The insect immune response demonstrates a number of structural and functional similarities to the innate immune system of mammals. As a result of these conserved features insects have become popular ...choices for evaluating the virulence of microbial pathogens or for assessing the efficacy of antimicrobial agents and give results which are comparable to those that can be obtained using mammals. Analysis of the cellular component of the insect and mammalian immune systems demonstrates many similarities. Insect hemocytes recognize pathogens and phagocytose material in a similar manner to neutrophils. The killing of ingested microbes is achieved in both cell types by the production of superoxide and by the release of enzymes in the process of degranulation. Insect hemocytes and mammalian neutrophils are sensitive to the same inhibitors. This review highlights the strong similarities between the phagocytic cells of both groups of animals and demonstrates the potential benefits of using selected insects as in vivo screening systems.
In piglets, it is observed that early weaning can lead to poor weight gain due to an underdeveloped gastrointestinal (GI) tract, which is unsuitable for an efficient absorption of nutrients. ...Short-chain fatty acids (SCFAs) such as butyrate have demonstrated their ability to improve intestinal development by increasing cell proliferation, which is vital during this transition period when the small and large intestinal tracts are rapidly growing. Previous reports on butyrate inclusion in feed demonstrated significantly increased feed intakes (FIs) and average daily gains (ADGs) during piglet weaning. Similar benefits in piglet performance have been observed with the inclusion of yeast cell wall in diets. A proprietary mix of yeast cell wall, SCFAs, and zinc proteinate (YSM) was assessed here in vitro to determine its impact on cellular growth, metabolism and appetite-associated hormones in ex vivo small intestinal pig cells and STC-1 mouse intestinal neuroendocrine cells. Intestinal cells demonstrated greater cell densities with the addition of YSM (150 ppm) compared to the control and butyrate (150 ppm) at 24 h. This coincided with the higher utilisation of both protein and glucose from the media of intestinal cells receiving YSM. Ghrelin (an appetite-inducing hormone) demonstrated elevated levels in the YSM-treated cells on a protein and gene expression level compared to the cells receiving butyrate and the control, while satiety hormone peptide YY protein levels were lower in the cells receiving YSM compared to the control and butyrate-treated cells across each time point. Higher levels of ghrelin and lower PYY secretion in cells receiving YSM may drive the uptake of protein and glucose, which is potentially facilitated by elevated gene transporters for protein and glucose. Greater ghrelin levels observed with the inclusion of YSM may contribute to higher cell densities that could support pig performance to a greater extent than butyrate alone.
Display omitted
•Mild stresses increase the resistance of Galleria mellonella larvae to infection.•Stress induces a short term increase in larval immunity.•Stress leads to increased haemocyte density ...at 24h but this declines thereafter.•Stress leads to a transient increase in the expression of immune related proteins.
Exposure of larvae of Galleria mellonella larvae to mild physical (i.e. shaking) or thermal stress for 24h increased their ability to survive infection with Aspergillus fumigatus conidia however larvae stressed in a similar manner but incubated for 72h prior to infection showed no elevation in their resistance to infection with A. fumigatus. Stressed larvae demonstrated an elevated haemocyte density 24h after initiation of the stress event but this declined at 48 and 72h. Larval proteins such as apolipophorin, arylophorin and prophenoloxidase demonstrated elevated expression at 24h but not at 72h. Larvae maintained at 37°C showed increased expression of a range of antimicrobial and immune-related proteins at 24h but these decreased in expression thereafter. The results presented here indicate that G. mellonella larvae are capable of altering their immune response following exposure to mild thermal or physical stress to mount a response capable of counteracting microbial infection which reaches a peak 24h after the initiation of the priming event and then declines by 72h. A short-term immune priming effect may serve to prevent infection but maintaining an immune priming effect for longer periods may be metabolically costly and unnecessary while living within the colony of another insect.
