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Microbiome > MHD4 Models to characterise host-microbial interaction > Flashcards

MHD4 Models to characterise host-microbial interaction Flashcards

1
Q

What are the three branches of types of study of host microbial interactions?

A
  • in vitro
  • in vivo
  • in silico
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2
Q

Describe in vitro, in vivo and in silico

A

in vitro: Performing a given procedure in a controlled environment outside of a living organism
in vivo: experiments carried out in a whole and living organism
in silico: Computational models, eg predicting how drugs interact with us and microbes

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3
Q

Order the list of simple to complex organisms for fundamental mechanistic to translational systemic experiments

A

Bacterial batch culture > dynamic bacterial culture models > mammalian cell culture > 3D cell culture > mammalian and bacterial co-culture > worms and flies > fish > cats/dogs/pigs > humans

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4
Q

What is basic/fundamental research, and give an example

A

It is used to gain a basic understanding of an organism or phenomena. e.g. to understand glycolysis

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5
Q

What is translational research, and give an example

A

Applying findings from fundamental research to enhance human health and wellbeing. e.g. how glycolysis is affected in patients with diabetes

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6
Q

What are the advantages of in vitro experiments

A
  • faster
  • easier
  • no ethics involved
  • easy to control environment
  • highly reproducible
  • very easy to obtain results (unlike gut biopsies etc.)
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7
Q

What is the biggest disadvantage of in vitro models compared to in vivo

A

The absence of a complete physiological environment and immune component

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8
Q

What is batch culturing

A

Microorganisms grown in a closed system with nutrients supplied at the beginning of the culture

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9
Q

What do you use batch culturing for?

A

Microorganisms can be grown either in isolation or combined. Used to study the bacterial metabolic activity, e.g. the ability to use certain substrates to produce other compounds. Offers a good model for a specific region of the gastrointestinal tract under the controlled conditions.

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10
Q

How do you use batch culturing?

A

The culture medium can be designed to replicate different conditions:

  • different pHs
  • different oxygen exposure
  • Normally 37 degrees
  • different nutrients
  • Normally 24-48 hours
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11
Q

Why should batch culture fermentation only be used for short periods of time?

A
  • Changes in pH
  • Changes in community structure
    Short incubations are therefore more accurate
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12
Q

Give an example of batch culture in practise

A

Analysing impact of antibiotics to e.coli over a set period of time, measuring samples’ 16S rRNA sequences every 2 hours. (to quantify number of cells - using qPCR)

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13
Q

What are chemostats?

A

continuous culture systems, carried out in a chamber

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14
Q

Why are chemostats more informative than batch culture?

A

It shows bacterial being grown in a microbial community in vitro under physiological conditions of the gut, rather than in isolation

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15
Q

What do chemostats intend to do?

A

The chamber maintains a continuously growing microbial culture, at exponential or log phase

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16
Q

How do chemostats work?

A
  • Continually supply fresh nutrients
  • Remove accumulated waste products at the same rate^
  • keeping all other conditions optimal
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17
Q

What can single-stage chemostats (single vessel), mimic?

A

Conditions are created that mimic physiological environment of specific sections of the gut and the culture vessel is seeded with faeces or the gut content

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18
Q

What does multistage chemostats mimic?

A

Consists of multiple vessels, each of which represents the physiological environment of a specific section of the gut

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19
Q

What can chemostats not accurately predict?

A

Cannot accurately model the gut microbiota present at the mucosal surface, which is known to be a distinct luminal community

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20
Q

What is added to the culture medium in order to maintain some gut microbiota that depend on this substrate? Describe it.

A

Mucin - a family of high molecular weight, heavily glycosylated proteins produced by epithelial tissues

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21
Q

What is the problem with bacterial in vitro systems?

A

Lack of host cell feedback into the system

E.g. intestinal epithelial cells and immune cells are missing, which are crucial players in host-microbial interactions

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22
Q

How do you overcome the main problem of in vitro systems?

A

Medium samples from the bacterial in vitro models can be combined with mammalian cell culture systems to better mimic the interaction with the host (co-culture)

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23
Q

Where do the host-microbial interactions occur?

