Intestinal organoids to better appreciate gut dynamics
Published on 12.11.2020 by Gut Health Consultancy
In-vitro models are invariably used in an attempt to develop knowledge and mechanistic understanding. This information is often then used in the product innovation and technical-marketing process. However, there are many challenges in interpreting and extrapolating results from these studies to the in-vivo situation. Many in-vitro models lack real-life complexity and key features, have altered molecule expression or inconvenient life spans, which compromise their physiological application (Kar et al., 2020).
Intestinal organoids offer a helpful step forward. Intestinal organoids can be derived from embryonic, induced or organ-specific stem cells to replicate the architecture, spatial orientation, differentiated cell types and features found in-vivo (Kar et al., 2020). Such intestinal organoids can be exposed to luminal factors (e.g. nutrients, additives, microbes, etc.) to investigate the array of interactions that occur. Organoids have been used to study cellular signalling and secretion (Rubert et al., 2020).
Intestinal organoids have been cultured from various host cells, including swine and chickens, to generate species-specific intestinal models to investigate responses to nutrients, pathogens, toxins or chemicals (Augustyniak et al., 2019). For example, Li et al. (2020) developed a porcine intestinal organoid culture system to investigate the infectivity of, and immune responses to, transmissible gastroenteritis virus. For chickens, the effect of various vitamins, toxins and chemicals on organoid morphology was recently assessed (Acharya et al., 2020).
These is much promise for species-specific intestinal organoids to provide more reliable mechanistic insight than traditional in-vitro approaches. Please contact us if you would like to know more!
NF-KB and target gene expression
Published on 02.11.2020 by Gut Health Consultancy
The NF-κB family is made up of five proteins/subunits that can form a range of homo- and hetero-dimers. It has been reported that different subunits regulate a different subset of the ~600 known NF-κB target genes, although, interestingly, NF-κB may interact with many more genes. Changes in gene transcription can be considered a function of the nuclear abundance of the specific NF-κB dimers, their affinity for, and the availability of, DNA regulatory binding sites, but some of the details remain to be resolved.
www.bu.edu/nf-kb/gene-resources/target-genes/
Numerous inhibitors of NF-κB signalling have been identified. In recent work, honokiol (polyphenol extracted from the Magnolia plant) and capsaicin (component of chilli peppers) modulated adjuvant-induced inflammation and increased IFN-γ and antibody responses to ovalbumin in mice. The effects were dose-dependent, but capsaicin did cause lethargy at the tested doses. Capsaicin activated transient receptor potential cation channel subfamily V member 1 (TRPV1) to dampen systemic inflammation, but a separate mechanism was responsible for antibody enhancement. Honokiol has been reported to impact multiple pathways. www.frontiersin.org/articles/10.3389/fimmu.2020.511513/full#B31
Often, general NF-κB inhibition is promoted to limit inflammatory responses, but, given the range of genes regulated, this approach is likely to compromise wider, beneficial cellular responses. Thus, knowledge of selectively inhibiting specific NF-κB subunits could be exploited and selective inhibition has been proposed to potentially enhance uninhibited functions. In additional work, a partially selective NF-κB (p50 subunit) inhibitor combined with common immune adjuvants regulated systemic inflammation while enhancing antigen presentation and adaptive responses in response to various antigens, and enhanced protection and tolerability in an influenza A mouse vaccine model.
advances.sciencemag.org/content/6/37/eaaz8700.full
Selective inhibition of NF-κB subunits could/will be important for vaccine and natural immunomodulator strategies. Please contact us if you would like to discuss NF-κB modulation further!
Unexpected microbiota results?
Published on 29.08.2020 by Gut Health Consultancy
Various factors that can influence microbiota analysis need to be kept in mind. For example:
Sampling - What has been sampled (e.g. ileal microbiota influence on colonic or faecal microbiota in chickens)? Exposure to oxygen? Transport and storage conditions?
In-vitro - Optimal DNA extraction protocol? 16S rRNA gene hypervariable region? Primer selection? Specifics of sequencing platform?
In-silico - Databases or algorithms used for sequence curation, clustering and taxonomic assignment? Realistic taxonomic resolution? Appropriate statistical analyses?
DNA extraction, sequencing methods and bioinformatic processing methods have been identified as particularly important, with the latter having the greatest impact in a recent study (Szamosi et al., 2020).
We have been engaged by several clients to retrospectively interpret unexpected microbiota data. This is challenging. Careful review of procedures beforehand can minimise potential problems. Please contact us to discuss more!
Intestinal inflammation models - being specific!
Published on 22.08.2020 by Gut Health Consultancy
There are numerous models available to study intestinal inflammation, which often use chemicals, pathogens and/or viscous diets as inducers. Whilst histopathological changes with these models may seem similar, such as inflammatory cell infiltrates and mucosal modifications, the specific changes with each model are important. Important factors include dose, duration, frequency of administration, animal genotype, and even the chemical's molecular weight. For example, some studies suggest that dextran sulfate sodium (DSS; a commonly used chemical) induces inflammation only/mainly in the large intestine, while others have found wider effects (e.g. small intestine); that broilers are less susceptible than layers; while some have found relatively low doses preferable (0.75% DSS in drinking water) for desired effects in broilers, others needed higher doses (2.5% DSS in drinking water) to observe pathology (references available).
Gut Health Consultancy have been advising clients on the use of inflammation models and appropriate partners for their work. Getting advice early in these projects can help ensure the validity of the data generated and their interpretation. Please contact us for further information!
