Bioactive properties and gut microbiota modulation by date seed polysaccharides extracted using ultrasound-assisted deep eutectic solvent.

Polysaccharides are abundant macromolecules. The study extracted date seed polysaccharides (UPS) using ultrasound-assisted deep eutectic solvent extraction to valorize date seeds. UPS were subjected to comprehensive characterization and evaluation of their bioactivity, prebiotic properties, and their potential to modulate the gut microbiome. Characterization revealed UPS's heteropolysaccharide composition with galactose, mannose, fructose, glucose, and galacturonic acid respectively in 66.1, 13.3, 9.9, 5.4, and 5.1%. UPS showed a concentration-dependent increase of radical scavenging and antioxidant activities, evidenced by FRAP, TAC, and RP assays. They also displayed antimicrobial efficacy against E. coli O157:H7, S. typhimurium, S. aureus, and L. monocytogenes. Rheological analysis showed UPS's elastic-dominant nature with thixotropic tendencies. UPS inhibited α-glycosidase, α-amylase, and ACE up to 86%, and reduced Caco-2 and MCF-7 cell viability by 70% and 46%, respectively. UPS favored beneficial gut microbiota growth, releasing significant SCFAs during fecal fermentation.

Oral administration of Manuka honey induces IFNγ-dependent resistance to tumor growth that correlates with beneficial modulation of gut microbiota composition.

Background: To investigate the potential of Manuka honey (MH) as an immunomodulatory agent in colorectal cancer (CRC) and dissect the underlying molecular and cellular mechanisms. Methods: MH was administered orally over a 4 week-period. The effect of MH treatment on microbiota composition was studied using 16S rRNA sequencing of fecal pellets collected before and after treatment. Pretreated mice were implanted with CRC cells and followed for tumor growth. Tumors and lymphoid organs were analyzed by flow cytometry (FACS), immunohistochemistry and qRT-PCR. Efficacy of MH was also assessed in a therapeutic setting, with oral treatment initiated after tumor implantation. We utilized IFNγ-deficient mice to determine the importance of interferon signaling in MH-induced immunomodulation. Results: Pretreatment with MH enhanced anti-tumor responses leading to suppression of tumor growth. Evidence for enhanced tumor immunogenicity included upregulated MHC class-II on intratumoral macrophages, enhanced MHC class-I expression on tumor cells and increased infiltration of effector T cells into the tumor microenvironment. Importantly, oral MH was also effective in retarding tumor growth when given therapeutically. Transcriptomic analysis of tumor tissue highlighted changes in the expression of various chemokines and inflammatory cytokines that drive the observed changes in tumor immunogenicity. The immunomodulatory capacity of MH was abrogated in IFNγ-deficient mice. Finally, bacterial 16S rRNA sequencing demonstrated that oral MH treatment induced unique changes in gut microbiota that may well underlie the IFN-dependent enhancement in tumor immunogenicity. Conclusion: Our findings highlight the immunostimulatory properties of MH and demonstrate its potential utilization in cancer prevention and treatment.

Characterizing date seed polysaccharides: A comprehensive study on extraction, biological activities, prebiotic potential, gut microbiota modulation, and rheology using microwave-assisted deep eutectic solvent.

This study investigated the biological activities, prebiotic potentials, modulating gut microbiota, and rheological properties of polysaccharides derived from date seeds via microwave-assisted deep eutectic solvent systems. Averaged molecular weight (246.5 kDa) and a monosaccharide profile (galacturonic acid: glucose: mannose: fructose: galactose), classifying MPS as a heteropolysaccharide. MPS, at concentrations of 125-1000 µg/mL, demonstrates increasing free radical scavenging activities (DPPH, ABTS, MC, SOD, SORS, and LO), potent antioxidant potential (FRAP: 51.2-538.3 µg/mL; TAC: 28.3-683.4 µg/mL; RP: 18.5-171.2 µg/mL), and dose-dependent antimicrobial activity against common foodborne pathogens. Partially-purified MPS exhibits inhibition against α-glucosidase (79.6 %), α-amylase (85.1 %), and ACE (68.4 %), along with 80 % and 46 % inhibition against Caco-2 and MCF-7 cancer cells, respectively. Results indicate that MPS fosters the growth of beneficial fecal microbiota, including Proteobacteria, Firmicutes, and Actinobacteria, supporting microbes responsible for major SCFAs (acetic, propionic, and butyric acids) production, such as Ruminococcus and Blautia.

Innate and adaptive immune-directed tumour microenvironment in pancreatic ductal adenocarcinoma.

