Final (human microbiology)

What is the role of alpha-synuclein in Parkinson's disease?

*Alpha-synuclein* is a protein that is toxic to neurons. Reactive oxygen species involvement. If build up too many, neurons die and get defects. - neurons are involved in dopamine production, so increase in alpha-synuclein leads to decrease in dopamine production.

Describe the study from Hsaio et al. What defects were observed in the MIA offspring? What effect did B. fragilis have on the MIA offspring?

*Hsaio study* boldly said the microbiota changes physiology and behavior associate with autism. -The study inject the pregnant mice with immunostimulant (polycytidylic acid) -> offspring mice have leaky gut and increase in serum metabolites in their blood = 4EPS + p-cresol + indolepyruvate (all three have been proposed to be potential markers for autism). -*Bacteriodes fragilis* did improve some of the symptoms and fix part of the immune system

*Explain deconjugation and 7α/β dehydroxylation activity. What are the potential problems with each of these for the host?

*Problem is that it increases the toxic effects of bile acid and could lead to colon cancer. -*Deconjugation* is the removal of the glycine or taurine from the conjugated bile acids. -*7a/B dehydroxylation* activity deals with producing secondary bile acids from deconjugated primary bile acids (CA to DCA and CDCA to LCA). -Both of these *can cause an increase of colon cancer*

What is the generalized process for tumor formation in colon cancer?

- Lack of control in the cell cycle caused by changes in DNA leads to uncontrolled growth of cells (less cell death) and a lack of differentiation. - With colon cancer, inflammation or reactive oxygen species can lead to mutations in DNA which can lead to malignant growths called polyps. Polyps are considered pre-cancerous and if it grows, it can develop into a tumor. If grows enough, can metastasize and spread to other parts of the body - Can take decades to develop. - normal epithelium --> hyperproliferative epithelium --> adenoma --> adenocarcinoma

How is the microbiota impacted by C. difficile infection? What about recurrent C. difficile infection?

- microbiota largely destroyed. Phyla composition changed drastically. Loss of richness. - Once have a single case of C. diff, have a 20% chance of getting another. Once have a second case of C. diff, have a 40% chance of another. Once a third, 60% chance of another infection.

How is the brain thought to influence the microbiota?

1. *Stress* causes compositional changes -*Decrease in Bacteroides and increase in Clostridium* -At the phylum level you see changes in GI tract 2. Mechanisms are largely unknown - Alterations to intestinal environment? - Stress *change hormones* and then lead to physical *changes in intestinal motility, function of epithelium layers and mucus*

What factors impact how a particular drug will alter the microbiota?

1. *different people* = different impact with same drug (genetics). 2. *different drug* = different impact. 3. *different dosage* = different impact

What is the proposed mechanism for how Clostridium scidens may be able to prevent C. difficile infection?

C. scidens contains 7 alpha/beta dehydroxylation so it can produce secondary bile acids from primary bile acids. This will make C. difficile not toxic anymore even if it is present.

What is the evidence in support of a possible single causative agent for IBD? Is this the predominant theory behind this chronic disease?

In the past, thought it was pathogen caused, but evidence today doesn't support a single cause. - *Mycobacterium avium subspecies paratuberculosis*: causes intestinal inflammation, diabetes, and weight loss. Found more in developed countries. Can be passed through milk. found in higher rates for people with IBD than those without, but in larger surveys, less correlation with larger populations. - *Adherent and Invasive E. coli (AIEC)*. Adheres to epithelial cells and then invades. Will cause problems even with healthy person. IBD more common in kids with AIEC than without. By itself can cause inflammation with mice. - Clostridium difficile. Not thought to be initial trigger. Can cause to go out of remission (healthy state to flare up). More susceptible to C. diff because of damage. Doesn't think is single cause.

Over the last 20 years (including recent data), how has the definition of the hygiene hypothesis shifted?

There is a revamped definition and assumption that the reduction in antigen exposure prevents the immune system from developing properly leading to imbalance and less microbiota diversity. These effects later translate into chronic inflammatory conditions like allergic diseases, inflammatory bowl diseases, and autoimmune diseases.

Are probiotics thought the help with the prevention of allergic diseases? The treatment?

Yes for prevention, no for treatment.

In terms of C. difficile colonization, what is known about asymptomatic carriers? Infants?

1 - 3 % Asymptomatic carriers have C. diff. (don't know if there for short-term of if just a snap shot) ~50% infants are colonized with C. diff, but rarely gets disease from C. diff. - Unsure why C. diff doesn't make infants sick.

Do antibiotics only impact the microbes they directly inhibit? Explain.

Bacteria in the GI tract are interdependent, therefore, the antibiotics will end up hurting the microbiota community as a whole regardless of how narrow the drug range is. One drug targeting one microbe can wipe out entire metabolic networks. Ex: if wipe out microbe B, then lactic acid builds up and kills microbe A.

What is the gut-brain axis?

Communication -> complex net work of communication with the immune system, enzymes, and hormones involved

What is the evidence for involvement of environmental factors in the development of allergic diseases?

Even though allergies can be found with genetics/within families, because there is a rapid increase of people with allergic diseases over time, something environmental has to be happening to make the allergies more frequent. They are found more in developed countries than developing countries. Possible correlated environmental factors include: 1. being fed breast milk vs infant formula or raw cows' milk 2. being delivered vaginally vs via a C-section 3. the diet of the mother when pregnant 4. the antibiotics of the mother 5. the family size 6. sibship order 7. daycare attendance 8. contact with pets and farm animals 9. infection history 10. antibiotic use 11. vaccination 12. "time window" 13. organ development 14. immune system development.

What are some of the difficulties with investigating possible microbial involvement in autism spectrum disorders?

It has a wide spectrum of disorders ranging from mild to severe symptoms, however, there is *no consistent risk factors and markers* and the *diagnostic method is in debate*.

Explain epithelial cell proliferation and migration (in the general way that was discussed in class).

Normally, there are stem cells in the small intestine that are partially in the crypts and villi that make new cells on a regular basis and migrate up the villi (5 - 8 days to move up). They differentiate, and at the tip, they are either sloughed off from peristalsis (movement of the GI tract) or go through apoptosis. (Different for germ free mice)

What are the functions of bile?

(bile acid is made by the liver and stored in the gallbladder) *1. absorption of fat and fat soluble vitamins*. (Mainly absorbed in the small intestine) - *Can act as a detergent* (how absorbs fats and cell envelopes) *2. role in colonization resistance*. (can promote or inhibit C. diff. infection) *3. Neutralization of gastric acid in small intestine* *4. Prevent small bowel growth (tumor?) and prevent abnormal growth of bacteria* (to prevent vitamin/mineral deficiency) *5. can act as a ligand for receptors* such as: - TGR5 - Farnesoid X (FXR) (these receptors affect everything from glucose and lipid metabolism, regulation of immune response, and other major body processes)

Explain the data involving Salmonella enterica serovar Typhimurium infection that support the concept of colonization resistance.