In swine intestinal barrier deterioration can be caused by exposure to harmful bacteria, toxins or contaminants that can lead to a leaky gut and post weaning diarrhoea. A leaky gut leads to increased ...infection, inflammation and poor nutrient absorption that can impair piglet growth and ultimately survival. Application of yeast cell wall (YCW) products may offer an opportunity to reduce the intestinal barrier damage caused by microbial challenge. A Mannan rich fraction (MRF) and three YCW products were compared by examining their impact on intestinal barrier function using a Jejunal model of intestine in response to a bacterial challenge using Salmonella LPS.
Trans epithelial electrical resistance (TEER) readings showed MRF had a significantly higher barrier function (P ≤ 0.05) over the positive control while YCW products A, B and C demonstrated no significant improvement to the positive control. Transcriptome analysis of the IPEC-J2 cells showed that differentially expressed genes associated with the gene ontology (GO) term for Structural molecule activity was significantly upregulated in the MRF treated cells over the positive control cells with 56 genes upregulated compared to product B (50 genes), Product C, (25 genes) and the negative control's 60 genes. Product A had no functional grouping under the structural molecule activity term. Both qPCR and western blotting analysis of tight junction associated genes showed that MRF treated cells demonstrated significantly higher Claudin 3 junctional gene expression (P ≤ 0.05) over the positive control and treatments A, B and C. Occludin expression was significantly higher in MRF treated cells (P ≤ 0.05) over the positive control and product B. A nonsignificant rise in TJP-1 gene expression was observed in the MRF treated cells when compared to the positive control. Protein abundances of Claudin 3, Occludin and TJP-1 were significantly (P ≤ 0.05) higher following MRF application to LPS challenged IPEC-J2 cells over the positive control.
The difference in each YCW products production and composition appeared to influence intestinal barrier integrity. The action of MRF demonstrates its potential ability to raise intestinal barrier integrity of IPEC-J2 intestinal cells on an in vitro level through significantly elevated intracellular connections.
Studies have endeavored to understand the cause for impaired antimicrobial killing by neutrophils of people with cystic fibrosis (PWCF). The aim of this study was to focus on the bacterial phagosome. ...Possible alterations in degranulation of cytoplasmic granules and changes in pH were assessed. Circulating neutrophils were purified from PWCF (n = 28), PWCF receiving ivacaftor therapy (n = 10), and healthy controls (n = 28). Degranulation was assessed by Western blot analysis and flow cytometry. The pH of phagosomes was determined by use of BCECF-AM-labelled
or SNARF labelled
. The antibacterial effect of all treatments tested was determined by colony forming units enumeration. Bacterial killing by CF and healthy control neutrophils were found to differ (p = 0.0006). By use of flow cytometry and subcellular fractionation the kinetics of intraphagosomal degranulation were found to be significantly altered in CF phagosomes, as demonstrated by increased primary granule CD63 (p = 0.0001) and myeloperoxidase (MPO) content (p = 0.03). In contrast, decreased secondary and tertiary granule CD66b (p = 0.002) and decreased hCAP-18 and MMP-9 (p = 0.02), were observed. After 8 min phagocytosis the pH in phagosomes of neutrophils of PWCF was significantly elevated (p = 0.0001), and the percentage of viable bacteria was significantly increased compared to HC (p = 0.002). Results demonstrate that the recorded alterations in phagosomal pH generate suboptimal conditions for MPO related peroxidase, and α-defensin and azurocidine enzymatic killing of
and
. The pattern of dysregulated MPO degranulation (p = 0.02) and prolonged phagosomal alkalinization in CF neutrophils were normalized
following treatment with the ion channel potentiator ivacaftor (p = 0.04). Our results confirm that alterations of circulating neutrophils from PWCF are corrected by CFTR modulator therapy, and raise a question related to possible delayed proton channel activity in CF.