One of the most deadly and aggressive cancers in the world, pancreatic ductal adenocarcinoma (PDAC), typically manifests at an advanced stage. PDAC is becoming more common, and by the year 2030, it is expected to overtake lung cancer as the second greatest cause of cancer-related death. The poor prognosis can be attributed to a number of factors, including difficulties in early identification, a poor probability of curative radical resection, limited response to chemotherapy and radiotherapy, and its immunotherapy resistance. Furthermore, an extensive desmoplastic stroma that surrounds PDAC forms a mechanical barrier that prevents vascularization and promotes poor immune cell penetration. Phenotypic heterogeneity, drug resistance, and immunosuppressive tumor microenvironment are the main causes of PDAC aggressiveness. There is a complex and dynamic interaction between tumor cells in PDAC with stromal cells within the tumour immune microenvironment. The immune suppressive microenvironment that promotes PDAC aggressiveness is contributed by a range of cellular and humoral factors, which itself are modulated by the cancer. In this review, we describe the role of innate and adaptive immune cells, complex tumor microenvironment in PDAC, humoral factors, innate immune-mediated therapeutic advances, and recent clinical trials in PDAC.

Gut microbiota modulation, prebiotic and bioactive characteristics of date pomace polysaccharides extracted by microwave-assisted deep eutectic solvent.

This study investigated the characteristics of polysaccharides from date pomace using microwave-assisted deep eutectic solvents. The impact on the gut microbiota and probiotics growth was examined in vitro. The study also examined its antioxidant properties, ability to inhibit enzymes linked to diabetes and high blood pressure, impact on cell growth, and physical properties. The isolated MPS had an average molecular weight of 8073.38 kDa and contained mannose, galacturonic acid, galactose, glucose, and fructose in specific proportions. At a concentration of 1000 mg/L, MPS showed strong antioxidant activity, with significant scavenging rates in various tests such as DPPH (57.0 ± 1.05 %) and ABTS (66.4 ± 2.48 %). MPS displayed 77 %, 80 %, and 43 % inhibition for α-amylase, α-glucosidase, and ACE-inhibition, respectively. MPS displayed significant antiproliferative effects, achieving 100 % and 99 % inhibition against Caco-2 and MCF-7 cells at 2500 mg/L, respectively. MPS showed broad-spectrum antibacterial properties against both Gram-positive and Gram-negative foodborne bacteria. Gemmiger formicilis, Blautia species, Collinsella aerofaciens, and Bifidobacterium longum showed strong positive correlations, suggesting increased SCFA production. Network analysis indicated species correlations, with 86 % showing negative correlations with Escherichia and Enterococcus saccharolyticus. MPS was abundant in Firmicutes, Actinobacteria, and Proteobacteria phyla. Date pomace could serve as a dietary fiber source, promoting better health.

Interplay between Microbiota and γδ T Cells: Insights into Immune Homeostasis and Neuro-Immune Interactions.

The gastrointestinal (GI) tract of multicellular organisms, especially mammals, harbors a symbiotic commensal microbiota with diverse microorganisms including bacteria, fungi, viruses, and other microbial and eukaryotic species. This microbiota exerts an important role on intestinal function and contributes to host health. The microbiota, while benefiting from a nourishing environment, is involved in the development, metabolism and immunity of the host, contributing to the maintenance of homeostasis in the GI tract. The immune system orchestrates the maintenance of key features of host-microbe symbiosis via a unique immunological network that populates the intestinal wall with different immune cell populations. Intestinal epithelium contains lymphocytes in the intraepithelial (IEL) space between the tight junctions and the basal membrane of the gut epithelium. IELs are mostly CD8+ T cells, with the great majority of them expressing the CD8αα homodimer, and the γδ T cell receptor (TCR) instead of the αß TCR expressed on conventional T cells. γδ T cells play a significant role in immune surveillance and tissue maintenance. This review provides an overview of how the microbiota regulates γδ T cells and the influence of microbiota-derived metabolites on γδ T cell responses, highlighting their impact on immune homeostasis. It also discusses intestinal neuro-immune regulation and how γδ T cells possess the ability to interact with both the microbiota and brain.

Correction: Farnesyl pyrophosphate is a new danger signal inducing acute cell death.

[This corrects the article DOI: 10.1371/journal.pbio.3001134.].

Cis-Nerolidol Inhibits MAP Kinase and NF-κB Signaling Pathways and Prevents Epithelial Tight Junction Dysfunction in Colon Inflammation: In Vivo and In Vitro Studies.

