Mouse Peritoneal Research Articles

SPHK1/S1P/S1PR pathway promotes the progression of peritoneal fibrosis by mesothelial-mesenchymal transition.

Long-term exposure to non-physiologically compatible dialysate inevitably leads to peritoneal fibrosis (PF) in patients undergoing peritoneal dialysis (PD), and there is no effective prevention or treatment for PF. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid produced after catalysis by sphingosine kinase (SPHK) 1/2 and activates signals through the S1P receptor (S1PR) via autocrine or paracrine. However, the role of SPHK1/S1P/S1PR signaling has never been elucidated in PF. In our research, we investigated S1P levels in peritoneal effluents and demonstrated the role of SPHK1/S1P/S1PR pathway in peritoneal fibrosis. It was found that S1P levels in peritoneal effluents were positively correlated with D/P Cr (r = 0.724, p < .001) and negatively correlated with 4 h ultrafiltration volume (r = -0.457, p < .001). S1PR1 and S1PR3 on peritoneal cells were increased after high glucose exposure invivo and invitro. Fingolimod was applied to suppress S1P/S1PR pathway. Fingolimod restored mouse peritoneal function by reducing interstitial hyperplasia, maintaining ultrafiltration volume, reducing peritoneal transport solute rate, and mitigating the protein expression changes of fibronectin, vimentin, α-SMA, and E-cadherin induced by PD and S1P. Fingolimod preserved the morphology of the human peritoneal mesothelial cells, MeT-5A, and moderated the mesothelial-mesenchymal transition (MMT) process. We further delineated that SPHK1 was elevated in peritoneal cells after high glucose exposure and suppression of SPHK1 in MeT-5A cells reduced S1P release. Overexpression of SPHK1 in MeT-5A cells increased S1P levels in the supernatant and fostered the MMT process. PF-543 treatment, targeting SPHK1, alleviated deterioration of mouse peritoneal function. In conclusion, S1P levels in peritoneal effluent were correlated with the deterioration of peritoneal function. SPHK1/S1P/S1PR pathway played an important role in PF.

Cultures of Human Skin Mast Cells, an Attractive In Vitro Model for Studies of Human Mast Cell Biology.

Studies of mast cell biology are dependent on relevant and validated in vitro models. Here, we present detailed information concerning the phenotype of both freshly isolated human skin mast cells (MCs) and of in vitro cultures of these cells that were obtained by analyzing their total transcriptome. Transcript levels of MC-related granule proteins and transcription factors were found to be remarkably stable over a 3-week culture period. Relatively modest changes were also seen for important cell surface receptors including the high-affinity receptor for IgE, FCER1A, the low-affinity receptor for IgG, FCGR2A, and the receptor for stem cell factor, KIT. FCGR2A was the only Fc receptor for IgG expressed by these cells. The IgE receptor increased by 2-5-fold and an approximately 10-fold reduction in the expression of FCGR2A was observed most likely due to the cytokines, SCF and IL-4, used for expanding the cells. Comparisons of the present transcriptome against previously reported transcriptomes of mouse peritoneal MCs and mouse bone marrow-derived MCs (BMMCs) revealed both similarities and major differences. Strikingly, cathepsin G was the most highly expressed granule protease in human skin MCs, in contrast to the almost total absence of this protease in both mouse MCs. Transcript levels for the majority of cell surface receptors were also very low compared to the granule proteases in both mouse and human MCs, with a difference of almost two orders of magnitude. An almost total absence of T-cell granzymes was observed in human skin MCs, indicating that granzymes have no or only a minor role in human MC biology. Ex vivo skin MCs expressed high levels of selective immediate early genes and transcripts of heat shock proteins. In validation experiments, we determined that this expression was an inherent property of the cells and not the result of the isolation process. Three to four weeks in culture results in an induction of cell growth-related genes accompanying their expansion by 6-10-fold, which increases the number of cells for in vitro experiments. Collectively, we show that cultured human skin MCs resemble their ex vivo equivalents in many respects and are a more relevant in vitro model compared to mouse BMMCs for studies of MC biology, in particular human MC biology.

TLR4 Ligands in Typhoid Vi Polysaccharide Subunit Vaccines Contribute to Immunogenicity.