Abstract
Mannan rich fractions were previously demonstrated to improve junctional gene expression which can reduce leaky intestinal tracts that facilitate bacterial infection in piglets. The work ...here assessed MRF and other yeast mannan products’ effects on intestinal barrier function in response to bacterial challenge from Salmonella LPS. Differentiated IPEC cells were grown to 14 days until a trans-epithelial electrical resistance reading (TEER) of ~ 4500 Ohms/cm2 was reached. Prior to lipopolysaccharide (LPS) (1µg/mL) challenge, cells were pre-treated and post-treated with MRF or yeast products A, B, and C (16mg/mL). Post treated cells were lysed in RLT buffer and RNA isolated (RNeasy). RIN values above 7 were used to synthesise cDNA (SuperScript®-III). Junctional genes Occludin, Claudin3 and Tight junction protein1 (TJP1) were assessed by qPCR (Applied Biosystems 7500 Fast). TNFα proinflammatory secretion was measured by ELISA. Three independent biological replicates were performed, with One-way ANOVA carried out unless stated otherwise. TEER results showed MRF significantly recovered barrier function (5090.3±187.0, P ≤ 0.05) over the positive control (PC) (3754.2±605.8) while product A (3502.5±182.7749), B (3414.289±733.8854) and C (3938.4±491.4) demonstrated no significant improvement to the PC. MRF treated cells were significantly higher for Claudin3 gene expression (1.33±0.18, P ≤ 0.05) over the control (0.671661±0.277) and treatments A (0.53±0.16, P ≤ 0.05), B (0.91±0.18, P ≤ 0.05) and C (0.69±0.25, P ≤ 0.05). Occludin expression was significantly higher in MRF treated cells (1.09±0.01, P ≤ 0.05) over the PC (0.89±0.09, P ≤ 0.05) and treatment B (0.88±0.04, P ≤ 0.05). TJP-1 gene expression was highest in the MRF treated cells (1.30±0.41) but not significantly, compared to the PC (1.00±0.14). TNFα (pg/mL) protein secretion was significantly lower in both the MRF treated (0.0809±0.86x10-3, P ≤ 0.05) and treatment C 0.081±0.18 x10-3, P ≤ 0.05) over the PC (0.0813±0.22x10-3). MRF augmented junctional expression improving TEER readings and potentially lessened LPS intracellular leakage that led to lower proinflammatory protein secretion. The present study highlights differences in efficacy of a variety of yeast cell wall products.
Abstract
Salmonella species are associated with post-weaning diarrhea, which results in poor weight gain, potential death, and economic cost. A yeast mannan rich fraction (MRF) was assessed alongside ...the industry standard treatment Zinc Oxide (ZO) in vitro to determine its impact on Salmonella Dublin infection of a pig intestinal cell line (IPEC). IPEC cells were exposed to MRF or ZO in the presence of S. Dublin (1x108/mL). IPEC cell RNA was isolated and cDNA synthesized. Gene expression for IL-1β, TNFα, IL-8 and cellular tight Junction genes Occludin, Claudin3 and Tight junction protein1 (TJP1) were assessed by qPCR. S. Dublin adhesion to IPEC cells (500:1) assessed in the presence or absence of either ZO or MRF for 1 hour at 37°C. IPEC cells with attached S. Dublin were lysed, diluted, plated and incubated overnight and enumeration. Three biological replicates were performed for all experiments and statistical analysis determined by One-way ANOVA. Proinflammatory gene TNFα was significantly reduced (P ≤ 0.001) following S. Dublin infection and treatment with MRF compared with ZO. IL-1β demonstrated no change between treatments although IL-8 gene expression was significantly reduced in both ZO (P≤0.01) and MRF (P ≤ 0.05) treated cells over the control. Significantly higher expression of Occludin (P ≤ 0.01), Claudin3 (P ≤ 0.001) and TJP1 (P ≤ 0.05) was observed in IPEC cells exposed to S. Dublin in the presence of MRF compared to ZO. Adhesion of S. Dublin to IPEC cells was significantly reduced in response to MRF addition compared to ZO treated cells (P ≤ 0.001) and the control cells (P ≤ 0.05). ZO treated cell demonstrated no improvement over the control cell levels of bacterial attachment. Both on a physical and molecular level bacterial infection of intestinal cells was more significantly impaired by MRF addition. With the ban on ZO, yeast MRF may prove to be a suitable alternative to support gut health in piglets.