(Bohnhoff et al, 1950's *Effect of antibiotic treatment on infectious dose of Salmonella enterica serovar Typhimurium*) - shows that if you have healthy mice, it's difficult to infect them with Salmonella. - However, if you first treat the mice with antibiotic you can easily infect them with Salmonella (*2008 Salmonella study* Streptomycin or vancomycin treatment followed by S. enterica serovar Typhimurium treatment) - looking at Salmonella in mice with different types of antibiotics - Unique: use lower concentration of antibiotics than previous studies, leading to composition changes Result: as you increase antibiotic concentration, did not see reduction in bacterial load, but you increase the amount of Salmonella load to enter and infect the GI tract. (Note: conventionalized mice have uniform infection of Salmonella because Schaedler flora was used = low level of complexity microbiota)

What is the evidence for microbial involvement in colon cancer?

(Colon cancer drastically changes area of colon and likely changes microbiota. Don't know if cause or effect) Have rodent models where rodents prone to developing tumors in intestinal tract. If you keep mice germ free, develop less tumors than if you colonize them. Decreased diversity. But inflammation often decreases diversity. Rodent models --> tumors (germ free = less). Within colon, most get tumors at distal end at the bottom of the colon. If go in and take samples throughout colon, pH different there. Increase SCFAs at beginning vs end of colon and microbial communities also assumed to be diff there. Maybe the microbes at the end lead to colon cancer.

Describe the ways in which pathogens might be able to infect despite having a healthy commensal microbiota (how do they subvert colonization resistance?).

*1. Antibiotic treatment* *2. Help from the normal microbiota* in non-ideal situation: Thetaiotamicron produces succinate (instead of fucose -> ideal situation), the EHEC e. coli comes in produces mucinases that will destroy your gut mucus layer -> colonize your gut and take over -> theta in return even produce more succinate in reaction to all of this. (Viruses may be able to infect normal gut rather than antibiotic gut) *3. Pathogen mechanisms* -*Virulence gene expression* (ex: shigatoxin eliminate competition, promote spread) -*Successful competition of niches and nutrients* ( some bacteria can cause inflammation as a lot of good bacteria cannot survive in the inflamed area) *Ability to thrive in the inflammatory environment* (product produced by inflammatory cells such as carbon sources (produced from host cell damage) used as resource) *Localization to specific areas* Additional supporting material: (Some bacteria can replicate inside macrophages and turn it into a pathogen machine) (some microbes able to dig through mucous layer) (able to go unrecognized by immune system like with Salmonella producing siderophores (prevent bacteria from getting iron and they will die -> Salmonella bring their own Siderophores and get the iron they need).* (able to use products in gut that others don't, like Ethanolamine) (thrive in place that is rarely colonized)

Explain the generalized mechanisms for microbial involvement in colon cancer formation.

*1. Bile acids:* If microbes are involved, secondary bile acids can be produced and if have more of these, more likely to develop cancer. *2. DNA damage:* leads to uncontrolled growth *3. Inflammation:* causes damage to cells, increases immune system presence and breaks down barrier function. Suppressed inflammation also leads to cancer formation. *4. SCFAs:* involved with barrier function, inflammation, and host cell gene expression. Fiber consumption and how changes colon cancer risk.

What are the future directions in trying to understand the connection between the GI microbiota and IBS?

*1. There is a need for a multifactorial approach to IBS* - we need to look at environmental factors, genetics, infections, healthy history, and the microbiota (composition and function) if we are going to have a chance at understanding this. Most studies to date have only looked at one or a few factors. 2. For the microbiome: most studies to date have looked at fecal samples (for all the reasons we have previously talked about), but we are well aware that the microbiota in the GI tract is different from the community in fecal samples. In an *ideal situation, we would not only be able to look at intestinal samples, but we might be able to differentiate between the microbes in the lumen and those in the mucus layer/close to epithelial cells*. It is entirely *possible that there are differences there that we are not picking up with fecal samples.* Finally, *we MUST divide studies by the subtypes of IBS* if we are going to have a chance. It is highly unlikely that IBS-C and IBS-D are caused by the exact same things. Unfortunately, this *means that the study sizes must increase greatly*. A study that previously was 100 healthy controls and 100 IBS patients could now become 100 healthy controls, 100 IBS-D patients, 100 IBS-C patients, 100 IBS-M patients, and 100 unsubtyped IBS patients. This would mean *better diagnostic tools and substantially more money and time would be required*

What are the new possible treatments for C. difficile infection? Explain each option.

*1. thuricin CD:* - Bacteriocin produced by Bacillus thuringiensis. - narrow spectrum drug ( since other organisms are unlikely to be codependent with C. diff, the narrow spectrum targeting it may get rid of C. diff without harming the other organisms) *2. probiotics* - for prevention, not treatment *3. Non-toxigenic C. diff strains* - colonizing people with this may be able to outcompete the toxin ones. - problem with this is that through horizontal gene transfer, the non-toxic ones may become pathogenic? *4. Fecal transplants or pills* - via pills or direct fecal transplant *5. Toxin inactivation* - Ebselen goes after virulence factors and inhibits the action of C. diff toxins - may allow microbiota and environment to reestablish themselves. - Once reestablished, may get rid of C. diff on its own

What are some of the mechanisms through which an undisturbed gastrointestinal microbiota might be able to prevent C. difficile infection?

*5 Theories:* 1. *destroy C. diff as a competitor* 2. *change bile composition* (so that C. diff is not triggered to reproduce/multiply) 3. *direct antagonism* - create bacteriocins to destroy C. diff. 4. *an alert immune system* normally, immune system is constantly being expressed and is on alert due to microbial presence, and if this were to decrease the immune system would relax and would result in a chance for C.diff to invade unnoticed. 5. *SCFAs* - increased butyrate levels inhibit certain aspects of C. diff lire

What possible impacts will antibiotic-induced changes have on the host?

*Based on the study that we reviewed, the antibiotic end up killing most of the bacteria within 3-4 days of administration - increase in resistance - more likely to be colonized by pathogens following treatment (decrease in colonization resistance) - change in functions of microbiota (nutrient absorption, intestinal lining, SCFAs)

What is the connection between bile and Clostridium difficile?

*Bile acids are thought to be involved in spore germination and outgrowth of C. diff* in the intestines (Taurocholate (conjugated) and free glycine are required for germination Cholate induces germination (higher % spores germinate here so need deconjugation) Chenodeoxycholate inhibits germination Deoxycholate induces germination but... (more spore germination but then vegetative cells cant germinate???))