Activation of B cells and T cells requires the engagement of costimulatory signaling pathways in addition to Ag receptor signaling for efficient immune responses. None of the typhoid Vi polysaccharide (ViPS) subunit vaccines contains adjuvants that could activate costimulatory signaling pathways, yet these vaccines are very immunogenic. I hypothesized that residual TLR ligands present in the ViPS preparation used for making typhoid subunit vaccines account for the robust immune response generated by these vaccines. I show the presence of endotoxin, a potent agonist of TLR4, in ViPS preparations and ViPS vaccines. Furthermore, I found that ViPS obtained from various sources induces the production of proinflammatory cytokines such as IL-6 from mouse peritoneal exudate cells. Unconjugated and tetanus toxoid-conjugated ViPS vaccines activate human and mouse TLR4. Mice deficient in TLR4 or the signaling adaptors MyD88 and Trif (Toll/IL-1R domain-containing adapter inducing IFN-β) are severely impaired in generating anti-ViPS responses to these vaccines. Elimination of the TLR4 agonist in ViPS preparation resulted in the loss of immunogenicity, and addition of lipid A, a known TLR4 agonist, restored the immunogenicity. These data highlight the importance of associated TLR ligands in the immunogenicity of ViPS subunit vaccines.

Antibacterial Activity and Proposed Mode of Action of Extracts from Selected Zimbabwean Medicinal Plants against Acinetobacter baumannii.

Acinetobacter baumannii was identified by the WHO as a priority pathogen in which the research and development of new antibiotics is urgently needed. Plant phytochemicals have potential as sources of new antimicrobials. The objective of the study was to determine the antibacterial activity of extracts of selected Zimbabwean medicinal plants against A. baumannii and determine their possible mode of action. Extracts were prepared from the leaves of the eight plants including the bark of Erythrina abyssinica using solvents of different polarities. Antibacterial activity was evaluated using the microbroth dilution method coupled with the in vitro iodonitrotetrazolium colorimetric assay. The effect of the extracts on membrane integrity was determined by quantifying the amount of protein and nucleic acid leaked from the cells after exposure to the extracts. The effects of the extracts on biofilms were investigated. Toxicity studies were carried out using sheep erythrocytes and murine peritoneal cells. Seven out of eight evaluated plant extracts were found to have antibacterial activity. The Combretum apiculatum acetonie (CAA) extract showed the highest inhibitory activity against A. baumannii with a minimal inhibitory concentration of 125 µg/mL. The minimum inhibitory concentration (MIC) of the CAA extract caused a protein leakage of 32 µg/mL from A. baumannii. The Combretum apiculatum acetonie (CAA), C. apiculatum methanolic (CAM), Combretum zeyheri methanolic (CZM), and Erythrina abyssinica methanolic (EAM) extracts inhibited A. baumannii biofilm formation. The EAM extract was shown to disrupt mature biofilms. The potent extracts were nontoxic to sheep erythrocytes and mouse peritoneal cells. The activities shown by the extracts indicate that the plants have potential as sources of effective antibacterial and antibiofilm formation agents against A. baumannii.

Human serous cavity macrophages and dendritic cells possess counterparts in the mouse with a distinct distribution between species.

In mouse peritoneal and other serous cavities, the transcription factor GATA6 drives the identity of the major cavity resident population of macrophages, with a smaller subset of cavity-resident macrophages dependent on the transcription factor IRF4. Here we showed that GATA6+ macrophages in the human peritoneum were rare, regardless of age. Instead, more human peritoneal macrophages aligned with mouse CD206+ LYVE1+ cavity macrophages that represent a differentiation stage just preceding expression of GATA6. A low abundance of CD206+ macrophages was retained in C57BL/6J mice fed a high-fat diet and in wild-captured mice, suggesting that differences between serous cavity-resident macrophages in humans and mice were not environmental. IRF4-dependent mouse serous cavity macrophages aligned closely with human CD1c+CD14+CD64+ peritoneal cells, which, in turn, resembled human peritoneal CD1c+CD14-CD64- cDC2. Thus, major populations of serous cavity-resident mononuclear phagocytes in humans and mice shared common features, but the proportions of different macrophage differentiation stages greatly differ between the two species, and dendritic cell (DC2)-like cells were especially prominent in humans.

