PIK3R2, RAC2, DMD, LATS2, YAP1, WWTR1, SMAD7, CTGF, FGF1, AXIN2, GLI2, ID2, and CCND2) who were enriched in 6 crosstalk pathways named viral myocarditis, insulin resistance, sphingolipid signaling pathway, hippo signaling pathway, chemokine signaling pathway, and leukocyte transendothelial migration were identified as the key candidate genes regulating the hypertrophy of goose pectoral muscles. In inter-breed bioinformatics analysis, abundant different expression genes (DEGs) related to lipid metabolism, immune response, and proliferation/apoptosis were identified between LD and SW geese too, and compared with SW geese, the expression level of MYL10 in LD geese was lower, while the expression levels of GLI2/CTGF/SMAD7 in LD geese were higher. These results suggested that the hypertrophy of goose pectoral muscles might be achieved through more lipid deposition and less leukocyte infiltration to promote the proliferation of cells within the muscles, and the low expression of MYL10 and high expressions of GLI2/CTGF/SMAD7 might the keys to induce the pectoral muscle hypertrophy of LD geese from 6 to 30 weeks of age over that of SW geese. All data the present study obtained will provide new insights into the molecular mechanisms regulating the hypertrophy of goose pectoral muscles.

Protective role of TRPV1+ nociceptive neurons communication to macrophages against T. cruzi infection in mice

Chagas’ disease is a life-threatening condition caused by Trypanosoma cruzi. Patients with chronic disease may develop gastrointestinal, neurological, or associated neuro-digestive dysfunctions. CNS invasion by T. cruzi can occur in the acute phase, and its presence in the brain and cerebrospinal fluid was reported. T. cruzi induces nociceptor neuron activation and pain. Nociceptive neurons and macrophage interact in diseases, and this neuroimmune communication has a pivotal role in disease outcome. We investigated, the role of TRPV1+ neurons in experimental T. cruzi infection in mice. T. cruzi forms were observed in the DRG and spinal cord in early stages of acute infection, and intrathecal administration of T. cruzi antigens into spinal cord induced pain. Trpv1 mRNA expression was increased in the DRG of infected mice and targeting TRPV1 reduced T. cruzi-induced pain. TRPV1 nociceptor ablation increased blood parasitemia while TRPV1 knockout mice presented 50% mortality upon infection in a normally non-lethal model. TRPV1 knockout also worsened clinical outcomes (hepatomegaly and megacecum), increased plasmatic Th2 cytokines and nitrite in cardiac tissue, and reduced heart leukocyte infiltration. Conditioned media of capsaicin-stimulated DRG neurons decreased macrophage internalization of T. cruzi, and CGRP receptor antagonism in infected mice reduced pain, increased early parasitemia and promoted 18% mortality. This indicates that soluble mediators released upon nociceptor activation such as CGRP increase macrophage ability to control disease outcome. These data unveil TRPV1+ neurons release CGRP to limit macrophage internalization of T. cruzi, triggering protective mechanisms against T. cruzi infection.

CD22 blockade exacerbates neuroinflammation in Neuromyelitis optica spectrum disorder