Published on 12.11.2020 by Gut Health Consultancy
In-vitro models are invariably used in an attempt to develop knowledge and mechanistic understanding. This information is often then used in the product innovation and technical-marketing process. However, there are many challenges in interpreting and extrapolating results from these studies to the in-vivo situation. Many in-vitro models lack real-life complexity and key features, have altered molecule expression or inconvenient life spans, which compromise their physiological application (Kar et al., 2020).
Intestinal organoids offer a helpful step forward. Intestinal organoids can be derived from embryonic, induced or organ-specific stem cells to replicate the architecture, spatial orientation, differentiated cell types and features found in-vivo (Kar et al., 2020). Such intestinal organoids can be exposed to luminal factors (e.g. nutrients, additives, microbes, etc.) to investigate the array of interactions that occur. Organoids have been used to study cellular signalling and secretion (Rubert et al., 2020).
Intestinal organoids have been cultured from various host cells, including swine and chickens, to generate species-specific intestinal models to investigate responses to nutrients, pathogens, toxins or chemicals (Augustyniak et al., 2019). For example, Li et al. (2020) developed a porcine intestinal organoid culture system to investigate the infectivity of, and immune responses to, transmissible gastroenteritis virus. For chickens, the effect of various vitamins, toxins and chemicals on organoid morphology was recently assessed (Acharya et al., 2020).
These is much promise for species-specific intestinal organoids to provide more reliable mechanistic insight than traditional in-vitro approaches. Please contact us if you would like to know more!
NF-KB and target gene expression
Published on 02.11.2020 by Gut Health Consultancy
The NF-κB family is made up of five proteins/subunits that can form a range of homo- and hetero-dimers. It has been reported that different subunits regulate a different subset of the ~600 known NF-κB target genes, although, interestingly, NF-κB may interact with many more genes. Changes in gene transcription can be considered a function of the nuclear abundance of the specific NF-κB dimers, their affinity for, and the availability of, DNA regulatory binding sites, but some of the details remain to be resolved.
www.bu.edu/nf-kb/gene-resources/target-genes/
Numerous inhibitors of NF-κB signalling have been identified. In recent work, honokiol (polyphenol extracted from the Magnolia plant) and capsaicin (component of chilli peppers) modulated adjuvant-induced inflammation and increased IFN-γ and antibody responses to ovalbumin in mice. The effects were dose-dependent, but capsaicin did cause lethargy at the tested doses. Capsaicin activated transient receptor potential cation channel subfamily V member 1 (TRPV1) to dampen systemic inflammation, but a separate mechanism was responsible for antibody enhancement. Honokiol has been reported to impact multiple pathways. www.frontiersin.org/articles/10.3389/fimmu.2020.511513/full#B31
Often, general NF-κB inhibition is promoted to limit inflammatory responses, but, given the range of genes regulated, this approach is likely to compromise wider, beneficial cellular responses. Thus, knowledge of selectively inhibiting specific NF-κB subunits could be exploited and selective inhibition has been proposed to potentially enhance uninhibited functions. In additional work, a partially selective NF-κB (p50 subunit) inhibitor combined with common immune adjuvants regulated systemic inflammation while enhancing antigen presentation and adaptive responses in response to various antigens, and enhanced protection and tolerability in an influenza A mouse vaccine model.
advances.sciencemag.org/content/6/37/eaaz8700.full
Selective inhibition of NF-κB subunits could/will be important for vaccine and natural immunomodulator strategies. Please contact us if you would like to discuss NF-κB modulation further!
Unexpected microbiota results?
Published on 29.08.2020 by Gut Health Consultancy
Various factors that can influence microbiota analysis need to be kept in mind. For example:
Sampling - What has been sampled (e.g. ileal microbiota influence on colonic or faecal microbiota in chickens)? Exposure to oxygen? Transport and storage conditions?
In-vitro - Optimal DNA extraction protocol? 16S rRNA gene hypervariable region? Primer selection? Specifics of sequencing platform?
In-silico - Databases or algorithms used for sequence curation, clustering and taxonomic assignment? Realistic taxonomic resolution? Appropriate statistical analyses?
DNA extraction, sequencing methods and bioinformatic processing methods have been identified as particularly important, with the latter having the greatest impact in a recent study (Szamosi et al., 2020).
We have been engaged by several clients to retrospectively interpret unexpected microbiota data. This is challenging. Careful review of procedures beforehand can minimise potential problems. Please contact us to discuss more!
Intestinal inflammation models - being specific!
Published on 22.08.2020 by Gut Health Consultancy
There are numerous models available to study intestinal inflammation, which often use chemicals, pathogens and/or viscous diets as inducers. Whilst histopathological changes with these models may seem similar, such as inflammatory cell infiltrates and mucosal modifications, the specific changes with each model are important. Important factors include dose, duration, frequency of administration, animal genotype, and even the chemical's molecular weight. For example, some studies suggest that dextran sulfate sodium (DSS; a commonly used chemical) induces inflammation only/mainly in the large intestine, while others have found wider effects (e.g. small intestine); that broilers are less susceptible than layers; while some have found relatively low doses preferable (0.75% DSS in drinking water) for desired effects in broilers, others needed higher doses (2.5% DSS in drinking water) to observe pathology (references available).
Gut Health Consultancy have been advising clients on the use of inflammation models and appropriate partners for their work. Getting advice early in these projects can help ensure the validity of the data generated and their interpretation. Please contact us for further information!