Background formation of lower levels of reactive oxygen species by neutrophils after hypomagnetic field exposure is not accompanied by the impairment of chemiluminescence response of neutrophils to respiratory-burst stimuli

This study shows that the background formation of lower levels of reactive oxygen species in mouse peritoneal neutrophils after short-term (40 minutes) exposure to hypomagnetic fields with ~10 nT residual field at physiological temperatures, which has been detected by use of lucigenin-dependent chemiluminescence, is not accompanied by the impairment of chemiluminescence response of neutrophils to respiratory-burst stimuli: the formylated tripeptide N-formyl-Met-Leu-Phe (fMLF) and phorbol ester phorbol-12-myristate-13-ace-tate (PMA). These results were obtained with lucigenin or luminol-enhanced activated chemiluminescence and various combinations of reactive oxygen species production stimuli (phorbol-12-myristate-13-acetate and/or N-formyl-Met-Leu-Phe). Based on the results of the present work as well as on those of previous studies, this study reveal that the systems that control the respiratory burst in neutrophils can be excluded from a list of main targets and acceptors that respond to short-term deprivation of the magnetic field.

Human placental extract suppresses mast cell activation and induces mast cell apoptosis

BackgroundHuman placental extract (HPE) has been documented to facilitate the healing of certain disorders including allergy. However, the effects of HPE on the functionality of mast cells, a critical cell type in allergic diseases, have not been reported.MethodsTo investigate the effects of HPE on the regulation of allergy with respect to the biological functions of mast cells, the mast cell line C57 or HMC-1 cells were treated with HPE followed by the assessment of cell proliferation, apoptosis, activation, chemotaxis and phagocytosis. Mouse peritoneal mast cells were also investigated for their responses to induction of apoptosis by HPE in vivo. Furthermore, the effect of HPE on mast cell degranulation was confirmed using the passive cutaneous anaphylaxis (PCA) assay, an acute allergy model.ResultsHPE was capable of suppressing mast cell proliferation and inducing mast cell apoptosis. Mast cell degranulation in response to compound 48/80- or anti-DNP IgE and DNP-mediated activation was suppressed. In addition, treatment with HPE compromised the production of cytokines by mast cells and cell chemotaxis. These observations were consistent with the dampened passive cutaneous anaphylaxis (PCA) assay following treatment with HPE.ConclusionThis study revealed a suppressive effect of HPE on overall mast cell activities, suggesting a potential regulatory role of HPE on the alleviation of allergic diseases through mast cells.

Fluorescence-guided photoimmunotherapy using targeted nanotechnology and ML7710 to manage peritoneal carcinomatosis.

Fluorescence-guided intervention can bolster standard therapies by detecting and treating microscopic tumors before lethal recurrence. Tremendous progress in photoimmunotherapy and nanotechnology has been made to treat metastasis. However, many are lost in translation due to heterogeneous treatment effects. Here, we integrate three technological advances in targeted photo-activable multi-agent liposome (TPMAL), fluorescence-guided intervention, and laser endoscopy (ML7710) to improve photoimmunotherapy. TPMAL consists of a nanoliposome chemotherapy labeled with fluorophores for tracking and photosensitizer immunoconjugates for photoimmunotherapy. ML7710 is connected to Modulight Cloud to capture and analyze multispectral emission from TPMAL for fluorescence-guided drug delivery (FGDD) and fluorescence-guided light dosimetry (FGLD) in peritoneal carcinomatosis mouse models. FGDD revealed that TPMAL enhances drug delivery to metastases by 14-fold. ML7710 captured interpatient variability in TPMAL uptake and prompted FGLD in >50% of animals. By combining TPMAL, ML7710, and fluorescence-guided intervention, variation in treatment response was substantially reduced and tumor control improved without side effects.

RACK1 mediates NLRP3 inflammasome activation during Pasteurella multocida infection