BackgroundNeuromyelitis optica spectrum disorder (NMOSD) is an autoantibody-triggered central nervous system (CNS) demyelinating disease that primarily affects the spinal cord, optic nerves and brainstem. Among the first responders to CNS injury, microglia are prominent players that drive NMOSD lesion formation. However, the key molecular switches controlling the detrimental activity of microglia in NMOSD are poorly understood. CD22 governs the activity of innate and adaptive immunity. In this study, we investigated to what extent and by what mechanisms CD22 may modulate microglial activity, neuroinflammation and CNS lesion formation.MethodsTo determine the expression profile of CD22 in NMOSD, we performed single-cell sequencing and flow cytometry analysis of immune cells from human peripheral blood. We investigated the potential effects and mechanisms of CD22 blockade on microglial activity, leukocyte infiltration and CNS demyelination in a mouse model of NMOSD induced by injection of NMOSD patient serum-derived AQP4-IgG and human complement.ResultsSingle-cell sequencing and flow cytometry analysis revealed that CD22 was expressed in B cells, neutrophils, monocytes and microglia-derived exosomes in human peripheral blood from NMOSD patients and controls (n = 5 per group). In a mouse model of NMOSD, CD22 was expressed in B cells, neutrophils, monocytes and microglia (n = 8 per group). In NMOSD mice, CD22 blockade significantly increased the number of CNS lesions, astrocyte loss and demyelination, accompanied by increased inflammatory activity and phagocytosis in microglia. Furthermore, the detrimental effects of CD22 blockade were significantly alleviated in NMOSD mice subjected to depletion of microglia or Gr-1+ myeloid cells, suggesting the involvement of microglia and peripheral Gr-1+ myeloid cells. Additionally, CD22 blockade also led to significantly reduced phosphorylation of SYK and GSK3β in NMOSD. Notably, the detrimental effects of CD22 blockade were greatly diminished in NMOSD mice receiving the phosphorylated SYK inhibitor R406.ConclusionsOur findings revealed a previously unrecognized role of CD22 as a key molecular switch that governs the detrimental effects of microglia and Gr-1+ myeloid cells in NMOSD, which paves the way for the future design of immune therapies for NMOSD.Graphical abstract

Therapeutic Potential of Hydrogen as a Radioprotective Agent for the Prevention of Radiation Dermatitis

Radiation dermatitis (RD) is a common side effect in patients receiving radiotherapy. Currently, clinical skincare approaches for acute RD vary widely among institutions and lack consensus. Hydrogen molecules, acting as radioprotective agents by selectively scavenging free radicals, have the potential to protect against RD. In this study, we demonstrate that hydrogen reduces double-strand breaks, mitochondrial depolarization, and inflammatory cytokines induced by irradiation damage in HaCaT cells. Furthermore, in vivo experiments reveal that exposing irradiated skin areas to a hydrogen gas environment alleviates RD. Assessment of skin appearance grade and histology staining revealed that direct transdermal application of hydrogen can prevent radiation-induced follicle damage, dermal thickening, and leukocyte infiltration, thereby reducing the severity of RD. In addition, hydrogen enhances the skin’s antioxidant capacity, leading to a reduction in the Bcl-2-associated X protein/B-cell lymphoma 2 (Bax/Bcl-2) ratio, the number of apoptotic cells, and the expression of pro-inflammatory cytokines. Our data demonstrate that hydrogen possesses antioxidant, anti-inflammatory, and anti-apoptotic properties, and could be a preventive strategy for RD.

Repositioning anthelmintics for the treatment of inflammatory-based pathological conditions.

Acute, uncontrolled and/or long-lasting inflammation causes a breakdown in immunological tolerance, leading to chronicity and contributing to a series of significant local or systemic tissue changes. Anti-inflammatory efficacy, fewer adverse effects, improved selectivity, and curative action are imminent issues for patients suffering from chronic inflammation-related pathologies. Then, we performed a complete and critical review about anthelmintics, discussing the main classes and the available preclinical evidence on repurposing to treat inflammation-based conditions. Despite low bioavailability, many benzimidazoles(albendazole and mebendazole), salicylanilides(niclosamide), macrocyclic lactones(avermectins), pyrazinoisoquinolones (praziquantel), thiazolides (nitazoxanide), piperazine derivatives, and imidazothiazoles(levamisole) indicate that repositioning is a promising strategy. They may represent a lower cost and time-saving course to expand anti-inflammatory options. Although mechanisms of action are not fully elucidated and well-delineated, in general, anthelmintics disrupt mitogen-activated protein kinases, the synthesis of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8, IL-12, and IFN-γ), the migration and infiltration of leukocytes, and decrease COX-2 expression, which impacts negatively on the release of prostanoids and leukotrienes. Moreover, some of them reduce nuclear accumulation of NF-κB (niclosamide, albendazole, and ivermectin), levels of nitric oxide (nitazoxanide and albendazole), and mucus, cytokines, and bronchoconstriction in experimental inflammatory pulmonary diseases (ivermectin and niclosamide). Considering the linking between cytokines, bradykinin, histamine, and nociceptors with algesia, anthelmintics also stand out for treating inflammatory pain disorders (ivermectin, niclosamide, nitazoxanide, mebendazole, levamisole), including for cancer-related pain status. There are obstacles, including the low bioavailability and the first-pass metabolism.

