Surface layer proteins from virulent Clostridium difficile ribotypes exhibit signatures of positive selection with consequences for innate immune response.

BACKGROUND: Clostridium difficile is a nosocomial pathogen prevalent in hospitals worldwide and increasingly common in the community. Sequence differences have been shown to be present in the Surface Layer Proteins (SLPs) from different C. difficile ribotypes (RT) however whether these differences influence severity of infection is still not clear. RESULTS: We used a molecular evolutionary approach to analyse SLPs from twenty-six C. difficile RTs representing different slpA sequences. We demonstrate that SLPs from RT 027 and 078 exhibit evidence of positive selection (PS). We compared the effect of these SLPs to those purified from RT 001 and 014, which did not exhibit PS, and demonstrate that the presence of sites under positive selection correlates with ability to activate macrophages. SLPs from RTs 027 and 078 induced a more potent response in macrophages, with increased levels of IL-6, IL-12p40, IL-10, MIP-1α, MIP-2 production relative to RT 001 and 014. Furthermore, RTs 027 and 078 induced higher expression of CD40, CD80 and MHC II on macrophages with decreased ability to phagocytose relative to LPS. CONCLUSIONS: These results tightly link sequence differences in C. difficile SLPs to disease susceptibility and severity, and suggest that positively selected sites in the SLPs may play a role in driving the emergence of hyper-virulent strains.

Adaptive Evolution as a Predictor of Species-Specific Innate Immune Response.

It has been proposed that positive selection may be associated with protein functional change. For example, human and macaque have different outcomes to HIV infection and it has been shown that residues under positive selection in the macaque TRIM5α receptor locate to the region known to influence species-specific response to HIV. In general, however, the relationship between sequence and function has proven difficult to fully elucidate, and it is the role of large-scale studies to help bridge this gap in our understanding by revealing major patterns in the data that correlate genotype with function or phenotype. In this study, we investigate the level of species-specific positive selection in innate immune genes from human and mouse. In total, we analyzed 456 innate immune genes using codon-based models of evolution, comparing human, mouse, and 19 other vertebrate species to identify putative species-specific positive selection. Then we used population genomic data from the recently completed Neanderthal genome project, the 1000 human genomes project, and the 17 laboratory mouse genomes project to determine whether the residues that were putatively positively selected are fixed or variable in these populations. We find evidence of species-specific positive selection on both the human and the mouse branches and we show that the classes of genes under positive selection cluster by function and by interaction. Data from this study provide us with targets to test the relationship between positive selection and protein function and ultimately to test the relationship between positive selection and discordant phenotypes.

Khaya grandifoliola C.DC: a potential source of active ingredients against hepatitis C virus in vitro.

In this study, we examined the antiviral properties of Khaya grandifoliola C.DC (Meliaceae) on the hepatitis C virus (HCV) life cycle in vitro and identified some of the chemical constituents contained in the fraction with the most antiviral activity. Dried bark powder was extracted by maceration in a methylene chloride/methanol (MCM) system (50:50; v/v) and separated on silica gel by flash chromatography. Infection and replication rates in Huh-7 cells were investigated by luciferase reporter assay and indirect immunofluorescence assay using subgenomic replicons, HCV pseudotyped particles, and cell-culture-derived HCV (HCVcc), respectively. Cell viability was assessed by MTT assay, and cellular gene expression was analysed by qRT-PCR. The chemical composition of the fraction with the most antiviral activity was analysed by coupled gas chromatography and mass spectrometry (GC-MS). Five fractions of different polarities (F0-F100) were obtained from the MCM extract. One fraction (KgF25) showed the strongest antiviral effect on LucUbiNeoET replicons at nontoxic concentrations. Tested at 100 µg/mL, KgF25 had a high inhibitory effect on HCV replication, comparable to that of 0.01 µM daclatasvir or 1 µM telaprevir. This fraction also inhibited HCVcc infection by mostly targeting the entry step. KgF25 inhibited HCV entry in a pan-genotypic manner by directly inactivating free viral particles. Its antiviral effects were mediated by the transcriptional upregulation of the haem oxygenase-1 gene and interferon antiviral response. Three constituents, namely, benzene, 1,1'-(oxydiethylidene)bis (1), carbamic acid, (4-methylphenyl)-, 1-phenyl (2), and 6-phenyl, 4-(1'-oxyethylphenyl) hexene (3), were identified from the active fraction KgF25 by GC-MS. Khaya grandifoliola contains ingredients capable of acting on different steps of the HCV life cycle.