Pasteurella multocida is a gram-negative bacterium that causes serious diseases in a wide range of animal species. Inflammasomes are intracellular multimolecular protein complexes that play a critical role in host defence against microbial infection. Our previous study showed that bovine P. multocida type A (PmCQ2) infection induces NLRP3 inflammasome activation. However, the exact mechanism underlying PmCQ2-induced NLRP3 inflammasome activation is not clear. Here, we show that NLRP3 inflammasome activation is positively regulated by a scaffold protein called receptor for activated C kinase 1 (RACK1). This study shows that RACK1 expression was downregulated by PmCQ2 infection in primary mouse peritoneal macrophages and mouse tissues, and overexpression of RACK1 prevented PmCQ2-induced cell death and reduced the numbers of adherent and invasive PmCQ2, indicating a modulatory role of RACK1 in the cell death that is induced by P. multocida infection. Next, RACK1 knockdown by siRNA significantly attenuated PmCQ2-induced NLRP3 inflammasome activation, which was accompanied by a reduction in the protein expression of interleukin (IL)-1β, pro-IL-1β, caspase-1 and NLRP3 as well as the formation of ASC specks, while RACK1 overexpression by pcDNA3.1-RACK1 plasmid transfection significantly promoted PmCQ2-induced NLRP3 inflammasome activation; these results showed that RACK1 is essential for NLRP3 inflammasome activation. Furthermore, RACK1 knockdown decreased PmCQ2-induced NF-κB activation, but RACK1 overexpression had the opposite effect. In addition, the immunofluorescence staining and immunoprecipitation results showed that RACK1 colocalized with NLRP3 and that NEK7 and interacted with these proteins. However, inhibition of potassium efflux significantly attenuated the RACK1-NLRP3-NEK7 interaction. Our study demonstrated that RACK1 plays an important role in promoting NLRP3 inflammasome activation by regulating NF-κB and promoting NLRP3 inflammasome assembly.

Semaphorin 3 C enhances putative cancer stemness and accelerates peritoneal dissemination in pancreatic cancer

PurposeSemaphorins, axon guidance cues in neuronal network formation, have been implicated in cancer progression. We previously identified semaphorin 3 C (SEMA3C) as a secreted protein overexpressed in pancreatic ductal adenocarcinoma (PDAC). We, therefore, hypothesized that SEMA3C supports PDAC progression. In this study, we aimed to investigate the clinical features of SEMA3C, especially its association with chemo-resistance and peritoneal dissemination.MethodsIn resected PDAC tissues, we assessed the relationship between SEMA3C expression and clinicopathological features by immunohistochemistry. In vitro studies, we have shown invasion assay, pancreatosphere formation assay, colony formation assay, cytotoxicity assay, and activation of SEMA3C downstream targets (c-Met, Akt, mTOR). In vivo, we performed a preclinical trial to confirm the efficacy of SEMA3C shRNA knockdown and Gemcitabine and nab-Paclitaxel (GnP) in an orthotopic transplantation mouse model and in peritoneal dissemination mouse model.ResultsIn resected PDAC tissues, SEMA3C expression correlated with invasion and peritoneal dissemination after surgery. SEMA3C promoted cell invasion, self-renewal, and colony formation in vitro. We further demonstrated that SEMA3C knockdown increased Gem-induced cytotoxicity by suppressing the activation of the Akt/mTOR pathway via the c-Met receptor. Combination therapy with SEMA3C knockdown and GnP reduced tumor growth and peritoneal dissemination.ConclusionsSEMA3C enhances peritoneal dissemination by regulating putative cancer stemness and Gem resistance and activates phosphorylation of the Akt/mTOR pathway via c-Met. Our findings provide a new avenue for therapeutic strategies in regulating peritoneal dissemination during PDAC progression.

Synthesis of Liposomes Conjugated with CpG Oligonucleotide and Loaded with a Set of T-Cell Epitopes of the SARS-CoV-2 Virus

The synthesis of lipid conjugate of immunostimulatory oligodeoxyribonucleotide CpG-ODN (PD-CpG-DOPE) is described. Liposomes loaded with a composition of T-cell epitopes of the SARS-CoV-2 virus (7 peptides) and carrying PD-CpG-DOPE conjugate in the membrane, including lyophilized liposomes suitable for long-term storage, were prepared. In vitro experiments on mouse peritoneal exudate cells showed a tendency to increase the immunogenicity of liposomes with peptides when PD-CpG-DOPE conjugate was introduced into the lipid bilayer, compared with the addition of the (commercial) phosphorothioate derivative of CpG-ODN in solution.