Inflammation of the GI tract leads to compromised epithelial barrier integrity, which increases intestine permeability. A compromised intestinal barrier is a critical event that leads to microbe entry and promotes inflammatory responses. Inflammatory bowel diseases that comprise Crohn's disease (CD) and ulcerative colitis (UC) show an increase in intestinal permeability. Nerolidol (NED), a naturally occurring sesquiterpene alcohol, has potent anti-inflammatory properties in preclinical models of colon inflammation. In this study, we investigated the effect of NED on MAPKs, NF-κB signaling pathways, and intestine epithelial tight junction physiology using in vivo and in vitro models. The effect of NED on proinflammatory cytokine release and MAPK and NF-κB signaling pathways were evaluated using lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophages. Subsequently, the role of NED on MAPKs, NF-κB signaling, and the intestine tight junction integrity were assessed using DSS-induced colitis and LPS-stimulated Caco-2 cell culture models. Our result indicates that NED pre-treatment significantly inhibited proinflammatory cytokine release, expression of proteins involved in MAP kinase, and NF-κB signaling pathways in LPS-stimulated RAW macrophages and DSS-induced colitis. Furthermore, NED treatment significantly decreased FITC-dextran permeability in DSS-induced colitis. NED treatment enhanced tight junction protein expression (claudin-1, 3, 7, and occludin). Time-dependent increases in transepithelial electrical resistance (TEER) measurements reflect the formation of healthy tight junctions in the Caco-2 monolayer. LPS-stimulated Caco-2 showed a significant decrease in TEER. However, NED pre-treatment significantly prevented the fall in TEER measurements, indicating its protective role. In conclusion, NED significantly decreased MAPK and NF-κB signaling pathways and decreased tight junction permeability by enhancing epithelial tight junction protein expression.

Characterization of immunomodulatory responses induced by manuka honey.

Manuka honey (MH) is known for its wound-healing, anti-microbial, anti-oxidant and anti-tumor properties. However, there is conflicting evidence regarding the role of MH in inflammatory responses, with some studies highlighting its pro-inflammatory capacity and others showing that it has a predominantly anti-inflammatory activity. The current study is aimed at characterizing the immunomodulatory capacity of MH using both in vitro and in vivo approaches, focusing on the underlying mechanisms. Treatment of RAW 264.7 macrophages with 1% MH (w/v) resulted in a significant increase in the gene expression (~26-fold) and secretion (~27-fold) of tumor necrosis factor-alpha (TNF-α). Similarly, an increase was observed in the gene expression of other inflammatory cytokines including interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS), as well as the chemokines; (C-X-C motif) ligand 2 (CXCL2) and (C-C) motif ligand 2 (CCL2). Using an in vivo model, intraperitoneal (i.p.) administration of MH in C57BL/6 mice elicited a peritoneal response characterized by a significant expansion in the number of peritoneal exudate cells (PECs), which was mainly due to a 35-fold increase in the recruitment of neutrophils. Importantly, this response was evident in toll-like receptor 4 (TLR4)-defective C3H/HeJ mice, indicating that the observed stimulatory effect occurs independently of TLR4 and unlikely to be mediated by any lipopolysaccharide (LPS) contaminant. MH administration also led to changes in the phenotypic expression and functional maturation of peritoneal macrophages, as evidenced by a shift towards the CD11blo F4/80lo phenotype and an increase in the expression of major histocompatibility complex (MHC) class II proteins. In contrast, the MH-initiated peritoneal response was largely abrogated in mice deficient in myeloid differentiation primary response 88 (MyD88) protein, a critical adaptor of most TLR signaling pathways. Thus, the current findings help to characterize the immunostimulatory properties of MH and their dependence on TLR signaling, and highlight the potential utility of MH as an immunomodulatory agent in a variety of disorders.

Bioactive properties and untargeted metabolomics analysis of bioaccessible fractions of non-fermented and fermented date fruit pomace by novel yeast isolates.

In this study, attempts were made to utilize date by-product (date fruit pomace; DFP). This study aimed to investigate the health-promoting benefits of the fermented and non-fermented DFP before in vitro digestion and after (bioaccessible fraction). Untargeted metabolomic analyses for bioaccessible fractions were performed by UPLC-QTOF. DPPH percentages were 89.7%-90.3%, 90.1%-91.3%, and 90.8%-91.3% in the control, I. orientalis, and P. kudriazevii samples, respectively, before digestion; α-glucosidase inhibition before digestion was 1.9%-24.4%, 16.3%-30.0%, and 21.3%-31.3%, respectively; antimicrobial activities were 6.1%-13.3%, 13.7%-25.7%, and 20.6%-28.0% against E. coli O157:H7 and 2.2%-11.9%, 7.2%-20.7%, and 11.9%-29.2% against L. monocytogenes, respectively. The DPPH scavenging percentages were ∼63% lower in the bioaccessible fraction. The differentially regulated metabolites classes were benzene and derivatives, amino acids, peptides and analogs, organic acids, and phenols. This study revealed that the fermented DFP exhibited higher health properties than control.