Adult vascular smooth muscle cells in culture express neural stem cell markers typical of resident multipotent vascular stem cells.

Differentiation of resident multipotent vascular stem cells (MVSCs) or de-differentiation of vascular smooth muscle cells (vSMCs) might be responsible for the SMC phenotype that plays a major role in vascular diseases such as arteriosclerosis and restenosis. We examined vSMCs from three different species (rat, murine and bovine) to establish whether they exhibit neural stem cell characteristics typical of MVSCs. We determined their SMC differentiation, neural stem cell marker expression and multipotency following induction in vitro by using immunocytochemistry, confocal microscopy, fluorescence-activated cell sorting analysis and quantitative real-time polymerase chain reaction. MVSCs isolated from rat aortic explants, enzymatically dispersed rat SMCs and rat bone-marrow-derived mesenchymal stem cells served as controls. Murine carotid artery lysates and primary rat aortic vSMCs were both myosin-heavy-chain-positive but weakly expressed the neural crest stem cell marker, Sox10. Each vSMC line examined expressed SMC differentiation markers (smooth muscle α-actin, myosin heavy chain and calponin), neural crest stem cell markers (Sox10(+), Sox17(+)) and a glia marker (S100ß(+)). Serum deprivation significantly increased calponin and myosin heavy chain expression and decreased stem cell marker expression, when compared with serum-rich conditions. vSMCs did not differentiate to adipocytes or osteoblasts following adipogenic or osteogenic inductive stimulation, respectively, or respond to transforming growth factor-ß1 or Notch following γ-secretase inhibition. Thus, vascular SMCs in culture express neural stem cell markers typical of MVSCs, concomitant with SMC differentiation markers, but do not retain their multipotency. The ultimate origin of these cells might have important implications for their use in investigations of vascular proliferative disease in vitro.

The former annotated human pseudogene dihydrofolate reductase-like 1 (DHFRL1) is expressed and functional.

Human dihydrofolate reductase (DHFR) was previously thought to be the only enzyme capable of the reduction of dihydrofolate to tetrahydrofolate; an essential reaction necessary to ensure a continuous supply of biologically active folate. DHFR has been studied extensively from a number of perspectives because of its role in health and disease. Although the presence of a number of intronless DHFR pseudogenes has been known since the 1980s, it was assumed that none of these were expressed or functional. We show that humans do have a second dihydrofolate reductase enzyme encoded by the former pseudogene DHFRP4, located on chromosome 3. We demonstrate that the DHFRP4, or dihydrofolate reductase-like 1 (DHFRL1), gene is expressed and shares some commonalities with DHFR. Recombinant DHFRL1 can complement a DHFR-negative phenotype in bacterial and mammalian cells but has a lower specific activity than DHFR. The K(m) for NADPH is similar for both enzymes but DHFRL1 has a higher K(m) for dihydrofolate when compared to DHFR. The need for a second reductase with lowered affinity for its substrate may fulfill a specific cellular requirement. The localization of DHFRL1 to the mitochondria, as demonstrated by confocal microscopy, indicates that mitochondrial dihydrofolate reductase activity may be optimal with a lowered affinity for dihydrofolate. We also found that DHFRL1 is capable of the same translational autoregulation as DHFR by binding to its own mRNA; with each enzyme also capable of replacing the other. The identification of DHFRL1 will have implications for previous research involving DHFR.

Novel Fermentates Can Enhance Key Immune Responses Associated with Viral Immunity.

Fermented foods have long been known to have immunomodulatory capabilities, and fermentates derived from the lactic acid bacteria of dairy products can modulate the immune system. We have used skimmed milk powder to generate novel fermentates using Lb. helveticus strains SC234 and SC232 and we demonstrate here that these fermentates can enhance key immune mechanisms that are critical to the immune response to viruses. We show that our novel fermentates, SC234 and SC232, can positively impact on cytokine and chemokine secretion, nitric oxide (NO) production, cell surface marker expression, and phagocytosis in macrophage models. We demonstrate that the fermentates SC234 and SC232 increase the secretion of cytokines IL-1ß, IL-6, TNF-α, IL-27, and IL-10; promote an M1 pro-inflammatory phenotype for viral immunity via NO induction; decrease chemokine expression of Monocyte Chemoattractant Protein (MCP); increase cell surface marker expression; and enhance phagocytosis in comparison to their starting material. These data suggest that these novel fermentates have potential as novel functional food ingredients for the treatment, management, and control of viral infection.