What are the causes of antibiotic-associated diarrhea?

*Causes of antibiotic associated Diarrhea:* 1. Damaged commensals (species that benefit from the host association—may obtain nutrients, shelter, support, or locomotion from the host species, which is unaffected.) - bulk of complex carbs no longer being degraded. - decrease SCFAs and screw up osmotic balance in the colon (more water in or less absorbed --> diarrhea) 2. Drugs 3. Specific Pathogens - is usually C. diff (25% chance) - can also be candida albicans or staph aureus

Explain the characteristics of Crohn's disease vs ulcerative colitis.

*Crohn's disease* - Generally more severe - Impacts any portion of GI tract - Affect all layers and sections of GI tract Inflammation is patchy - Can remove inflamed patches to help cure crohn's *Ulcerative Colitis* - Begins in rectum and can extend to involve the entire colon - Continuous in inflammation - Only on the surface of the epithelial lining - Can remove the entire colon to fully cure colitis

What are some of the defects found in germ-free rodents (in terms of gastrointestinal tract development)?

*Defects found in Germ Free Rodents*: 1. have a large cecum 2. Crypts and villi structure is altered so don't absorb nutrients as well. (Villi get longer and thinner with shallower crypts --> less absorption) 3. Decreased peristalsis - Things move through the GI tract at a different rate (ex: dyes) 4. Decreased barrier function (easier for things to get out of the gut, leaky gut) 5. Reduced presence of the immune system. 6. epithelial cells turn over at a different/lower rate 7. Decreased mucous in the GI tract 8. Different mucous composition 9. Fever blood vessels in GI tract

Briefly describe the findings from the two studies presented in class (what antibiotic treatments were used, what subjects were investigated, what were the overall findings?)

*Dethlefsen, Relman 2011*: - Three participants - two 5-day courses Cipro spaced six months apart Conclusions: -Interpersonal variation prior to treatment matters, especially in terms of recovery, -major loss in diversity, most microbes lost were recovered, but never fully *Antonopoulos, Huse 2009*: - Mice - Amoxicillin + metronidazole + bismuth - Cefoperazone - vancomycin (second study? added into own test later) Conclusion: different people and different drugs have different impacts, normal donor microbiota (through coprophagy in mice) can help gut microbiota recover

How is the microbiota thought to influence brain development? The already developed brain?

*Developing Brain*: 1. Germ-free animals *They'll react different to stress -> some overreaction nd explore more than usual 2. Defects in responses to stress; altered behavior *Early colonization = normalization *Later colonization = incomplete normalization Brain chemistry is different *Neural pathways are developed differently *Developed Brain*: 3. Behavior is altered (the already developed brain) *Dietary changes 50% lean ground beef diet -> this group has better memory, better learning, reduced anxiety without looking at their vascular health *Antibiotic administration: Variable effects of behavior -> different antibiotics change the GI in different ways and lead to different changes *Number of changes based on different genetic background +Swiss Webster mice: less anxious and explore more +Mice B: tend to be more anxious +Cross fecal transplant effect their behavior Sw->b = calmer B and B->Sw = more anxious Sw *Transfer studies Behavioral traits were passed on with the microbiota transfers

Explain enterohepatic circulation

*Enterohepatic circulation* - process by which bile is normally processed and how 95% of it circles from the liver to the gallbladder to the small intestine back to the liver. 1. Primary bile acids are synthesized from cholesterol in the liver. 2. These bile acids are secreted into the gallbladder. 3. When a meal is eaten, choleystokinin (hormone) is released from the duodenum of the small intestine and causes the gallbladder to contract and release the bile acids into the small intestine. 4. the bile acids act on the food in the small intestine, aiding in digestion and absorption of fats and fat soluble vitamins. (also prevents bacterial overgrowth) 5. the bile acids are absorbed by the distal ileum. 6. the majority of bile acids are absorbed into the bloodstream and are sent back to the liver with a 95% recycling. 7. The other 5% goes to the colon or are modified by bacteria. The majority of bile modification occurs in the colon. 8. the 5% is likely excreted in fecal samples

What is the evidence for microbial involvement in autism? Be able to describe the evidence involving environmental factors, the gut-brain axis, fermentation profiles, and microbial dysbiosis.

*Evidence for environmental factors* -Rapid increase of autism disorder within the population -> the disorder have tripled since 2000. This may be due to more awareness and better diagnostic tools -Concordance rates indicates room for environmental factors -Diet: some suggestion that avoidance of certain food will decrease symptoms of autism, associate with absorbing carbohydrates and put on diet similar to IBS *Gut-brain axis* -Germ-free mice have altered behavior and response to stress more highly -Microbes are direct barrier to the gut function and can cause systemic dysbiosis -Leaky gut leads to larger amount of microbial metabolites in the blood, leading to systemic inflammation *Changes in fermentation profiles* -problems with carbohydrates absorption and transport: 95% of SFCA are absorbed in the gut +some people with autism have larger total amounts of SFCA form their stool, possibly either from secretion or result from decrease absorption +hexose sugar is not well absorbed in certain autism subgroup -Altered SCFA levels +Propionate -> some studies suggest an increase overall in propionate production in autism individual +issue: there has been studies suggest higher amount of propionate in fecal samples and blood stream +if you inject rats with propionate, they get seizures and avoid social problems. -Altered fecal ammonia levels -> potentially cause a leaky gut *Changes in the microbial composition* -There are some reports that antibiotics and probiotics help autism but they are not big study -Increased in Firmicutes to Bacteroidetes -Presence of Sutterella genus: a lot of autistics versus non-autistics individuals -Decrease in Akkermansia muciniphila -Very little consistency

What types of specific organisms might be involved in colon cancer formation?

*Fusobacterium nucleatum*: role in oral biofilm formation. In most people if find this, find in mouth, but in gut, healthy people don't have large amounts of it. - In people with IBD, can find F. nucleatum in gut. - In people with colon cancer, can also find in colon. (Could possibly be moved from mouth to colon somehow) *E. coli and Bacteroides fragilis*(not all types): Important subsets where they produce certain toxins. - PKs island. Genes in this island are responsible for producing the colibactin (toxin) - Same thing true with B. fragilis. Gene produces enterotoxin (toxin) - Both toxins damage DNA and are correlated with cancer formation. Without right repair, increases mutation rate and if in tumor suppression genes for example, leads to cancer.

Explain the differences between Type 1 and Type 2 diabetes.