#4662 MSC-DERIVED EXOSOMES PREVENT PERITONEAL FIBROBLAST TRANSDIFFERENTIATION IN PERITONEAL DIALYSIS-ASSOCIATED PERITONEAL FIBROSIS

Abstract Background and Aims Peritoneal dialysis (PD) as a long-term renal replacement therapy is limited by peritoneal fibrosis. The purpose of our study was to identify the mechanism of mesenchymal stem cell-derived exosomes (MSC-Exos) in regulating the transdifferentiation of fibroblasts (FBs) to myofibroblasts (MFBs) in peritoneal dialysis-associated peritoneal fibrosis. Method The experimental mice were divided into 3 groups according to treatment: control group (Saline), peritoneal injury group (2.5% glucose peritoneal dialysate + LPS), and exosome group (2.5% glucose peritoneal dialysate + LPS + MSC-Exos). After six weeks of modeling, the parietal peritoneum was collected for histological analyses and transcriptomic analysis. And we verified the effects of MSC-Exos on fibroblasts in vitro and in vivo experiments referred to immunofluorescence, quantitative real-time RT–PCR assay, and western blotting analysis. Results First, tracing study of DiI-labeled exosome showed that exosomes can enter peritoneal tissue and peritoneal cells in vivo and in vitro. Our studies have found that MSC-Exos can alleviate peritoneal fibrosis in peritoneal injury mouse model. MSC-Exos treatment significantly decreased the expression of mouse peritoneal MFBs markers (α-SMA), while maintaining the phenotype of fibroblasts (Collagen I), indicating that MSC-Exos might inhibit the transdifferentiation of FBs to MFBs. The in vitro results further confirmed these findings. So we supported that MSC-Exos could alleviate peritoneal fibrosis by inhibiting the transdifferentiation of FBs into MFBs. Transcriptomic analysis results revealed that SPIC gene which is related to cell differentiation ranked first in the differential expression genes. The expression of Spi-C was significantly increased in peritoneal injury group, while were significantly down-regulated in MSC-Exos group. The results showed that Spi-C overexpression or knockdown had no significant impact in MSC-Exos group, while the transdifferentiation of FBs into MFBs greatly increased with Spi-C overexpression and significantly decreased with Spi-C knockdown in peritoneal injury group. The results indicated that Spi-C might be a critical molecule to promote peritoneal FBs to differentiate into MFBs in peritoneal injury mice. Moreover, the miRNA sequencing results showed that miR-34b-3p is one of the main components of MSC-Exos, and it can specifically bind to SPIC gene sequence. The dual-luciferase reporter assay revealed that miR-34b-3p mimic significantly reduced the luciferase activity of Spi-C reporter. And miR-34b-3p mimics could down-regulate the expression of Spi-C, and the expression of Spi-C was not affected after miR-34b-3p inhibitor intervention. The above results suggested that MSC-Exos-derived miR-34b-3p could inhibit the transdifferentiation of FBs into MFBs by targeting Spi-C. Conclusion MSC-Exos-derived miR-34b-3p targeting Spi-C can alleviate peritoneal fibrosis by inhibiting the differentiation of subperitoneal mesothelial FBs into MFBs.

Giant Peritoneal Loose Body Causing Acute Intestinal Obstruction: Case Report

Peritoneal loose bodies are a rare condition. They are caused by the peritoneal appendages that become inflamed and cause a buildup of material in the peritoneal cavity. This can be hard to spot because it's so rare. The availability of CT scans makes this diagnosis easier. A 77-year-old patient with hypertension and diabetes underwent peritoneal mouse challenge. The patient suffers from acute intestinal sub obstruction. CT scan revealed a mobile calcified density lesion approximately 5 cm in diameter in the peritoneal cavity. Peritoneal loose bodies are a rare complication of omental appendages that have distorted due to ischemic conditions. Laparoscopy excised a firm mass suggestive of primary omental appendicitis. Diagnostic terms for acute or chronic abdominal pain can be found in the general medical literature. Photographs of the affected individual's body show a large greasy or chalky substance.

FOXO3 Deficiency in Neutrophils Drives Colonic Inflammation and Tumorigenesis.

Inflammatory bowel disease (IBD), characterized by infiltration of polymorphonuclear neutrophils (PMNs), increases the risk of colon cancer. PMN activation corresponds to the accumulation of intracellular Lipid Droplets (LDs). As increased LDs are negatively regulated by transcription factor Forkhead Box O3 (FOXO3), we aim to determine the significance of this regulatory network in PMN-mediated IBD and tumorigenesis. Affected tissue of IBD and colon cancer patients, colonic and infiltrated immune cells, have increased LDs' coat protein, PLIN2. Mouse peritoneal PMNs with stimulated LDs and FOXO3 deficiency have elevated transmigratory activity. Transcriptomic analysis of these FOXO3-deficient PMNs showed differentially expressed genes (DEGs; FDR < 0.05) involved in metabolism, inflammation, and tumorigenesis. Upstream regulators of these DEGs, similar to colonic inflammation and dysplasia in mice, were linked to IBD and human colon cancer. Additionally, a transcriptional signature representing FOXO3-deficient PMNs (PMN-FOXO3389) separated transcriptomes of affected tissue in IBD (p = 0.00018) and colon cancer (p = 0.0037) from control. Increased PMN-FOXO3389 presence predicted colon cancer invasion (lymphovascular p = 0.015; vascular p = 0.046; perineural p = 0.03) and poor survival. Validated DEGs from PMN-FOXO3389 (P2RX1, MGLL, MCAM, CDKN1A, RALBP1, CCPG1, PLA2G7) are involved in metabolism, inflammation, and tumorigenesis (p < 0.05). These findings highlight the significance of LDs and FOXO3-mediated PMN functions that promote colonic pathobiology.