Syntaxin-4 is essential for IgE secretion by plasma cells.

The humoral immune system provides a crucial first defense against the invasion of microbial pathogens via the secretion of antigen specific immunoglobulins (Ig). The secretion of Ig is carried out by terminally differentiated B-lymphocytes called plasma cells. Despite the key role of plasma cells in the immune response, the mechanisms by which they constitutively traffic large volumes of Ig out of the cell is poorly understood. The involvement of Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in the regulation of protein trafficking from cells has been well documented. Syntaxin-4, a member of the Qa SNARE syntaxin family has been implicated in fusion events at the plasma membrane in a number of cells in the immune system. In this work we show that knock-down of syntaxin-4 in the multiple myeloma U266 human plasma cell line results in a loss of IgE secretion and accumulation of IgE within the cells. Furthermore, we show that IgE co-localises with syntaxin-4 in U266 plasma cells suggesting direct involvement in secretion at the plasma membrane. This study demonstrates that syntaxin-4 plays a critical role in the secretion of IgE from plasma cells and sheds some light on the mechanisms by which these cells constitutively traffic vesicles to the surface for secretion. An understanding of this machinery may be beneficial in identifying potential therapeutic targets in multiple myeloma and autoimmune disease where over-production of Ig leads to severe pathology in patients.

A role for TLR4 in Clostridium difficile infection and the recognition of surface layer proteins.

Clostridium difficile is the etiological agent of antibiotic-associated diarrhoea (AAD) and pseudomembranous colitis in humans. The role of the surface layer proteins (SLPs) in this disease has not yet been fully explored. The aim of this study was to investigate a role for SLPs in the recognition of C. difficile and the subsequent activation of the immune system. Bone marrow derived dendritic cells (DCs) exposed to SLPs were assessed for production of inflammatory cytokines, expression of cell surface markers and their ability to generate T helper (Th) cell responses. DCs isolated from C3H/HeN and C3H/HeJ mice were used in order to examine whether SLPs are recognised by TLR4. The role of TLR4 in infection was examined in TLR4-deficient mice. SLPs induced maturation of DCs characterised by production of IL-12, TNFα and IL-10 and expression of MHC class II, CD40, CD80 and CD86. Furthermore, SLP-activated DCs generated Th cells producing IFNγ and IL-17. SLPs were unable to activate DCs isolated from TLR4-mutant C3H/HeJ mice and failed to induce a subsequent Th cell response. TLR4⁻/⁻ and Myd88⁻/⁻, but not TRIF⁻/⁻ mice were more susceptible than wild-type mice to C. difficile infection. Furthermore, SLPs activated NFκB, but not IRF3, downstream of TLR4. Our results indicate that SLPs isolated from C. difficile can activate innate and adaptive immunity and that these effects are mediated by TLR4, with TLR4 having a functional role in experimental C. difficile infection. This suggests an important role for SLPs in the recognition of C. difficile by the immune system.

Targeted Application of Functional Foods as Immune Fitness Boosters in the Defense against Viral Infection.

In recent times, the emergence of viral infections, including the SARS-CoV-2 virus, the monkeypox virus, and, most recently, the Langya virus, has highlighted the devastating effects of viral infection on human life. There has been significant progress in the development of efficacious vaccines for the prevention and control of viruses; however, the high rates of viral mutation and transmission necessitate the need for novel methods of control, management, and prevention. In recent years, there has been a shift in public awareness on health and wellbeing, with consumers making significant dietary changes to improve their immunity and overall health. This rising health awareness is driving a global increase in the consumption of functional foods. This review delves into the benefits of functional foods as potential natural means to modulate the host immune system to enhance defense against viral infections. We provide an overview of the functional food market in Europe and discuss the benefits of enhancing immune fitness in high-risk groups, including the elderly, those with obesity, and people with underlying chronic conditions. We also discuss the immunomodulatory mechanisms of key functional foods, including dairy proteins and hydrolysates, plant-based functional foods, fermentates, and foods enriched with vitamin D, zinc, and selenium. Our findings reveal four key immunity boosting mechanisms by functional foods, including inhibition of viral proliferation and binding to host cells, modulation of the innate immune response in macrophages and dendritic cells, enhancement of specific immune responses in T cells and B cells, and promotion of the intestinal barrier function. Overall, this review demonstrates that diet-derived nutrients and functional foods show immense potential to boost viral immunity in high-risk individuals and can be an important approach to improving overall immune health.