*Type 1* -Autoimmune disorder: the body destroys the beta cells in the pancreas and the body loose the ability to produce insulin. The body can compensate and keep up for only sometimes until the majority of the pancreas is destroyed. (Pregnant women can loose their ability to control blood sugar during pregnancy (we call this gastrointestinal diabetes), they will recover from this but have higher risk of developing type 2 diabetes later on.) *Type 2* -It is associated with obesity and ties with heart disease. -Cells in the body loose their insulin tolerance and stops responding to insulin, while the pancreas keep on producing more insulin which cause a person to require more and more insulin to control his/her blood sugar. If goes untreated, the pancreas will be damaged.

What impacts do microbes have on epithelial cell proliferation, migration, and differentiation? Gut motility? Mucus? Barrier function? GALT? Blood vessel formation? *Basic structures in the GI tract (crypts and villi)?

*IN GERM FREE MICE:* *Decreased cell proliferation* (stem cells not as active and maturation of cells is slower) *Slower and reduced migration* (repair is slower) *Different timing with differentiation* (can predict timing of differentiation in normal mice but not germ free mice ex: Paneth cells and lysozyme production by them can be predicted in normal mice) *Slower Motility, thereby less efficient at absorbing nutrients:* - reduced peristalsis thought to be due to either lower serotonin from less active endocrine cells or from a decreased ability to respond to serotonin. (affects the nervous system thereby affecting peristalsis.) *Breached Mucus layers leading to inflammation:* - if microbe get into single layer of mucus in small intestine or second layer (last layer) of mucus in large intestine, triggers inflammation. - Thinner layers of mucus *Decreased Barrier function:* - since decreased or lack of immune system presence, can't repair damage as well, so breaches in the epithelial are easier. disruptions in tight junctions (normally prevent things from going between epithelial cells). "leaky gut" *GALT (gut-associated lymphoid tissue) is underdeveloped:* - impacts immune system (ex: Peyer's patches) *Decreased blood vessel formation:* - if colonized, increase in formation from increased oxygen demand from microbes *Basic structures in GI tract (crypts and villi):* if colonized: villi shorten and widen, crypts get deeper, Surface area increases.

Describe the study from Kim et al. What is the effect of the maternal microbiota on the MIA offspring? What data support this?

*Kim et al* paper focused on the mother microbiome to see changes and the effect of these changes 6 C57B mice obtained from two different vendors either form Treat mothers with saline, immuno-stimulant, and stimulant with vancomycin See *no difference between treatment groups and saline solution (control) mothers* 1. *Maternal bacteria promote abnormal behaviors associated with neurodevelopmental disorders in MIA offspring* 2. Also looked at were the mice were from (Taconic or Jackson lab) Noticed that *Taconic induced IL-17 production* +If mother is prone to IL-17 production there is a *greater chance* that the offspring have behavioral abnormalities → increased risk of *autism in offspring if mother gets infection during pregnancy*

How does circulating levels of LPS potentially tie in to diabetes? What about the exact structure of LPS?

*Metabolic endotoxemia show people have low level of LPS, but individual with high LPS level -> chronic inflammation -> then systemic level of high inflammation -> autoimmune disease -> Studies show that LPS mice become obese Bacteria have variation within their LPS layers. Studies in T1D between Finland and Russia -> T1D individuals from Finland are dominated with Bacteriodetes and T1D individuals from Russian are dominated with E. coli -> the E. coli have LPS that can induce endotoxin tolerance in its host and decreased host autoimmune activity *Finland are more prone to developing T1D

Be able to explain the proposed mechanisms behind colonization resistance (both microbe-microbe and host-microbe).

*Microbe-Microbe interaction* 1. Competition for niches and nutrients (since there is a max carrying capacity, there is competition for territory and resources) 2. SCFA production - used as signals to direct virulence genes. Some things can down regulate gene expression in pathogens 3. Oxygen consumption -> facultative use up oxygen when it gets to the colon 4. Direct attack -> produce compounds that kill other bacteria in the competition for nutrient and space in your gut ex: bacteriocins made by bacteria like lactobacillus *Microbe-Host interaction* (complex) 1. Stimulation of innate and adaptive immune responses (how alert is your immune system on my GI microbiota and how the microbiota influence this (vice versa)) - Toll-like receptors - Balance of T cells 2. SCFAs - Butyrate and epithelial barrier function (If you don't have large amount of butyrate, it may be easier for pathogens to colonize the gut) (keeps lining healthy) 3. Antimicrobial production -> microbiota can influence defensins produced by Paneth cells in the small intestine -> has to go through cleavage to become active 4. Bile acid modification -> microbiota use bile acid as a signal (ratio of certain bile acid) to influence whether C. diff activates and become vegetative in the gut.

Be able to explain the experiments and results from the 2013 study by Ridaura et al.

*Ridaura et al*: The study recruited 750 pairs of twins (one lean and one obese), 4 pairs made the final cut which made it an incredibly small study. The twin human microbiota was transplanted into germ-free mice. Results: the mouse with the lean twin fecal sample became thinner, while the mouse with the obese twin fecal sample became obese. the study continued to see whether the obese microbiota can be passed from an individual within the same family -> they conducted the *cohousing experiments* where have they add another mouse to a cage full of mice: -> obese to obese mice cage -> obese to lean mice cage -> lean mouse to a lean mice cage (control) -> lean mouse to an obese mice cage -> results: the lean microbiota is relatively stable and the obese mice became lean after the lean mouse is added to the cage

What are the risk factors for C. difficile infection? Explain the problem with antibiotics (hint: think of risk factors and treatment options).

*Risk factors:* 1. Being older than 65 (elderly) 2. Being in a healthcare setting/receiving medical care 3. having IBD and obesity 4. received antibiotics within the last few months *Problem with antibiotics in relation to C. diff*: If stopping antibiotic does not treat C. diff infection, we treat C. diff with antibiotics such as Vancomycin and Metronidazole, and yet being treated with antibiotics is a risk factor for obtaining C. diff. Could make a C. diff infection worse.

Explain the results from the Wheeler et al study (fungal dysbiosis).*

*Wheeler ML, et al* Immunological Consequences of Intestinal Fungal Dysbiosis: Contained evidence that the mycobiota can alter immune responses. The antifungals changed composition of the the fungal communities (as opposed to the actual amount). Lost some good? Or gained some bad? Antifungals allows some to increase and those ones seem to promote allergies and asthma. Shows that not just bacteria is involved but also fungi.