Salmonella species are associated with post-weaning diarrhea, which results in poor weight gain, potential death, and economic cost. A yeast mannan rich fraction (MRF) was assessed alongside the ...industry standard treatment Zinc Oxide (ZO) in vitro to determine its impact on Salmonella Dublin infection of a pig intestinal cell line (IPEC). IPEC cells were exposed to MRF or ZO in the presence of S. Dublin (1x108/mL). IPEC cell RNA was isolated and cDNA synthesized. Gene expression for IL-1β, TNFα, IL-8 and cellular tight Junction genes Occludin, Claudin3 and Tight junction protein1 (TJP1) were assessed by qPCR. S. Dublin adhesion to IPEC cells (500:1) assessed in the presence or absence of either ZO or MRF for 1 hour at 37°C. IPEC cells with attached S. Dublin were lysed, diluted, plated and incubated overnight and enumeration. Three biological replicates were performed for all experiments and statistical analysis determined by One-way ANOVA. Proinflammatory gene TNFα was significantly reduced (P < 0.001) following S. Dublin infection and treatment with MRF compared with ZO. IL-1β demonstrated no change between treatments although IL-8 gene expression was significantly reduced in both ZO (P≤0.01) and MRF (P < 0.05) treated cells over the control. Significantly higher expression of Occludin (P ≤ 0.01), Claudin3 (P ≤ 0.001) and TJP1 (P ≤ 0.05) was observed in IPEC cells exposed to S. Dublin in the presence of MRF compared to ZO. Adhesion of S. Dublin to IPEC cells was significantly reduced in response to MRF addition compared to ZO treated cells (P ≤ 0.001) and the control cells (P ≤ 0.05). ZO treated cell demonstrated no improvement over the control cell levels of bacterial attachment. Both on a physical and molecular level bacterial infection of intestinal cells was more significantly impaired by MRF addition. With the ban on ZO, yeast MRF may prove to be a suitable alternative to support gut health in piglets.
The antimicrobial drug candidate 1,3-dibenzyl-4,5-diphenyl-imidazol-2-ylidene silver(I) acetate (SBC3) was evaluated for its ability to function in vivo using larvae of
Galleria mellonella
. A SBC3 ...concentration of 25 μg/ml inhibited the growth of
Staphylococcus aureus
by 71.2 % and
Candida albicans
by 86.2 % in vitro. Larvae inoculated with 20 μl of SBC3 solution showed no ill effects up to a concentration of 250 μg/ml but administration of 500 μg/ml resulted in a 40 % reduction in larval survival and administration of a dose of 1,000 μg/ml resulted in total larval death at 24 h. Larvae inoculated with
S. aureus
or
C. albicans
and subsequently administered SBC3 showed increased survival. Administration of SBC3 to larvae did not boost the insect immune response as indicated by lack of an increase in the density of circulating haemocytes (immune cells). The abundance of a number of proteins involved in the insect immune response was reduced in larvae that received 20 μl SBC3 solution of 100 μg/ml. This is the first demonstration of the in vivo activity of SBC3 against
S. aureus
and
C. albicans
and demonstrates that SBC3 does not stimulate a non-specific immune response in larvae.
Larvae of Galleria mellonella are widely used to study the virulence of microbial pathogens and for assessing the potency of antimicrobial agents. This work examined the effect of nutritional ...deprivation on the ability of larvae to withstand infection in order to establish standardized conditions for the treatment of larvae for in vivo testing. Larvae deprived of food for seven days demonstrated an increased susceptibility to infection by the yeast Candida albicans. These larvae displayed a lower density of hemocytes compared with controls but hemocytes from starved and control larvae demonstrated the same ability to kill yeast cells. Hemolymph from starved larvae demonstrated reduced expression of a range of antimicrobial peptides (e.g., lipocalin) and immune proteins (e.g., apolipophorin and arylphorin). Deprivation of G. mellonella larvae of food leads to a reduction in the cellular and immune responses and an increased susceptibility to infection. Researchers utilizing these larvae should ensure adequate food is provided to larvae in order to allow valid comparisons to be made between results from different laboratories.
PDF