Bioactive Ingredients from Dairy-Based Lactic Acid Bacterial Fermentations for Functional Food Production and Their Health Effects.

Lactic acid bacteria are traditionally applied in a variety of fermented food products, and they have the ability to produce a wide range of bioactive ingredients during fermentation, including vitamins, bacteriocins, bioactive peptides, and bioactive compounds. The bioactivity and health benefits associated with these ingredients have garnered interest in applications in the functional dairy market and have relevance both as components produced in situ and as functional additives. This review provides a brief description of the regulations regarding the functional food market in the European Union, as well as an overview of some of the functional dairy products currently available in the Irish and European markets. A better understanding of the production of these ingredients excreted by lactic acid bacteria can further drive the development and innovation of the continuously growing functional food market.

Steps toward a digital twin for functional food production with increased health benefits.

Lactobacillus rhamnosus (L. rhamnosus) is a commensal bacterium with health-promoting properties and with a wide range of applications within the food industry. To improve and optimize the control of L. rhamnosus biomass production in batch and fed-batch bioprocesses, this study proposes the application of artificial neural network (ANN) modelling to improve process control and monitoring, with potential future implementation as a basis for a digital twin. Three ANNs were developed using historical data from ten bioprocesses. These ANNs were designed to predict the biomass in batch bioprocesses with different media compositions, predict biomass in fed-batch bioprocesses, and predict the growth rate in fed-batch bioprocesses. The immunomodulatory effect of the L. rhamnosus samples was examined and found to elicit an anti-inflammatory response as evidenced by the inhibition of IL-6 and TNF-α secretion. Overall, the findings of this study reinforce the potential of ANN modelling for bioprocess optimization aimed at improved control for maximising the volumetric productivity of L. rhamnosus as an immunomodulatory agent with applications in the functional food industry.

Colon cancer associated genes exhibit signatures of positive selection at functionally significant positions.

BACKGROUND: Cancer, much like most human disease, is routinely studied by utilizing model organisms. Of these model organisms, mice are often dominant. However, our assumptions of functional equivalence fail to consider the opportunity for divergence conferred by ~180 Million Years (MY) of independent evolution between these species. For a given set of human disease related genes, it is therefore important to determine if functional equivalency has been retained between species. In this study we test the hypothesis that cancer associated genes have different patterns of substitution akin to adaptive evolution in different mammal lineages. RESULTS: Our analysis of the current literature and colon cancer databases identified 22 genes exhibiting colon cancer associated germline mutations. We identified orthologs for these 22 genes across a set of high coverage (>6X) vertebrate genomes. Analysis of these orthologous datasets revealed significant levels of positive selection. Evidence of lineage-specific positive selection was identified in 14 genes in both ancestral and extant lineages. Lineage-specific positive selection was detected in the ancestral Euarchontoglires and Hominidae lineages for STK11, in the ancestral primate lineage for CDH1, in the ancestral Murinae lineage for both SDHC and MSH6 genes and the ancestral Muridae lineage for TSC1. CONCLUSION: Identifying positive selection in the Primate, Hominidae, Muridae and Murinae lineages suggests an ancestral functional shift in these genes between the rodent and primate lineages. Analyses such as this, combining evolutionary theory and predictions - along with medically relevant data, can thus provide us with important clues for modeling human diseases.

Autoantibodies - enemies, and/or potential allies?