A

At the interface of the gastrointestinal mucosa

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24
Q

What does the interface of the gastrointestinal mucosa contain?

A

mucus, glycocalyx (glycoproteins and lipid-linked carbohydrates that spans the plasma membrane forming a slimy layer), epithelium & lamina propria (a layer of loose areolar connective tissue)

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25
Q

What does PAMPs stand for?

A

Pathogen associated molecular patterns: The innate immune system recognises and responds to these patterns (BAD)

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26
Q

What does MAMPs for?

A

Microbial associated molecular patters: Produced from friendly bacteria. They are sensed by pattern recognition receptors (PRRs)

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27
Q

What are AMPs?

A

Anti-microbial peptides: They are mainly secreted by enterocytes and Paneth cells found in the inner mucus layer, and they play an important part in the barrier against enteric pathogens

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28
Q

Name two types of pattern recognition receptors (PRRs)

A
  • TLRs: toll-like receptors

- NLRs: NOD-like receptors

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29
Q

What do pattern recognition receptors (PRRs) do?

A

They trigger the production of AMPs and other immunological responses

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30
Q

What do AMPs do?

A

They are involved in modulating host responses towards intestinal homeostasis

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31
Q

What are primary cells?

A

Cells isolated from human or animal tissue, they can maintain their functionality for a few-24 hours, during this time examinations can be carried out on the cells

32
Q

What is the problem using primary cells?

A

The reproducibility varies depending on the donor

33
Q

Name two cell lines used widely to study the effects of microbial metabolites on human intestinal epithelial cells

A

T-29 and Caco-2: derived from human colon cancer tumours

34
Q

What are the advantages of cell lines

A

relatively easy to maintain in laboratories and can offer reproducible results. It also doesn’t require ethics

35
Q

What is the disadvantage of using cell lines in studies?

A

Cancer cells exhibit altered cell properties, such as specific glycosylation profiles, which may affect the cell responses to various treatments. Therefore, it is crucial to choose the most appropriate cell lines for your studies

36
Q

What are the positives about 2D models?

A

inexpensive and easier in terms of assessing the cells and conducting measurements.

37
Q

What is the negative about 2D models?

A

they are not representative of real physiological environments and lack transferability

38
Q

What are the benefits of 3D culture systems?

A

The major advantage of 3D culture models is that they are more physiologically relevant due to a higher degree of structural complexity. Cell functions can also be maintained for longer.

39
Q

Simply describe the structure of organoids

A

A semi-solid, laminin/collagen-rich Matrigel as a scaffold and a cell culture medium that mimics the stem cell niche to achieve long-term culture of the stem cells. They contain central lumen surrounded by “buds” that represent the intestinal crypts

40
Q

What is the function of the crypt like domains, why is this helpful?

A

very similar to those of the adult intestine; therefore, these organoids represent a highly physiologically relevant phenotype

41
Q

What does the co-culture model combine?

A

different intestinal epithelial cells, immunecompetent cells (primary or macrophage cell lines, such as PBMC monocytes and TLT) and bacterial cells

42
Q

What is the purpose of co-culture models?

A

To study the interactions between enterocytes, immune competent cells, microbial cells and their metabolites

43
Q

What are immunecompetent cells?

A

cells capable of producing antibodies in response to an antigenic stimulus

44
Q

What’s wrong with 2D cell culture systems? What should be used instead?

A

they are not representative of real physiological environments and lack transferability (i.e. drugs tested in 2D system might not behave in the same way as in vivo).
- 3D models

45
Q

Why are 3D models better than 2D models?

A

3D cell culture systems are more representative of tissue outside our body and closer to the physiological environment.
More physiologically relevant due to a higher degree of structural complexity.
Cell functions can also be maintained for longer.

46
Q

Describe the structure of 3D intestinal and colonic organoids

A

semi-solid, laminin/collagen-rich Matrigel as a scaffold and a cell culture medium that mimics the stem cell niche to achieve long-term culture of the stem cells. These “organoids” contain a central lumen surrounded by “buds” that represent the intestinal crypts

47
Q

What is the co-culture method?