TMT quantitative proteomics and network pharmacology reveal the mechanism by which asiaticoside regulates the JAK2/STAT3 signaling pathway to inhibit peritoneal fibrosis.

Traditional Chinese medicine, Centella asiatica (L.) Urb., has been extensively utilized in clinics to treat a variety of fibrotic disorders. Asiaticoside (ASI), as an important active ingredient, has attracted much attention in this field. However, the effect of ASI on peritoneal fibrosis (PF) is still unclear. Therefore, we evaluated the benefits of ASI for PF and mesothelial-mesenchymal transition (MMT) and revealed the underlying mechanisms. The objective of this investigation was to anticipate the potential molecular mechanism of ASI against peritoneal mesothelial cells (PMCs) MMT employing proteomics and network pharmacology, and to confirm it using in vivo and in vitro studies. The mesentery of peritoneal fibrosis mice and normal mice were analyzed quantitatively for proteins that were differentially expressed using a technique tandem mass tag (TMT). Next, the core target genes of ASI against PF were screened through network pharmacology analysis, and PPI and C-P‒T networks were constructed by Cytoscape Version 3.7.2. According to the findings of a GO and KEGG enrichment analysis of differential proteins and core target genes, the signaling pathway with a high correlation degree was selected as the key signaling pathway of ASI inhibiting the PMCs MMT for further molecular docking analysis and experimental verification. TMT-based quantitative proteome analysis revealed the identification of 5727 proteins, of which 70 were downregulated and 178 were upregulated. Among them, the levels of STAT1, STAT2, and STAT3 in the mesentery of mice with peritoneal fibrosis were considerably lower than in the control group, indicating a role for the STAT family in the pathogenesis of peritoneal fibrosis. Then, a total of 98 ASI-PF-related targets were identified by network pharmacology analysis. JAK2 is one of the top 10 core target genes representing a potential therapeutic target. JAK/STAT signaling may represent a core pathway mediating PF effects by ASI. Molecular docking studies showed that ASI had the potential to interact favorably with target genes involved in the JAK/STAT signaling pathway, such as JAK2 and STAT3.The experimental results showed that ASI could significantly alleviate Chlorhexidine Gluconate (CG)-induced peritoneal histopathological changes and increase JAK2 and STAT3 phosphorylation levels. In TGF-β1-stimulated HMrSV5 cells, E-cadherin expression levels were dramatically reduced whereas Vimentin, p-JAK2, α-SMA, and p-STAT3 expression levels were considerably increased. ASI inhibited the TGF-β1-induced HMrSV5 cell MMT, decreased the activation of JAK2/STAT3 signaling, and increased the nuclear translocation of p-STAT3, which was consistent with the effect of the JAK2/STAT3 pathway inhibitor AG490. ASI can inhibit PMCs MMT and alleviate PF by regulating the JAK2/STAT3 signaling pathway.

Upregulating the Expression of F-CAR-T Cells by Increasing theAmount of Tcm Cells in F-CAR-T Cells