Autoantibodies are well known as potentially highly harmful antibodies which attack the host via binding to self-antigens, thus causing severe associated diseases and symptoms (e.g. autoimmune diseases). However, detection of autoantibodies to a range of disease-associated antigens has enabled their successful usage as important tools in disease diagnosis, prognosis and treatment. There are several advantages of using such autoantibodies. These include the capacity to measure their presence very early in disease development, their stability, which is often much better than their related antigen, and the capacity to use an array of such autoantibodies for enhanced diagnostics and to better predict prognosis. They may also possess capacity for utilization in therapy, in vivo. In this review both the positive and negative aspects of autoantibodies are critically assessed, including their role in autoimmune diseases, cancers and the global pandemic caused by COVID-19. Important issues related to their detection are also highlighted.

Exopolysaccharides of Lactic Acid Bacteria: Production, Purification and Health Benefits towards Functional Food.

Lactic acid bacteria (LAB) are capable of synthesising metabolites known as exopolysaccharides (EPS) during fermentation. Traditionally, EPS plays an important role in fermented dairy products through their gelling and thickening properties, but they can also be beneficial to human health. This bioactivity has gained attention in applications for functional foods, which leads them to have prebiotic, immunomodulatory, antioxidant, anti-tumour, cholesterol-lowering and anti-obesity activity. Understanding the parameters and conditions is crucial to optimising the EPS yields from LAB for applications in the food industry. This review provides an overview of the functional food market together with the biosynthesis of EPS. Factors influencing the production of EPS as well as methods for isolation, characterisation and quantification are reviewed. Finally, the health benefits associated with EPS are discussed.

Modulation of gene expression in U251 glioblastoma cells by binding of mutant p53 R273H to intronic and intergenic sequences.

Missense point mutations in the TP53 gene are frequent genetic alterations in human tumor tissue and cell lines derived thereof. Mutant p53 (mutp53) proteins have lost sequence-specific DNA binding, but have retained the ability to interact in a structure-selective manner with non-B DNA and to act as regulators of transcription. To identify functional binding sites of mutp53, we established a small library of genomic sequences bound by p53(R273H) in U251 human glioblastoma cells using chromatin immunoprecipitation (ChIP). Mutp53 binding to isolated DNA fragments confirmed the specificity of the ChIP. The mutp53 bound DNA sequences are rich in repetitive DNA elements, which are dispersed over non-coding DNA regions. Stable down-regulation of mutp53 expression strongly suggested that mutp53 binding to genomic DNA is functional. We identified the PPARGC1A and FRMD5 genes as p53(R273H) targets regulated by binding to intronic and intra-genic sequences. We propose a model that attributes the oncogenic functions of mutp53 to its ability to interact with intronic and intergenic non-B DNA sequences and modulate gene transcription via re-organization of chromatin.

Adaptive Evolution in TRIF Leads to Discordance between Human and Mouse Innate Immune Signaling.

The TIR domain-containing adapter inducing IFN-ß (TRIF) protein is an innate immune system protein that mediates the MyD88-independent toll-like receptor response pathway in mice and humans. Previously, we identified positive selection at seven distinct residues in mouse TRIF (mTRIF), as compared with human and other mammalian orthologs, thus predicting protein functional shift in mTRIF. We reconstructed TRIF for the most recent common ancestor of mouse and human, and mutated this at the seven sites to their extant mouse/human states. We overexpressed these TRIF mutants in immortalized human and mouse cell lines and monitored TRIF-dependent cytokine production and gene expression induction. We show that optimal TRIF function in human and mouse is dependent on the identity of the positively selected sites. These data provide us with molecular data relating observed differences in response between mouse and human MyD88-independent signaling in the innate immune system with protein functional change.

Attenuation of inflammation and cellular stress-related pathways maintains insulin sensitivity in obese type I interleukin-1 receptor knockout mice on a high-fat diet.