Diabetes Mellitus Alters the Immuno-Expression of Neuronal Nitric Oxide Synthase in the Rat Pancreas.

Nitric oxide is generated from nitric oxide synthase following hyperglycemia-induced oxidative stress during the course of diabetes mellitus (DM). We examined the temporal immuno-expression of neuronal nitric oxide synthase (nNOS) in the pancreas of diabetic and non-diabetic rats using immunohistochemical, immunofluorescence and western blot techniques 12 h, 24 h, 1 week, 2 weeks, 1, 8 and 15 months after induction of DM. nNOS co-localized with pancreatic beta cells but disappears 12 h after the onset of DM. In contrast, the nNOS content of pancreatic nerves increased significantly (p < 0.001) 24 h after the induction of DM, and decreased sharply thereafter. However, nNOS-positive ganglion cells were observed even 15 months post-diabetes. ROS increased by more than 100% two months after the onset of DM compared to non-diabetic control but was significantly (p < 0.000001) reduced at 9 months after the induction of DM. The pancreatic content of GSH increased significantly (p < 0.02) after 9 months of DM. Although, TBARS content was significantly (p < 0.009; p < 0.002) lower in aged (9 months) non-diabetic and DM rats, TBARS rate was markedly (p < 0.02) higher 9 months after the induction of DM when compared to younger age group. In conclusion, nNOS is present in pancreatic beta cell, but disappears 12 h after the onset of diabetes. In contrast, the tissue level of nNOS of pancreatic nerves increased in the first week of diabetes, followed by a sharp reduction. nNOS may play important roles in the metabolism of pancreatic beta cell.

Lactic Acid Bacteria Isolated from Fresh Vegetable Products: Potential Probiotic and Postbiotic Characteristics Including Immunomodulatory Effects.

The ability to perform effectively in the gastrointestinal tract (GIT) is one of the most significant criteria in the selection of potential probiotic bacteria. Thus, the present study aimed to investigate the potential probiotic characteristics of some selected lactic acid bacteria (LAB) isolated from vegetable products. Probiotic characteristics included tolerance to acid and bile, cholesterol-removing ability, bile salt hydrolysis, resistance against lysozyme and antibiotics, production of exopolysaccharides (EPS), antimicrobial and hemolytic activities, and cell surface characteristics (auto-aggregation, co-aggregation, and hydrophobicity). The survival rate of isolates after G120 ranged from 8.0 to 8.6 Log10 CFU/mL. After the intestinal phase (IN-120), the bacterial count ranged from 7.3 to 8.5 Log10 CFU/mL. The bile tolerance rates ranged from 17.8 to 51.1%, 33.6 to 63.9%, and 55.9 to 72.5% for cholic acid, oxgall, and taurocholic acid, respectively. Isolates F1, F8, F23, and F37 were able to reduce cholesterol (>30%) from the broth. The auto-aggregation average rate increased significantly after 24 h for all isolates, while two isolates showed the highest hydrophobicity values. Moreover, isolates had attachment capabilities comparable to those of HT-29 cells, with an average of 8.03 Log10 CFU/mL after 2 h. All isolates were resistant to lysozyme and vancomycin, and 8 out of the 17 selected isolates displayed an ability to produce exopolysaccharides (EPS). Based on 16S rRNA sequencing, LAB isolates were identified as Enterococcus faecium, E. durans, E. lactis, and Pediococcus acidilactici.

Attenuated Salmonella potentiate PD-L1 blockade immunotherapy in a preclinical model of colorectal cancer.

The use of immune checkpoint inhibitors to treat cancer resulted in unprecedented and durable clinical benefits. However, the response rate among patients remains rather modest. Previous work from our laboratory demonstrated the efficacy of using attenuated bacteria as immunomodulatory anti-cancer agents. The current study investigated the potential of utilizing a low dose of attenuated Salmonella typhimurium to enhance the efficacy of PD-L1 blockade in a relatively immunogenic model of colon cancer. The response of MC38 tumors to treatment with αPD-L1 monoclonal antibody (mAb) was variable, with only 30% of the mice being responsive. Combined treatment with αPD-L1 mAb and Salmonella resulted in 75% inhibition of tumor growth in 100% of animals. Mechanistically, the enhanced response correlated with a decrease in the percentage of tumor-associated granulocytic cells, upregulation in MHC class II expression by intratumoral monocytes and an increase in tumor infiltration by effector T cells. Collectively, these alterations resulted in improved anti-tumor effector responses and increased apoptosis within the tumor. Thus, our study demonstrates that a novel combination treatment utilizing attenuated Salmonella and αPD-L1 mAb could improve the outcome of immunotherapy in colorectal cancer.