CAR-T cells are most known for protecting one’s immune system by attacking cancer cells. Usually, it is infused intothe patient’s body to provide treatment. When comparing F-CAR-T cells with C-CAR-T cells, F-CAR-T cells typicallyhave more Tscm cells. Thus, this study investigates the effect of upregulating Tscm cells in F-CAR-T cells using FACSfor CD45RO- and CD62L+. The experiments will use the expression of Tim3, LAG3, and PD1 and measure if F-CAR-Tcells can kill CD19-expressing RAJI cells by FACS for CD19 and Annexin V/PI through various durations and withvarious numbers of injected CarT. Then Tscm cells are measured by FACS for CD45RO- and CD62L+. Moreover,two special diets will be used as an experimental control to regulate the increase and decrease of Tscm cells. There aretwo most possible results: (1) Both the Tscm receptor and the diet will have a positive effect in terms of upregulatingTscm cells in F-CAR-T cells; (2) Both the mouse peritoneal macrophage and the diet will inhibit the growth of Tscmcells. The result of our study will provide important information for the future clinical trial of Tscm cell application andimprove CAR-T cells’ persistence. Future studies should focus on shortening the CAR-T cells manufacturing processesas well as exploring more applicable usages of Tscm cells in detail

Anti-inflammatory and Antimicrobial Potential of Three Natural Polyketides Isolated from Endophytic Fungus Phomopsis sp CAM212 against to Dysenteric Causing Pathogens

Aims: The present work aimed to evaluate the anti-amoebic, antibacterial, and anti-inflammatory potential of three natural polyketides from Phomopsis sp. CAM212.&#x0D; Study Design: Clinical isolates of E.histolytica, E.coli ATCC25922 strain, primary peritoneal mouse macrophages and three polyketides were used.&#x0D; Places and Duration of Study: Laboratory of Pharmacology and Toxicology, Laboratory of Medical Microbiology, Faculty of Science, University of Yaounde 1 between May and December 2022.&#x0D; Methodology: During this work, we evaluated the ability of three natural polyketides from Phomopsis sp to inhibit the growth of germs responsible for amoebic and bacillary dysentery. First, the anti-amoebic activity was carried out on clinical isolates of E. histolytica in polyxenic culture. Subsequently, we evaluated the antibacterial potential on a strain of E. coli ATCC25922. Finally, the anti-inflammatory potentials were evaluated on a primary culture of SC activated macrophages through inhibition of nitric oxide (NO) production, activation of phosphatase alcaline (ALP) and inhibition of 5-lipoxygenase (5-LOX).&#x0D; Results: It emerges from this work that among compounds, phomopsinin B, presented the highest anti-amoebic potential (84.4 % inhibition after 72h) and the highest antibacterial potential (MIC=12.5µg/mL and MBC/MIC=2). Phomopsini A and phomopsini A acetate showed moderate anti amoebic and antibacterial potentials. However, all these activities remain lower than that of metronidazole and ciprofloxacin (90% of amoebic inhibition after 72h; MIC=0.72µg/mL and MBC/MIC=4). Subsequently, all tested compounds were nontoxic on primary macrophages. Phomopsinin B exhibited a great anti-inflammatory potential through the inhibition of NO production (IC50=1.72±0.91µg/mL); inihibition of 5-LOX activity (IC50=36.97±7.12µg/mL) and activation of ALP activity (IC50=0.13±0.01µg/mL) as compared to Baicalin the standard. The anti-inflammatory potential of phomopsinin A and phomopsinin A acetate were lower compared to baicalin.&#x0D; Conclusion: Ultimately, among compounds tested, phomopsinin B exhibited the best anti-amoebic, antibacterial and ant-inflammatory potential similar to the respective standards within the limits of the tests carried out.

Knockdown of AK142426 suppresses M2 macrophage polarization and inflammation in peritoneal fibrosis via binding to c-Jun.

Peritoneal fibrosis is a common complication of peritoneal dialysis, which may lead to ultrafiltration failure and ultimately treatment discontinuation. LncRNAs participate in many biological processes during tumorigenesis. We investigated the role of AK142426 in peritoneal fibrosis. The AK142426 level in peritoneal dialysis (PD) fluid was detected by quantitative real-time-PCR assay. The M2 macrophage distribution was determined by flow cytometry. The inflammatory cytokines of TNF-α and TGF-β1 were measured by ELISA assay. The direct interaction between AK142426 and c-Jun was evaluated by RNA pull-down assay. In addition, the c-Jun and fibrosis related proteins were assessed by western blot analysis. The PD-induced peritoneal fibrosis mouse model was successfully established. More importantly, PD treatment induced M2 macrophage polarization and the inflammation in PD fluid, which might be associated with exosome transmission. Fortunately, AK142426 was observed to be upregulated in PD fluid. Mechanically, knockdown of AK142426 suppressed M2 macrophage polarization and inflammation. Furthermore, AK142426 could upregulate c-Jun through binding c-Jun protein. In rescue experiments, overexpression of c-Jun could partially abolish the inhibitory effect of sh-AK142426 on the activation of M2 macrophages and inflammation. Consistently, knockdown of AK142426 alleviated peritoneal fibrosis in vivo. This study demonstrated that knockdown of AK142426 suppressed M2 macrophage polarization and inflammation in peritoneal fibrosis via binding to c-Jun, suggesting that AK142426 might be a promising therapeutic target for patients of peritoneal fibrosis.