Study on toxicity responses in Xenopus tropicalis long-term exposure to norfloxacin, oxytetracycline and arsenic

The presence of residues of antibiotics and heavy metals in the global aquatic environment is a widespread potential environmental risk. Here, we studied their effect on Xenopus tropicalis by analysing the hepatotoxic effects of norfloxacin (NOR), oxytetracycline (OTC), and arsenic (As) on its histology, lipidomics, proteases, and cytokines. The results showed that development was inhibited, and additional vacuolation, sinusoids, pyknosis, nuclei, cell lysis, and leukocyte infiltration were observed in the liver after 72 days of exposure to NOR (0.1–4.0 mg L−1), OTC (0.1–4.0 mg L−1), and/or As (0.3–3.5 μg L−1). In addition, the size and number of lipid droplets increased with the superposition of drugs, disrupting lipid droplet homeostasis. Lipidomics proved that the intensity of lipid responses related to lipid metabolism was disrupted, especially for CerP and TAG. In addition, the lipotoxicity induced by joint exposure was more potent than that induced by a single exposure. Compared with the controls, the ROS levels in the liver were 7.21%–37.18% greater, which promoted oxidative stress damage. By revealing the underlying mechanisms involved, we found that the liver can resist oxidative stress and lipid peroxidation by regulating the expression of multiple cytokines. Our study provides new insights into the hepatotoxicity and underlying mechanisms in aquatic amphibians caused by long-term exposure to low concentrations of NOR, OTC, and/or As.

Qingke Pingchuan granules alleviate airway inflammation in COPD exacerbation by inhibiting neutrophil extracellular traps in mice

BackgroundChronic obstructive pulmonary disease (COPD) imposes a significant global health and socioeconomic burden. Exacerbations of COPD (ECOPD), characterized by heightened airway inflammation and mucus hypersecretion, adversely affect patient health and accelerate disease progression. Qingke Pingchuan (QKPC) granules, a formulation from Traditional Chinese Medicine initially prescribed for acute bronchitis, have shown unexplored potential in ECOPD management, with mechanisms of action yet to be clarified. PurposeThis study investigates the therapeutic effects of QKPC in a mouse model of ECOPD, focusing on underlying molecular mechanisms. MethodsCOPD was induced in mice through chronic cigarette smoke (CS) exposure, followed by intratracheal administration of Pseudomonas aeruginosa lipopolysaccharide (LPS) to trigger exacerbation, after which mice were treated with QKPC granules. Major compounds in QKPC were identified via UHPLC-QE-MS, and high-throughput RNA sequencing of lung tissue samples identified differentially expressed genes. Transcriptomic data were integrated with network pharmacology analysis to pinpoint potential pathways, bioactive compounds, and target genes through which QKPC might attenuate ECOPD. Molecular docking, protein-small molecule binding assays, and in vitro analyses further validated interactions between key compounds and target genes, shedding light on plausible signaling pathways. ResultsQKPC treatment led to significant reductions in airway leukocyte infiltration and goblet cell metaplasia in CS- and LPS-exposed mice, accompanied by decreased levels of inflammatory cytokines (IL-6, IL-1β, CXCL1, and TNF-α) and mucin MUC5AC in bronchoalveolar lavage fluid. The integrative transcriptomic and network pharmacology analysis identified the neutrophil extracellular trap (NET) formation pathway as a key mechanism underlying QKPC's protective effect against ECOPD. In vitro assays demonstrated that epigallocatechin-3-gallate (EGCG) and quercetin, two important bioactive compounds in QKPC, significantly inhibited NETosis induced by cigarette smoke extract (CSE) plus LPS in human neutrophils. The two compounds were found to interact directly with the reactive oxidative species (ROS)-generating enzyme NOX2 and its regulatory subunit p47phox. Subsequent in vitro studies further confirmed EGCG and quercetin's capacity to reduce ROS production and downregulate NOX2 and p47phox protein levels in neutrophils stimulated with CSE and LPS. Additionally, in vivo studies confirmed QKPC's efficacy in reducing NET formation, oxidative stress, and NOX2/p47phox protein expression in the lung tissue of ECOPD mice. ConclusionThese findings suggest that QKPC granules alleviate airway inflammation in ECOPD, potentially through inhibition of pulmonary NET formation via the NOX2/p47phox-ROS pathway, underscoring their potential therapeutic application for ECOPD management in clinical settings.