The development of insulin resistance in the obese is associated with chronic, low-grade inflammation. We aimed to identify novel links between obesity, insulin resistance and the inflammatory response by comparing C57BL/6 with type I interleukin-1 receptor knockout (IL-1RI(-/-)) mice, which are protected against diet-induced insulin resistance. Mice were fed a high-fat diet for 16 wk. Insulin sensitivity was measured and proteomic analysis was performed on adipose, hepatic and skeletal muscle tissues. Despite an equal weight gain, IL-1RI(-/-) mice had lower plasma glucose, insulin and triacylglycerol concentrations, compared with controls, following dietary treatment. The higher insulin sensitivity in IL-1RI(-/-) mice was associated with down-regulation of antioxidant proteins and proteasomes in adipose tissue and hepatic soluble epoxide hydrolase, consistent with a compromised inflammatory response as well as increased glycolysis and decreased fatty acid beta-oxidation in their muscle. Their lower hepatic triacylglycerol concentrations may reflect decreased flux of free fatty acids to the liver, decreased hepatic fatty acid-binding protein expression and decreased lipogenesis. Correlation analysis revealed down-regulation of classical biomarkers of ER stress in their adipose tissue, suggesting that disruption of the IL-1RI-mediated inflammatory response may attenuate cellular stress, which was associated with significant protection from diet-induced insulin resistance, independent of obesity.

The Fasciola hepatica tegumental antigen suppresses dendritic cell maturation and function.

Parasitic worms and molecules derived from them have powerful anti-inflammatory properties and are shown to have therapeutic effects on inflammatory diseases. The helminth Fasciola hepatica has been reported to suppress antigen-specific Th1 responses in concurrent bacterial infections, thus demonstrating its anti-inflammatory ability in vivo. Here, F. hepatica tegumental antigen (Teg) was shown to significantly suppress serum levels of gamma interferon (IFN-gamma) and interleukin-12p70 (IL-12p70) in a model of septic shock. Since dendritic cells (DCs) are a good source of IL-12p70 and critical in driving adaptive immunity, we investigated the effects of F. hepatica Teg on the activation and function of murine DCs. While Teg alone did not induce cytokine production or cell surface marker expression on DCs, it significantly suppressed cytokine production (IL-12p70, IL-6, IL-10, tumor necrosis factor alpha, and nitrite) and cell surface marker expression (CD80, CD86, and CD40) in DCs matured with a range of Toll-like receptor (TLR) and non-TLR ligands. Teg works independently of the TLR4 pathway, since it still functioned in DCs generated from TLR4 mutant and knockout mice. It impaired DC function by inhibiting their phagocytic capacity and their ability to prime T cells. It does not appear to target the common components (extracellular signal-regulated kinase, Jun N-terminal protein kinase, or p38) of the TLR pathways; however, it suppressed the active p65 subunit of the transcription factor NF-kappaB in mature DCs, which could explain the impairment of proinflammatory cytokine production. Overall, our results demonstrate the potent anti-inflammatory properties of F. hepatica Teg and its therapeutic potential as an anti-inflammatory agent.

A conjugated linoleic acid-enriched beef diet attenuates lipopolysaccharide-induced inflammation in mice in part through PPARgamma-mediated suppression of toll-like receptor 4.

Conjugated linoleic acid (CLA) is a PUFA found in beef and dairy products that has immunoregulatory properties. The level of CLA in beef can be enhanced by feeding cattle fresh grass rather than concentrates. This study determined the effect of feeding a high-CLA beef diet on inflammation in an in vivo model of septic shock. Mice were fed a high-CLA beef (4.3% total fatty acid composition) or low-CLA beef diet (0.84% total fatty acid composition) for 6 wk. Lipopolysaccharide (LPS; 3 microg) or sterile PBS was injected i.v. and serum was harvested 6 h after injection. Serum interleukin (IL)-1beta, IL-12p70, IL-12p40, and interferon-gamma concentrations were significantly reduced in response to the LPS challenge in the high-CLA beef diet group. Bone marrow-derived dendritic cells (BMDC) from the high-CLA beef diet group had significantly less IL-12 and more IL-10 in response to ex vivo LPS stimulation. Furthermore, toll-like receptor 4 (TLR4) and CD14 protein and mRNA expression on BMDC was significantly attenuated in the high-CLA compared with the low-CLA beef diet group. Complimentary in vitro experiments to determine the specificity of the effect showed that synthetic cis9, trans11-CLA suppressed surface expression of CD14 and TLR4 on BMDC. Treatment with the PPARgamma inhibitor GW9662 partially reversed TLR4 expression in immature BMDC. The results of this study demonstrate that feeding a diet enriched in high-beef CLA exerts profound antiinflammatory effects in vivo within the context of LPS-induced sepsis. In addition, downregulation of BMDC TLR4 is mediated through induction of PPARgamma.