Immunomodulatory properties of recombinant human granulocyte-macrophage colony-stimulating factor

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a myelopoietic growth factor that exerts pleiotropic effect not only on the differentiation of immature progenitor cells into polymorphonuclear neutrophils, monocytes/macrophages and dendritic cells, but also controls the functioning of differentiated cells. GM-CSF is currently being investigated in clinical trials as an immunomodulator and adjuvant. However, a wide range of biological activities and, sometimes, paradoxical effects of this cytokine require more thorough studies of its action, in order to predict its efficacy under different conditions of immunotherapy. In this work, we have studied the effect of recombinant human GM-CSF on metabolic activity of mouse peritoneal exudate cells in primary cell cultures. Metabolic (redox) activity of the cells was assessed by their ability to reduce nitroblue tetrazolium (NBT) in the course of MF- and Fc-dependent phagocytosis triggered by addition of opsonized zymosan, or sheep erythrocytes to the culture medium. We have shown the dose-dependent stimulatory effect of GM-CSF on the oxidative metabolism of phagocytic peritoneal macrophages and neutrophils. Upon culturing the pepton-elicited cells at wide range of GM-CSF concentrations (5 to 40,000 ng/mL) for 2 and 24 hours, a more pronounced effect of the substance was observed for neutrophils. The GM-CSF preparation caused a significant increase (by 13-17%) in the redox activity of neutrophils induced by opsonized zymosan that persisted at a low dose range, and was retained after 24 hours. The stimulatory effect of GM-CSF on macrophages with NBT index increase by 16% was observed in the short-term cultures. In general, the elicited cells of both types showed a more pronounced response to lower concentrations of GM-CSF (5-125 ng/mL), and weaker effect at higher doses of the preparation. A similar dependence was found when studying the resident macrophages. Culturing of resident cells with GM-CSF at the doses of 5,000 to 40,000 ng/mL for 24 hours caused a significantly increased redox activity of the cells induced by zymosan, or sheep erythrocytes (by 33-52%). In both cases, the maximal response was detected at a dose of 5,000 ng/mL and decreased with increasing dose. The stimulatory effect of GM-CSF upon resident macrophages was more pronounced as compared to elicited cells, which was characterized by the prolonged period of cell activation (up to 24 hours of culture). The data obtained are of interest, in view of prospective usage of GM-CSF as a component of immunomodulatory and adjuvant therapy for various infectious diseases.

Berberine Induces Combined Cell Death in Gastrointestinal Cell Lines.

Berberine (BBR) is a plant alkaloid that has various biological activities. The effects of BBR on gastrointestinal cancer (GIC) have also been investigated and anti-tumor effects such as induction of cell death have been reported. However, the mechanism of BBR-induced cell death has not been fully elucidated. To this end, we investigated the effects of BBR using three GIC cell lines. Our analyses revealed that BBR inhibited cell proliferation, invasion, sphere formation, and anticancer drug resistance in all of the cell lines. BBR also induced an increase in mitochondrial superoxide, lipid peroxide and Fe2+ levels, decreased mitochondrial membrane potential and respiration, decreased glutathione peroxidase 4 expression and glutathione and induced Parkin/PINK1-associated mitophagy. BBR, as well as rotenone, inhibited mitochondrial complex I and enhanced complex II, which were associated with autophagy, reactive oxidative species production, and cell death. Inhibition of complex II by malonate abrogated these changes. BBR-induced cell death was partially rescued by ferrostatin-1, deferoxamine, Z-VAD-FMK, and ATG5 knockdown. Furthermore, oral administration of BBR significantly reduced tumor weight and ascites in a syngeneic mouse peritoneal metastasis model using CT26 GIC cells. These findings suggest that BBR induced a combined type of cell death via complex I inhibition and autophagy. The marked anti-tumor and anti-stemness effects are expected to be useful as a new cell death-inducing agent for the treatment of GIC.