Abstract B034: Engineering the tumor microenvironment: Patient-specific microphysiological systems of the head and neck cancer tumor microenvironment

Abstract Introduction: Survival rates in advanced head and neck cancer (HNC) remain poor with no clinically approved biomarkers to predict treatment efficacy. HNC is a highly heterogenous disease and the lack of available models that capture patient heterogeneity and the complexity of the tumor microenvironment (TME) contributes to these poor patient outcomes. Microphysiological systems (MPS) are complex multicellular in vitro models that can recapitulate three-dimensional organ structure and function. The low cell number requirements for MPS permits the creation of patient-specific TME models, where all the cell types can be isolated from a single piece of tumor tissue. These patient-specific MPS represent a promising strategy for elucidating the contribution of the TME to treatment resistance, predicting the best treatment for each patient and improving patient outcomes in HNC. Here, we report a patient-specific MPS that contains multiple TME compartments and recapitulates patient heterogeneity. Methods: Tumor tissue was collected from patients with oral cavity carcinoma undergoing standard of care surgery followed by radiation or chemoradiation. Tissue from each patient was digested and tumor infiltrating leukocytes (TIL) were isolated using CD45 magnetic beads, while epithelial cells and fibroblasts were expanded in culture using specific media formulations. Tissue was also snap frozen for single cell RNA sequencing analysis. To build the MPS, a custom designed microfluidic device was filled with a hydrogel matrix containing patient-matched TIL, fibroblasts and epithelial spheroids to mimic the solid tumor. Lumens molded from the hydrogel were seeded with blood or lymphatic endothelial cells to create vasculature. Multiple replicate MPS can be built from each patient, which are cultured in an optimized media formulation to support the multiple primary cell types. Patient-specific MPS were treated with radiation or cisplatin + radiation to mimic the treatment the patient received. Multiple orthogonal endpoints were measured including tumor cell migration and viability, cytokine secretion and angiogenesis. Single cell sequencing was also performed on the original tumor tissue and matched MPS. Results: We have successfully created patient-specific models of the HNC TME. Single cell RNA sequencing analysis of the MPS suggests that cellular heterogeneity is being retained. Patient characteristics varied between MPS with some patient TME MPS showing highly migratory tumor cells or angiogenic responses. Treatment responses were measured for each patient-specific MPS and ongoing work will characterize the contributions of the TME to treatment resistance. These data demonstrate the feasibility of using patient-specific MPS to investigate the contribution of the TME to treatment response and resistance and lays important groundwork for using MPS to identify patient treatment responses. Citation Format: Adeel Ahmed, Marcos Lares, Fauzan Ahmed, Adam R. Burr, Paul M. Harari, David J. Beebe, Sheena C. Kerr. Engineering the tumor microenvironment: Patient-specific microphysiological systems of the head and neck cancer tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B034.

Histopathological Alterations in Placentas of Severe and Non-Severe Preeclamptic Patients

Preeclampsia is a significant pregnancy complication associated with vascular lesions and trophoblastic invasion, leading to substantial maternal and fetal risks. This study compares histopathological changes in placental structures among severe and non-severe preeclamptic patients. Placental tissues were analyzed, revealing that preeclampsia groups showed notable villous degeneration, increased fibrin deposition, vascular dilatation, congestion, and syncytial node formation. Severe preeclampsia further exhibited intensified hemorrhage and leukocyte infiltration. These findings underscore the link between preeclampsia severity and placental dysfunction, providing insights into the pathological mechanisms affecting perinatal outcomes. Keywords: placenta, preeclampsia, HELLP, histology

A sex-dependent role of Kv1.3 channels from macrophages in metabolic syndrome.