Describe IBS in general (symptoms, cause, etc)

- Chronic functional disorder (most widespread GI disorder) Unknown cause - Suspect multiple types, don't know if categorized correctly - Not everybody has symptoms at at one time. Possible symptoms: 1. inflammation 2. constipation 3. diarrhea 4. don't tend to see significant damage with the GI tract like with IBD 5. things in the gut may move to fast or slow 6. severe abdominal pain

What types of microbial changes are potentially associated with allergic diseases? (either cause or effect)

- Varies among studies - *Decreased Lactobacillus, Bifidobacterium, and Helicobacter pylori*. - *Increased or Decreased C. difficile* depending on the study. (Don't know if is an increase of good or a gain of bad microbes, so don't know if cause or effect) 1 and 2. Haemophilus and Monaxella (respiratory)- increase associated with asthma in children and adults. 3. Neiseeria (Resp)- Increase associated with asthma in adult 4. Streptococcus (Resp) - Increase associated with asthma in children 5. Lactobacillus (GI, resp) - Decrease associated with asthma in children and adults 6, 7, 8. Bifidobacterium, Facecalibacterium, Akkermansia (GI) - Decrease associated with risk for asthma development in childhood. 9. Morganella morganii (GI) - Increase associated with asthma in non-obese adults

What types of compositional and functional changes are proposed for dysbiosis as the possible cause/trigger/exacerbating factor for IBD?

- decreased diversity and richness in people with IBD (also true with Obesity) - don't know if increase in bad or decrease in good or which is more important.

What is the cecum?

- pouch that forms the first part of the large intestine. - connects the small intestine to the colon. - Absorbs fluids and salts that remain after completion of intestinal digestion and absorption and mixes contents with mucus. (Doesn't have major functional importance in humans but is full of bacteria that helps with fermentation in mice)

What general compositional changes are associated with IBS? What additional problem are IBS patients with a "normal" microbiota at risk for?

- temporal instability; decreased diversity - Increase in Firmicutes; decreases in Bacteriodetes - Increase in lactobacillus spp. but is counterintuitive because people tend to consider lactobacillus to be good bacteria and is in probiotics like yogurt. - IBS patients with a "normal" microbiota may have double the risk of depression than the normal population.

What is the purpose of the figure from the 2016 Sampson study?*

- to show that if microbes are not involved (germ free mice) there is no difference between wild type mice and defected germ free mice. They exhibit the same behaviors which shows that: there is microbial involvement in Parkinson's disease - (When GF mice were inoculated with healthy microbiota vs PD microbiota, the ones with PD microbiota developed motor defects. - antibiotics decreased sx)

What is the evidence for microbial involvement in Type 2? Be able to explain several points.

-Based on connection with obesity *Conventionalized (previously) germ-free mice shows an increase in body fat and loose their ability to control their blood sugar -> develop something similar to obesity and diabetes *Metabolic endotoxemia show people have low level of LPS, but individual with high LPS level -> chronic inflammation -> then systemic level of high inflammation -> Studies show that LPS mice become obese *However, obesity and Type 2 is a complicated interaction since some lean individuals can also develop T2D, leading to confuse/complicate research -Human studies: *show increase in Bacteroidetes to Firmicutes but this is shown to be reversed in some studies *decrease in butyrate producers *fecal transplant study -> 18 overweight men with metabolic syndrome divided into two groups, one without the experimental factors (9 got fecal transplant from their own species) and the 9 got fecal transplant with the lean donor results -> no visible difference in the control group, while the experimental group show weight loss and blood glucose regulation return to normal with no signal of diabetes in the pre-diabetic individuals *the issue is the microbiota did not persist for a long period and the experimental group began to gain their weight back along with the pre-diabetic markers.

What is infectobesity? What data support this concept?

-In animals, there are studies found some infectious agent that can induce obesity in animals and these comes from viruses, bacteria, parasites, and even prions -Majority of literature reference adenoviruses -> found in decent amount of population -> studies try to correlate these with obesity while some don't find such correlation -The idea is not well supported

What types of GI disorders are thought to be associated with autism? What unanswered questions remain in terms of GI disorders and autism?

-People with autism disorders have chronic inflammation and diarrhea, which correlate with diet or the consumption of the same food overtime -9-91% of individuals have GI disorders, depending on the severity of the disorder -It is unknown whether autism cause the GI disorders or the GI disorders cause autism

What is the evidence for microbial involvement in Parkinson's?

-less than 10% genetic risk -90% appear to have environmental influence - cause/effect of difference in the gut function - Gut-brain axis (Primary diagnostic tool used has to do with defects in brain in terms of aggregates but papers say starts in gut like constipation 30 years prior to PD. Hypothesis: starts in gut and misfolded proteins get to brain. (Metabolic ways bacteria can influence dopamine too). Microbiome of them vs others different.

In general, what have animal studies suggested in terms of the compositional changes in obese vs lean animals? Human studies?

1. *Animal studies* -> obese mice have increased the ratio of Firmicutes to Bacteroidetes. However, the ratio may flip-flop in diabetic patients *Obese animal* -> overall have decreased in diversity, richness, and evenness *Human Studies* -> complex in human with factors such as hormones, overeating, genetics, and many more, the connection to between Firmicutes/Bacteroidetes ratio is still not certain, and it is difficult to find the obese individual that does have multiple factors. Bariatric surgery increase in Bacteroides, prevotella, E. coli, and F. prausnitzii Obese Children Increase in S. aureas found in feces of overweight pregnant women→ introduction can lead to onset of obesity later in child's life

What are the potential mechanisms through which the microbiota may impact obesity? Be able to explain the ideas/data behind each of these.

1. *Energy extraction* -> a. fermentation of non-digestible carbohydrates: production of SCFAs -> b. regulation of host genes: fasting-induced adipocyte protein (fiaf) is a suppressor of energy storage which reduces the amount of energy that your fat cell are programmed to store and. Some microbes turn off this suppressor, leading to higher energy storage. 2. *short chain fatty acids* -> conflicting data -> a. energy source -> b. anti-inflammatory potential which keeps epithelial cells healthy through barrier function -> c. signals 3. *signaling* -> a. hormones (satiety): particularly leptin, a protein that regulates fat storage -> b. bile acts as ligand for a variety of important receptors such as TGR 5 and Farnesoid X (FXR), FXR is important in regulating the development of diet induced obesity (mice with FXR did not gain as much weigh as WT and GF mcie) -> the gut microbiota increases crown-like structure and expression of pro-inflammatory markers in white adipose tissue (WAT) through farnesoid X receptor. (ask teacher) -> FXR signaling alters the gut microbiota leading to modulation of glucose metabolism 4. *circulating levels of LPS* -> keep in mind everyone has low level of LPS -lead to constant low grade inflammation -lead to a theory called *metabolic endotoxemia* -> primary theory in regard to type II diabetes and obesity -> if you feed mouse high fat diet: LPS level increase in the blood stream -> A study: separate out changes in microbiota with changes in LPS level -> hook tiny pumps in mice and pump them with saline or diluted solution of LPS -> mice with LPS has increased -> mice with the LPS pump has increased of fasting glucose, triglycerides, fat deposits, and inflammation

Over the last 20 years (including recent data), What microbiological data has been reported?