Integration of Salmonella into Combination Cancer Therapy.

Current modalities of cancer treatment have limitations related to poor target selectivity, resistance to treatment, and low response rates in patients. Accumulating evidence over the past few decades has demonstrated the capacity of several strains of bacteria to exert anti-tumor activities. Salmonella is the most extensively studied entity in bacterial-mediated cancer therapy, and has a good potential to induce direct tumor cell killing and manipulate the immune components of the tumor microenvironment in favor of tumor inhibition. In addition, Salmonella possesses some advantages over other approaches of cancer therapy, including high tumor specificity, deep tissue penetration, and engineering plasticity. These aspects underscore the potential of utilizing Salmonella in combination with other cancer therapeutics to improve treatment effectiveness. Herein, we describe the advantages that make Salmonella a good candidate for combination cancer therapy and summarize the findings of representative studies that aimed to investigate the therapeutic outcome of combination therapies involving Salmonella. We also highlight issues associated with their application in clinical use.

CD40L modulates transcriptional signatures of neutrophils in the bone marrow associated with development and trafficking.

Neutrophils are produced in the BM in a process called granulopoiesis, in which progenitor cells sequentially develop into mature neutrophils. During the developmental process, which is finely regulated by distinct transcription factors, neutrophils acquire the ability to exit the BM, properly distribute throughout the body, and migrate to infection sites. Previous studies have demonstrated that CD40 ligand (CD40L) influences hematopoiesis and granulopoiesis. Here, we investigate the effect of CD40L on neutrophil development and trafficking by performing functional and transcriptome analyses. We found that CD40L signaling plays an essential role in the early stages of neutrophil generation and development in the BM. Moreover, CD40L modulates transcriptional signatures, indicating that this molecule enables neutrophils to traffic throughout the body and to migrate in response to inflammatory signals. Thus, our study provides insights into the complex relationships between CD40L signaling and granulopoiesis, and it suggests a potentially novel and nonredundant role of CD40L signaling in neutrophil development and function.

Probiotic survival, biological functionality and untargeted metabolomics of the bioaccessible compounds in fermented camel and bovine milk after in vitro digestion.

This study investigated probiotic survival and biological functionality of the bioaccessible fraction of fermented camel milk (FCM) and fermented bovine milk (FBM) after in vitro digestion. Furthermore, untargeted metabolomic analysis was performed to identify the bioaccessible compounds in FCM and FBM, which were produced using starter bacteria (SC), a potential probiotic (Pro) or a combination thereof (SC + Pro), followed by storage (21 d). Survival of Pro and SC + Pro bacteria in FCM was higher than in FBM throughout in vitro digestion. The antioxidant activities of the bioaccessible fractions differed slightly between culture types, whereas the antiproliferative activity of SC was highest, followed by SC + Pro. Antiproliferative activity of the bioaccessible fractions of FCM was greater than for FBM. Untargeted metabolomics of FCM demonstrated discrimination between cultures and from FBM. FCM produced with SC + Pro and Pro had closer clustering than with SC. The bioaccessible fraction of FCM exhibited higher biological functionality compared to FBM.

Farnesyl pyrophosphate is a new danger signal inducing acute cell death.

Cell death is a vital event in life. Infections and injuries cause lytic cell death, which gives rise to danger signals that can further induce cell death, inflammation, and tissue damage. The mevalonate (MVA) pathway is an essential, highly conserved and dynamic metabolic pathway. Here, we discover that farnesyl pyrophosphate (FPP), a metabolic intermediate of the MVA pathway, functions as a newly identified danger signal to trigger acute cell death leading to neuron loss in stroke. Harboring both a hydrophobic 15-carbon isoprenyl chain and a heavily charged pyrophosphate head, FPP leads to acute cell death independent of its downstream metabolic pathways. Mechanistically, extracellular calcium influx and the cation channel transient receptor potential melastatin 2 (TRPM2) exhibit essential roles in FPP-induced cell death. FPP activates TRPM2 opening for ion influx. Furthermore, in terms of a mouse model constructing by middle cerebral artery occlusion (MCAO), FPP accumulates in the brain, which indicates the function of the FPP and TRPM2 danger signal axis in ischemic injury. Overall, our data have revealed a novel function of the MVA pathway intermediate metabolite FPP as a danger signal via transient receptor potential cation channels.