Coronary artery disease (CAD) is the foremost single cause of mortality and disability globally. Patients with type 2 diabetes (T2DM) have a higher incidence of CAD, and poorer prognosis. The low-grade inflammation associated to T2DM contributes to increased morbidity and worst outcomes after revascularization. Inflammatory signaling in the vasculature supports endothelial dysfunction, leukocyte infiltration, and macrophage activation to a metabolic disease (MMe) specific phenotype, which could contribute to the metabolic disorders and ascular damage in T2DM. We have previously found that Kv1.3 blockers inhibit the development of intimal hyperplasia, thereby preventing restenosis. This inhibition was enhanced in a mouse model of T2DM, where systemic Kv1.3 blockers administration also improve metabolic dysfunction by acting on unidentified cellular targets other than vascular smooth muscle. Here we characterize the MMe phenotype in our T2DM model with a focus on macrophage Kv1.3 channels, to explore their contribution to vascular disease and their potential role as targets to ameliorate T2DM vascular risk. Male and female BPH mice fed on high-fat diet (HFD) develop metabolic syndrome (MetS) and T2DM. mRNA levels of several K+ channels (KV1.3, KCa3.1, Kir2.1) and macrophage markers (TNFα, NOS2, CD36) were analyzed. The MMe phenotype associated with increased CD36 expression. Channel-specific fingerprinting highlights a gender-specific increase of KV1.3 mRNA fold change in LPS stimulated macrophages from HFD compared to standard diet (SD). KV1.3 functional expression was also significantly increased after LPS stimulation in female HFD macrophages compared to SD. Functional studies showed that macrophage's KV1.3 channels of BPH female mice did not contribute to phagocytosis or metabolic profile but were relevant in cell migration rate. Altogether, our data suggest that by inhibiting macrophage infiltration, Kv1.3 blockers could contribute to disrupt the vicious cycle of inflammation and insulin resistance, offering a novel approach to prevent MetS, T2DM and its associated cardiovascular complications in females.

Identifying an Inversin as a Novel Prognostic Marker in Patients with Clear-Cell Renal Cell Carcinoma.

Precision medicine is a developing trend in oncology, and it includes the prognosis and treatment of advanced-stage ccRCC. New predictive factors and therapeutic targets for this disease are steadily needed. The aim of this study was to explore the tumor expression of inversin as a potential prognostic factor and/or therapeutic target in ccRCC. We compared the expression of inversin between primary ccRCC and normal renal tissues by using immunohistochemistry and rtPCR in our cohort, and we also analyzed publicly available data from the TCGA-KIRC cohort. We found that the expression of inversin was significantly lower in primary tumor tissue, in comparison to solid normal tissue. Data from the KIRC study confirmed that a lower INVS expression level in ccRCC was significantly related with the overall and disease-specific survival, as well as with a shorter progression-free interval (p < 0.05). Four out of ten inversin interactome partners were significantly related with the overall and disease-specific survival in ccRCC. A lower expression of ANKS6 was a negative survival predictor, while a higher expression of NPHP3, DVL1, or DVL3 was related with a lower survival. The expression of INVS and its interactome partners in ccRCC was correlated with the differentiation of the tumor and metastasis. The expression of INVS and its partners was also correlated with tumor leukocyte infiltration and the expression of immune checkpoint genes. The results of this study point to inversin and a distinguished group of its interactome partners as potential prognostic factors in ccRCC, with their predominant involvement in the modulation of the inflammatory infiltration of the tumor microenvironment and a strong relationship with the metastatic potential of the tumor.