1. *Exposure to Hepatitis A, Toxoplasma gondii, and Helicobacter pylori* can lead to suppression of inflammatory, a mechanism that allow these pathogen to persist. -> hookworms: produce major compounds to suppress inflammation -> Rates of H. pylori and hookworms infection overall decreased in developing countries 2. *Antibiotics*: early exposure to antibiotics in infant correlate with higher rate of allergy/asthma later in life. 3. *immunomodulatory capabilities* -> bacteroides fragilis: production of capsular polysaccharide A (PSA) -> probiotics: prevent the development of allergy but once you have it, probiotics will not help

What are the possible mechanisms of communication between the microbiota, the brain, and the gut?

1. *Neural pathways: Vagus nerve* -> control wide range of things throughout your body: *Lactobacillus rhamnosus* administration promoted exploration in mice (changed behavior and strain specific) +The behavior change is based on the Vagus nerve: one group normal + vn -> changed behavior and second group normal + damaged vn -> no changed behavior associated +Matter in some cases and is the not the only factor that matters +Only when the vagus nerve was fully functional 2. Bacterial metabolites: *Fermentation end-products: +*Lactic acid* +*SCFAs* - Propionate and butyrate #Largely based on people with depression fecal and altered ratio of SCFAs in their sample #Behavior changes of mice when injected into mice -> however, can they cross blood brain barrier *Tryptophan metabolites* +*Kynurenic acid*: connection with schizo with kynurenic acid (from amount of tryptophan produced by bacteria) +*Schizo have too much kynurenic acid* *Neurotransmitters:* +*GABA* +*Noradrenaline, dopamine, acetylcholine* +They are species in your gut that produce functional neurotransmitters 3. Immunological and endocrine mechanisms *Circulating levels of cytokines* -> microbes shifting the ratio of T cells *Enteroendocrine cells* -> serotonin levels

Over the last 20 years (including recent data), What epidemiological data has been reported?

1. Data on *household size and structure* such as: *Family size*: bigger family are less likely to have allergy and asthma *Birth order*: the youngest are least likely to have allergy *Gender*: having brothers lead to better protective effect against allergy *Genetics*: could influence risk of having allergy but can be override by environmental influences 2. *Day care* study suggested both decrease and increase in allergy and asthma 3. *Variations in socioeconomic status* -> Certain lifestyles can lead to lower allergy *Affluent*: wealthier lifestyle can lead to higher risk in developing allergic diseases *Urban poor*: may have the same risks as people in the same area, suggested that it may related to geographical area and not totally lifestyle. 4. *Animal exposure* *Farmers*: have less allergy than any other groups due to spending large amount of time outside *Pets*: increase microbial sharing and having certain type of pets lower incidence of allergy and asthma (dog is a big one) *Prenatal*: pregnant women goes to farm -> pre-natal antigen exposure can lead to development of autism, which we seen in mother mice.

What are the effects of antibiotics on the microbiota?

1. Decrease in richness 2. Decrease in diversity and overall bacterial load and absolute abundance 3. Larger presence of antibiotic resistant genes in the GI tract which can be passed down to pathogenic bacteria (increase in resistant organisms due to selecting for the resistance by killing those that are not resistant) 4. Can get permanent loss of some microbes. 5. Can get changes in microorganism's function. ex: lose the ability to degrade certain carbs and SCFAs

Are probiotics a possible treatment for certain disorders of the central nervous system? Why or why not?

1. Effects seem to be extremely strain specific 2. Some evidence of impacts on depression, anxiety, and perhaps stress-associated problems *It's difficult how FT effect these things 3. Some evidence of impacts on pain perception? *IBS -> large percentage claim to have sever abdominal pain and it's hard to determine they have actual more pain or higher pain perception *Few studies in mice show probiotics changes pain "perception" in the intestine. +GI pain perception study for mice/people-> small tube in the rear end and blow it up until the subject reacts -> longer endurance = higher lower pain perception

What is the evidence that suggests a link between the microbiota and the gut-brain axis? (what evidence is there that it should perhaps be called the microbiota-gut-brain axis)?

1. Encephalopathy associated with late stage liver disease *Confusion, comma, trouble with communication *To a certain point antibiotics can reverse encephalopathy -> suggest self role microbiota 2. Co-occurrence of certain GI conditions and changes in the microbiota *Disruption/changes in microbiota *IBS (not the same as IBD): functional disorder and not always associated with inflammation -> chronic constipation or chronic diarrhea or a mixed +50% have suffer from depression and anxiety +Autism: suffer some form of intestinal inflammation -> the exact percentages based on study and severity level of autism 3. Side effects of antibiotic treatment *We see alter in gut microbiome

What is the evidence for microbial involvement in both animal studies and humans?*

1. Germ-free mice are more prone to allergies, anaphylaxsis, asthma, and inflammatory disorders. If they are colonized early enough, they can reverse allergies for the most part. 2. Antibiotic treatment tends to shift to TH2 response which is associated with allergies. Certain microbes reduce allergies. Like Mycobacteria ad H. pylori which reduce incidence in mice. Ex: Experiment in which there were neonatal and adult mice were given two different antibiotics, Streptomycin or Vancomycin. Saw that Streptomycin in neonatal and adult mice had little impact on the microbiota. While Vancomycin had widespread effects on microbiota of both. When given to neonatal mice, caused increased incidence and severity of allergies. No effect on allergies in adult mice. Conclusion: Different antibiotics have different effects on the microbiota, and the age of when the antibiotics were given seem to matter with allergies and immune system responses. 3. C. albicans in mice seems to promote allergic diseases. This can break down the barrier function of the gut and make it more leaky leading to more antigens going out to the blood stream, leading to inflammation. Is normal to find in humans in the GI tract and Urogenital tract in small numbers but shouldn't be the primary bacteria to be found. Is documented to shift the immune response and to take advantage of when the community is shifted. 4. probiotics - could be used as a preventative measure, but not as a treatment. Would have to give probiotics to a pregnant woman and give to the baby within the first 6 weeks of life (which does back to the idea of immune system development). Ex: When given Lactobacillus rhamnosus GG to pregnant women or babies immediately after birth, and looked out 4 years, there was an overall reduction in skin allergies like dermatitis and eczema. Ex 2: with the treatment of L. rhamnosus GG, Bifidobacterium species, and L. reuteri in probiotics, there were variable results and no evidence to support using for treatment

What are the subtypes of IBS? How might these complicate the understanding of microbial involvement?