A

A model that combines different intestinal epithelial cells, immune-competent cells (primary or macrophage cell lines, such as PBMC monocytes and TLT) and bacterial cells to provide us with opportunities to study the interactions among enterocytes, immune-competent cells (cells capable of producing antibodies in response to an antigenic stimulus) and microbial cells and their metabolites. Bacterial cells can also be added to study these interactions

48
Q

If you aim to investigate the metabolism of glucose by several gut bacteria. Which model would you use initially?

A

Batch culture of each of the bacteria: To achieve the aims of the study, you would use batch culture of each of these bacteria with glucose as a substrate to look at how bacteria metabolise glucose because batch culture is simple and the experimental duration is short.
Continuous culture could be used but it is not the best for this research aim since it is more difficult to set up and takes much longer too. In order to check if each bacteria are able to metabolise glucose, it is better to culture them separately.

49
Q

If you are going to study how a bacterial community responds to an antibiotic treatment, which is the most appropriate model to use?

A

Continuous culture of a bacterial community
- Continuous culture provides a stable bacterial community and it is better than batch culture in terms of investigating the effects of antibiotics on bacterial changes. It also provides a possibility to look at the bacterial recovery from the treatment.

50
Q

Name three non-mammalian in vivo model used for studying host-microbial interactions?

A 
Drosophila melanogaster (fruit fly)
Caenorhabditis elegans (worm) (c.elegans)
Danio rerio (zebra fish)
51
Q

What is the major advantage of the fruit fly model?

A

the host genetics have been relatively well studied. It is feasible and relatively easy to conduct large-scale experiments. However, the complexity of its gut microbiome is far simpler than that of the human’s. The gut microbial community of the fruit fly only consists of about 20 species, which are aerobes. This is because the gut of fruit flies is permeable to oxygen, thus providing a favourable environment for aerobic bacteria. This poses a problem for using this model system to study human microbiome since the human gut microbiota is dominated by anaerobes.

52
Q

What is included in the natural microbiome of c.elegans?

A

Enterobacteriacea, Pseudomonadaceae, Xanthomonadaceae and Sphingobacteriaceae.

53
Q

What metabolites modulate the lifespan of c.elegans?

A

Metabolites produced by the bacteria in the culture c.elegans live on produce nitric oxide, folate (vitamin B9), and vitamin B12 which have been reported to modulate C. elegans’ lifespan.

54
Q

How can you study how particular bacteria contribute to the lifespan and fitness of c.elegans?

A

Using mutations that inactivate 21 of the identified host genes: then using the genetic screening tools, to identify candidate genes of the host that are differentially regulated in response to different bacterial strains.

55
Q

What are the advantageous features of using zebra fish as a model to study the gut microbiome?

A

transparent gut prior to adulthood, rapid development and techniques for genetic manipulation.It is also easy to carry out large-scale experiments in Zebrafish. Zebrafish embryos can be maintained in 96-well plates, allowing quick screening of the host or environmental factors that influence symbiosis. Zebrafish can be kept germ-free or with a defined microbial component (gnotobiotic) to further study the interactions between the host and the specific bacteria. Another advantage is the existence of adaptive immunity in this model. The cells of the adaptive immune system, such as B and T lymphocytes, express antigen-specific receptors which allows zebrafish to recognise and remember microbes. In addition, the microbiome composition is more complex compared with fruit fly and C. elegans worms, and is dominant by γ-Proteobacteria and Fusobacteria classes.

56
Q

What bacteria are rodent guts dominated by?

A

Two bacterial phyla, namely, Firmicutes (Gram-positive) and Bacteroidetes (Gram-negative), both of which are strict anaerobes.

57
Q

What is the advantage of using rodents?

A

Similar microbial composition (at least at the phylum level), small size of the host, large litters, relatively short generation time and manipulating certain host genes by gene-editing techniques. Owing to these advantages, rodent models are the most widely used systems to study the handshake between the microbiome and the host. There are a few ways to manipulate the gut microbiota in rodent models.

58
Q

What are the two approaches to generate germ free mice?

A

1) approach involves a caesarean procedure and a cross-foster onto a lactating germ-free host mother.
2) transfer an embryo from a non-germ free, or conventional, animal into a pseudo-pregnant germ-free female animal (a germ-free female who is mated to a vasectomised male) under sterile conditions

59
Q

How are germ free conditions maintained?