Morphological characteristics of wound healing with aloe extract, hydrogel, and their combination: experimental research

BACKGROUND: Currently, there remains a high incidence of skin damage due to trauma, chronic diseases, burns, so it is important to study the issues of regeneration and treatment of skin wounds. AIM: To conduct a morphological analysis of wound healing using aloe extract, hydrogel, and their combination in the experiment. MATERIALS AND METHODS: The study involved 40 sexually mature guinea pigs, which were divided into a control group and 4 experimental groups (independent wound healing, application of aloe extract, hydrosorb gel, layer-by-layer application of drugs). Histological skin preparations were prepared on days 3, 7, 14, and 28 of the experiment. In each microslide, in 10 fields of view of the microscope at a magnification of ×50, morphometric parameters were measured in micrometers (µm): the thickness of the purulent-necrotic scab, inflammatory (leukocyte) infiltrate, granulation tissue, the length of the newly formed epithelium on days 3, 7, 14; the thickness of the epithelium, its length and the thickness of the connective tissue in the central part of the regenerate on the 28th day. All data were subjected to statistical processing. RESULTS: The medications led to a decrease in the inflammatory reaction, acceleration of the formation of granulation tissue, and partial wound epithelization by day 7 of the experiment; however, more pronounced signs of wound healing were observed with layer-by-layer application of aloe extract and hydrogel. By day 14, regardless of the application method, the use of medications demonstrated signs of enhanced wound healing. Thus, the length of the epithelium increased by 1.2–1.6 times, the depth of granulation tissue increased by 1.0–1.2 times compared to the control group. By the end of the experiment, all animals had complete closure of the wound with scar formation, while in the experimental groups showed signs of skin remodeling with appendages. CONCLUSION: Morphological analysis showed that aloe extract, hydrosorb gel, and their combination accelerated the wound healing processes of full-thickness skin wounds in the experiment.

Protective effect of beta-carotene on hepato-nephrotoxicity of gentamicin in male Wistar rats

BackgroundDespite causing significant tissue damage at the molecular and cellular levels, partly due to its induction of oxidative stress, it remains of interest in medical applications. Beta-carotene, found in fruits and vegetables, is being studied for its antioxidant properties. This study aimed to explore beta-carotene's protective effects against gentamicin-induced hepatorenal toxicity. MethodThirty male Wistar-rats were divided into five groups. Control group received normal-saline, while the canola group received canola oil (beta-carotene solvent). Gentamicin group received 100 mg/kg gentamicin injections for seven days. Beta-carotene groups were treated with beta-carotene at doses of 10 and 20 mg/kg for 10 days, along with gentamicin from the fourth day for 7 days. Serum and tissue hepatorenal function tests were performed at the end of the study. ResultsGentamicin resulted in hepatorenal damage. Beta-carotene alongside gentamicin significantly decreased serum SGOT (152.3 ± 12.7 vs. 264.8 ± 9.3 IU/L), SGPT (65.7 ± 2.5 vs. 98.0 ± 4.8 IU/L), creatinine (0.74 ± 0.0 vs. 1.5 ± 0.1 mg/dL), and urea (78.1 ± 10.7 vs. 207.4 ± 23.6 mg/dL) in comparison to gentamicin alone (p < 0.05). Beta-carotene caused a significant decrease in vacuolar degeneration, interstitial nephritis and infiltration of lymphocytes in kidney, and cell necrosis, vacuolar degeneration and infiltration of leukocytes compared to the gentamicin group; additionally, beta-carotene prevented increase in oxidative stress in gentamicin group. ConclusionAdministration of gentamicin alone resulted in hepatorenal toxicity, whereas beta-carotene could prevent gentamicin-induced oxidative stress imbalance and tissue damage. Therefore, beta-carotene could serve as an adjunctive therapy to mitigate hepatorenal toxicity in patients undergoing gentamicin treatment.