1. IBS-D (typically present with diarrhea) 2. IBS-C (constipation) 3. Mixed IBS (diarrhea or constipation 4. Unsubtyped IBS (no functional disorder. No constipation or diarrhea, normal bowel movement, but several days a month with abnormal bowel pain which can be relieved by a bowel movement for example. - This complicates the understanding of IBS because we don't know if these subtypes are separated properly or if they should be combined. Moreover many studies have not divided the groups by these subtypes. So the results from studies are difficult to compare to one another. And the results might not be represented in an accurate manner if they are supposed to be divided into specific groups.

How are studies involving the microbiota-gut-brain axis conducted? What are some limitations to this research?

1. Light-dark preference: *Normal mice prefer dark area and enclosed area *How much time they spent in dark area and how much time they'll go explore 2. Step-down: *Open area -> waiting to see how much time mice take to get off the platform 3. Tail suspension *Hanged mouse by its tail a-> normal they'll repeatedly curl themselves up and free the tail vs depress -> hang there and not tried 4. Forced swim: *Hang the mouse and dipped in water -> the longer they'll try to swim to save themselves the less depress they are 5. Mazes *If they are not depress -> they have the faster memory 6. Learning *System of rewards and punishment set up -> board with wells some wells have food and others deliver small electric shock -> how quickly does the mice learn where things are Limitations are: 1. Germ-free animals are widely used *Inoculate GF with depressed person fecal plant -> mouse get depression *Inoculate GF with normal person fecal plant -> do not get depression 2. Mechanisms for studying influence of alterations in the microbiota *Antibiotic themselves can change the behavior *Diet can change behavior *All of these induce changes in microbiota -> changes in the gut 3. Mice vs humans (ask teacher)

How does the immune system control the microbiota? (think containment, including stratification and compartmentalization, and composition)

1. Must control contact with epithelial cells and invasion of tissues: a. Mucus: *Colon -> 2 mucus layer *Small intestine -> 1 mucus layer +Inflammation is when their invasion in the 1st mucus layer +Antimicrobial peptides found in different areas of mucus layer 2. Secreted IgA (immunoglobin A) *Along the epithelial cells are dendritic cells that can lead to activation of IgA once pathogen is detected -> bind to what pathogen bacteria -> make bacteria bulkier and less likely to enter pass the cells 3. Stratification: what in the gut should be kept in proper layer +Microbes that can stay in lumen, some on top of the mucus layer, but there should be no microbes on top of your epithelial cells 4. What about when a breach does occur? *Compartmentalization: keep everything in the gut *Mucus play role both in stratification and compartmentalization *However things will sometimes get through the mucus layer -> systemic immune system will get involved -> the epithelial lining and pass that huge level of immune system present 5. Control of composition: *Antimicrobial proteins are perhaps most well studied at this time +Alpha-defensins -> different types target different microbes -> luminal area +RegIII- gamma -> stay in the mucus and protect against pathogen

What are some of the possible mechanisms behind IBS? (focus on those with possible microbial involvement)

1. SCFA production imbalance (most studies suggest acetate and propionate are increased. Most have a reduction in butyrate, but too much butyrate in mice increases pain perception. represents 5% in fecal sample, so unlikely to be indicative of what goes on in the gut) 2. Barrier function (leaky gut) 3. Inflammation 4. Enteroendocrine system alteration (maturation of enteroendocrine cells? Seratonin levels?)* 5. CNS involvement (Gut-brain axis...)*

How does the microbiota impact immune system development and function?

1. more lymphoid structure and epithelial function -> wound healing and mucus production and normal level of antimicrobial, protein, and peptide just by colonizing the mice *Antimicrobial protein production 2. T cell subsets: -Balance of TH1, TH2, TH17, and Treg cells -Production of particular cytokines -> some cytokines can promote and some are anti-inflammatory. These ratio are impacted by microbes -> prone or not prone to inflammation (ex: IBD due to disruption of T cells and cytokines) *T cells subset and cytokine are both though to influence by the microbiota 3. Systemic immunity -> skin disorders, eczema *note specific antigens from the LPS layer of the bacterial can trigger the production of specific antibodies *PSA: polysaccharide A directly interacts with the dendritic cells and drive the T helper cells subset ratio -> PSA is responsible for a variety of modulating activity of T cell subset *SCFAs is the number one energy source for epithelium cells in the gut and promote barrier -> without it, you have starved cells which lead to difference in cytokines, T cells, activation of dendritic cells -> inflammation in the central nervous system gut. *SCFA also interact with goblet cells and drive programmed cell death *SFB (segmented filamentous bacteria): commonly found in mice and cannot be cultured, but is ties with the TH17 cells in the body -> play a big role in inflammatory response

How does age matter in terms of microbial exposure and immune system development/responsiveness? Does it always matter? Be able to explain the three specific examples discussed where age matters.

Based on GF mice studies, some effects of microbial exposure and the immune system development are age-dependent -> HOWEVER, some mechanisms can be repaired through microbial inoculation regardless of when inoculation occur For age-dependent influence exposures: there is a time frame where you can colonize the mice to successfully make a correction, otherwise the mice will be screwed up with their T cell balances and inflammatory response 3 examples discussed: 1. *iNKT (natural killer T cells)* cell colonization is regulated by the microbiota during early life and influences susceptibility to colitis and asthma later in life. Age-dependent defect -> where timing of colonization matter Figure 1. Mice with excess level of INKT -> airway = *asthma* -> gut = *colitis* -> This study looks at the colon and lungs of GF mice: a) If you don't inoculate the mice -> prone to colitis b) Colonize the mice after 2 weeks -> still prone to colitis (too late) c) (Colonize the mice within the first 2 weeks -> no longer prone to colitis* -> 2 mechanism = 1.cytokines production and 2. spingolipid d) If you colonize mice after 2 weeks e) *Colonize the mice within the 2 weeks -> no longer prone to asthma* -> B. fragilis only works in the gut and not the lungs Figure 2. Exposure to a microbiota during early life regulates *IgE levels* in serum of adult mice and their *sensitivity to orally induced anaphylaxis* The time frame is *within 4 weeks to fix* the exaggerated sensitivity to anaphylaxis (acute allergic reaction to an antigen) and the inoculation of fecal samples from conventionalized mice worked better than fecal samples from low diversity colonized mice Fig. 3 *Treg cells* colonize the skin of neonatal mice and induce tolerance to commensal bacteria. Use T regulatory cells to analyze *psoriasis (skin inflammation)* Result shows that early microbial *exposure (within 2 weeks of life) of Treg cells -> increase in tolerance in bacterial exposure -> less skin inflammation* This has to be down within 2 weeks of age!

Why is microbial colonization problematic for the immune system?

Because it's the it is challenging for our immune system to recognize a pathogen within a heavily colonized area (such as the GI tract). It's difficult to recognize between self and foreign microbes

What are the proposed functions for bile salt hydrolase activity in terms of how it benefits bacteria?