A

The germ-free animals are maintained in flexible plastic film isolators and food and water are strictly sterilised before feeding (Figure 9). The sterile condition is also monitored routinely using bacterial culture or 16s RNA sequencing.

60
Q

How can you study the contribution of the gut microbiota to host phenotype?

A

compared the germ-free animals with their conventional compartments or with the same animals post colonisation of the gut microbiota

61
Q

What have germ free mice compared to wild-type mice found?

A

This evidence showed that the gut microbiota contribute to the development of the host phenotype: germ-free animals have different morphological features of many organs, such as the gut. Specifically, the germ-free animals have smaller caecum, size and villus length compared with conventional ones, as well as dysregulated immune system and deficiency of certain vitamins.

62
Q

Why do we have to be careful interpreting results from germ free mice?

A

they can be biased because of underdeveloped organ function rather than being from the direct impact of gut microbial activity.

63
Q

How could you overcome the problem of germ-free mice having under-developed organs/not widely available/ expensive?

A

Antibiotics-treated animals become a great alternative for research in this field.

64
Q

What do typically protocols to achieve mice

A

Use of oral intake of wide-spectrum antibiotics and proton pumping inhibitors in drinking water or through oral gavage for a few days before transplanting the targeted microbiota of the interest of studies. This model system is easy to maintain and relatively cheap to run, and equally provides valuable information about the host-microbial interactions. The disadvantages of this model are that bacteria cannot be totally removed by the antibiotics and the residue bacteria could compete with the transplanted bacteria, resulting a failure of the colonisation of the targeted bacteria.

65
Q

Why would swine be used to study the gut microbiome?

A

Swine have high genome and protein sequence homology with humans, which serve as a closer-to-human model for studying host-microbial interactions. Pigs also share more similarities in immunology and gastrointestinal anatomy and physiology with humans compared with rodents. For example, both human and pigs are colon fermenters, whereas rodents are cecum fermenters. Furthermore, similar to the human brain, the major brain growth spurt of the pig extends from late prenatal to the early postnatal period. Therefore, swine could serve as a robust model for host-microbial research.

66
Q

What are the variations in human studies often due to?

A

a diverse range of lifestyle and environmental factors, such as dietary habits, exercise, pollutants, medication, etc.

67
Q

How can you control or minimise these variations between human samples?

A 
By selecting sub-groups of populations or patients exhibiting a lower degree of heterogeneity
 longitudinal studies (an observational research method where data is gathered for the same subjects repeatedly over a period of time) are preferred to the cross-sectional studies (an observational study that analyses data from a population, or a representative subset, at a specific point in time).
68
Q

Why do these controls work to minimise variations?

A

(1) the biological changes (e.g. metabolite changes and microbial shifts) are dynamic and
(2) inter-personal variations are often larger than the intra-personal variations. Therefore, it is best (where possible) to compare post-intervention to pre-intervention of the same individual.

69
Q

What have typical studies

A

Typical studies conducted in humans in the field of host-microbial interactions are to investigate dietary interventions, effects of prebiotics/probiotics, clinical trials, faecal transplant as a therapeutic treatment and various human diseases such as cancer, diabetes, obesity and depression.

70
Q

What are the three Rs

A
  • Replacement
  • Reduction
  • Refinement
71
Q

Define the contemporary replacement (3Rs)

A

Accelerating the development and use of models and tools, based on the latest science and technologies, to address important scientific questions without the use of animals

72
Q

Define the contemporary Reduction (3Rs)

A

Appropriately designed and analysed animal experiments that are robust and reproducible, and truly add to the knowledge base

73
Q

Define the contemporary Refinement (3Rs)

A

Advancing research into animal welfare by exploiting the latest in vivo technologies and by improving understanding of the impact of welfare on scientific outcomes

74
Q

Define the standard for replacement (3R)

A

Methods that avoid or replace the use of animals

75
Q

Define the standard for reduction (3R)

A

Methods that minimise the number of animals used per experiment

76
Q

Define the standard for refinement (3R)

A

Methods that minimise animal suffering and improve

Microbiome (10 decks)

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