OTUB1 regulation of ferroptosis and the protective role of ferrostatin-1 in lupus nephritis

Lupus nephritis (LN) is a prevalent and severe manifestation of systemic lupus erythematosus (SLE), leading to significant morbidity and mortality. OTUB1, a deubiquitinating enzyme, has emerged as a potential therapeutic target due to its role in cellular protection and regulation of ferroptosis, a form of cell death linked to LN. Our study revealed significantly reduced OTUB1 expression in the glomeruli of LN patients and podocytes, correlated with disease severity. CRISPR/Cas9-mediated OTUB1 knockout in podocytes resulted in pronounced injury, indicated by decreased levels of nephrin and podocin. Ferrostatin-1 treatment effectively mitigated this injury, restoring SLC7A11 expression and significantly reducing MDA levels, Fe2+ levels, BODIPY C11 expression, and normalized cysteine and glutathione expression. In the MRL/lpr mouse model, Ferrostatin-1 significantly improved renal function decreased proteinuria, and ameliorated renal histopathological changes, including reduced glomerular endothelial swelling, mesangial cell proliferation, and leukocyte infiltration. These results underscore the protective role of Ferrostatin-1 in modulating the pathogenesis of LN. OTUB1 plays a crucial protective role against podocyte injury in LN by regulating ferroptosis. Ferrostatin-1 effectively mitigates podocyte damage induced by OTUB1 deficiency, suggesting that targeting ferroptosis could be a promising therapeutic strategy for LN.

Acetyl 11-Keto Beta-Boswellic Acid Improves Neurological Functions in a Mouse Model of Multiple Sclerosis.

Acetyl-11-keto-β-boswellic acid is one of the main active components of Boswellia sp. resin with the most potent anti-inflammatory activity. In recent years, herbal therapy has received considerable attention for the treatments of inflammatory and demyelinating diseases such as Multiple sclerosis (MS). Studies have shown that herbal compounds could enhance myelin repair and suppress inflammation. This study was designed to investigate the therapeutic effects of intraperitoneal administration of AKBA in Experimental Autoimmune Encephalomyelitis (EAE), as an animal model of MS. Following EAE induction in female C57BL/6J mice, animals were treated with AKBA and the levels of different serum inflammatory mediators, as well as motor functions, myelination, and inflammatory cell infiltration were assessed. Our results revealed that the application of AKBA alleviated EAE clinical severity, and suppressed inflammation, demyelination, leukocyte infiltration, and gliosis in EAE mice. Our findings suggest that the therapeutic effects of AKBA are likely a consequence of its neuroprotective and anti-inflammatory properties. The beneficial effects of AKBA may therefore provide new insights in various neuroinflammatory diseases such as MS and thereby could serve as a potential treatment candidate.

Lysophosphatidylcholine Acyltransferase 2 Contributes to Increased Allergic and Irritant Inflammation in Mice.

Platelet-activating factor (PAF) is an important chemical mediator in the field of inflammation, but its function in the skin is unclear. To unravel the role of PAF, we focused on lysophosphatidylcholine acyltransferase 2 (LPCAT2 also called LPLAT9), a biosynthetic enzyme involved in PAF production, and investigated the role of PAF in allergic contact dermatitis (ACD) and irritant contact dermatitis (ICD). We measured the amount of PAF in the skin and investigated the ear swelling responses and leukocyte infiltration into the skin following the application of 2,4,6-trinitro-1-chlorobenzene (TNCB) or croton oil in wild-type (WT) and LPCAT2 knockout (LPCAT2-KO) mice. The amount of PAF was increased in the skin of WT mice after TNCB or croton oil application but not detected in LPCAT2-KO mice. The ear swelling response was decreased in LPCAT2-KO mice compared with that in WT mice. In the ACD model, the numbers of lymphocytes, eosinophils, macrophages, mast cells and neutrophils were smaller in LPCAT2-KO mice than in WT mice. In the ICD model, the ear swelling response was also decreased in LPCAT2-KO mice compared with that in WT mice. When double staining of each inflammatory cell type and LPCAT2 was performed in ACD tissue, marked co-staining of the eosinophil marker and LPCAT2 was observed. In addition, LPCAT2 expression was observed in the epidermis. These results indicate that PAF is involved in the infiltration of several cell types into the sites of allergic and non-allergic skin inflammation. Furthermore, eosinophils and keratinocytes are primarily responsible for PAF production in skin inflammation.