Functions for bile salt hydrolase activity: 1. *Colonization*: Interact with bile to successfully establish colony (Some: disrupt bile salt genes than they cannot colonize, although others do not have an impact) 2. *Detoxify bile* Problem: In deconjugated bile acids, they're much more toxic (Theory: b/c deconjugation has to happen first before anything, maybe there's a short term toxicity survival until they turn over and modify it) 3. *Potential nutrient source*

Explain the evidence for microbial involvement in the development or exacerbation of IBD (include genetic risk, environmental factors, plus any other supporting evidence).

Genetic risk: 25%. Bacterial recognition by the immune system: TLR4, TLR2, TLR9 (Toll-like receptors) NOD2 Bacterial killing and degradation by the immune system: NOD2 ATG16L1 - Some people don't have any of these. Think that there are genetic markers not yet identified. Environmental factors: 1. 50% concordance rate between monozygotic twins, 10% in dizygotic twins. 2. higher rates in developed countries than developing countries. IBD rates match the place where people move to. - means that areas with low IBD rates have people with genetic risk factors that aren't getting an environmental trigger, and when move to areas with high IBD, the genetic risk factors are triggered. 3. Rates of IBD overall have increased in numbers and younger and younger kids are getting diagnosed with it. Other evidence: 1*. Genetic risk factors and microbial involvement*: immune system defects related to recognition. The Gi tract is one of the most colonized areas in the body. *2. Antibiotics*: not curative, but can control flare ups in some people. *3. Fecal diversion therapy*: If damaged area of intestinal tract, we can divert the stream of feces around that area temporarily or permanently. When diverting, that part of the GI tract heals, but when food goes that route again if temporary diversion, will become inflamed again. *4. Fecal bacteriotherapy*: Not well established, but attempted with Ulcerative colitis (UC). Preliminary evidence that might help with some people. Don't think long term solution. Don't know if we can do over and over without substantial risk *5. Animal models*: if germ free, never develop intestinal inflammation. After colonization of GF mice, in less than 3 weeks, many will have severe chronic inflammation, and more prone to getting colon cancer if live long enough. Single organisms like H. hepaticus and E. coli can trigger inflammation in mice. *6. Specific pathogens*: - Mycobacterium avium subspecies paratuberculosis - Adherent and Invasive E. coli (AIEC) - Clostridium difficile.

What is the evidence for microbial involvement in Type 1? Be able to explain several points.

Human studies: "Diabetes Prediction and Prevention Study" Finland -> children with T1D show an increase in Bacteroidetes to Firmicutes "The Environmental Determinants of Diabetes in the Young (TEDDY) Study" and many others -> show that kids in Russia and Finland have exact genetic risk factors but one is more likely to develop diabetes than the other. -> recruited 500,000 individuals that may develop T1D due to genetic or other factors, they only ended up with 800 confirmed cases with T1D that shown increase in Bacteroidetes -> specially Bacteroides before the diabetes onset. -> the best indicator we have currently have is the appearance of antibodies against the beta cells in the pancreas -> How can the appearance of Bacteriodetes increase before the appearance of antibodies? Can this be a clinical trigger/maker? -> Other studies T2D patients show an increase in Bacteroides and a decrease in Prevotella. Bacterial role: lactic acid bacteria -> lactate -> producers of non-butyrate SCFAs -> propionate, acetate, succinate -> decrease in mucin synthesis + tight junctions -> permeable gut -> autoimmunity

Are fecal transplants potentially going to be successful in treating or preventing diabetes? Why or why not? (think a bit beyond the particular study mentioned in class)

In T2D, the fecal transplant can reset the microbiota within the gut of the patient, however, this is only a short-term method. - Healthy lifestyle and diet must be followed with the treatment because diet/exercise play important roles in the formation of the gut microbiota. The fecal microbiome cannot persist if old habits remain.

How was the hygiene hypothesis initially received by the scientific community? Why?

It was met with skepticism since many thought infectious disease were the cause of allergic disease and not a preventative measure. For these who accept the theory, they assumed that the less childhood infections may lead to lower TH1 and higher TH2, which is responsible for increased allergies , and autoimmune diseases.

Why was the hygiene hypothesis originally proposed (what trend was seen? What data was analyzed?)

Longitudinal study of 17,414 British children that overlooked the correlations with between hay fever, eczema, and allergic rhinitis. Shows that these illnesses are associated with family size and birth order The bigger the family and the latter the series of children the child was born, the lower the risk of having allergic diseases Recent declines in family size and increases in sanitation, and hygiene have led to decreased childhood infections -> increase in allergic diseases Particularly within families

What disorders are associated with microbial involvement in the gut-brain axis?

Multiple sclerosis? Late-onset autism spectrum disorders? Depression? Schizophrenia?

What are the potential mechanisms for microbial involvement in Parkinson's disease?

Potential connection with vagus nerve. - Evidence from animal models where if damage vagus nerve, alleviate symptoms for PD. - Onset of PD is where gut microbiota could be involved

What are the primary bile acids? The secondary bile acids? Conjugated bile acids? Deconjugated bile acids? Which of these bile acids are produced directly through bacterial activity?

Step 1. *Primary bile acids* (only major class of bile acids made from cholesterol in liver): - cholic acid (CA) and chenodeoxycholic acid (CDCA) Step 2. *Conjugated bile acids from primary bile acids* (conjugated with amino acids such as taurine or glycine by the liver) - Glycocholic acid (GCA) and taurocholic acid (TCA) -Glycochendeoxycholate (GCDCA) -Taurochenodeoxycholate (TCDCA) OR *secondary bile acids* (formed through bacterial partial dehydroxylation and the removal and glycine and taurine in the small intestine): - deoxycholic acid (DCA) and lithocholic acid (LCA) - Produced directly through bacterial activity Or *conjugations from secondary bile acids*: - glycodeoxycholic acid (GDCA) and taurodeoxycholic acid (TDCA) -glycolithocholic acid (GLCA) and taurolithocholic acid (TLCA) Step 3. *Deconjuaged bile acids* (made by bacteria through 7a/B dehydroxylation and the removal of glycine or taurine)

What were the initial data that suggested that the microbiota might be associated with obesity?*

Study shows that germ free mice eat more food* and higher amount of the energy is store as body fat compared to conventionalized mice. -suggest that the gut microbiota influence the amount of food one consume, energy extract, and fat storage. GF mice eat 30% less, but have 45% more body fat GF mice had 25% slower metabolism and altered leptin levels.

Over the last 20 years (including recent data), How have the proposed immune mechanisms changed?

Th1/Th2 mechanism was very popular in the 1980s because TH1 is associated with cytokine alleviated symptoms of allergies and other disorders lead to the increase of TH1 -> if TH1 is not stimulated correctly, the Th2 cells expand instead This support has weakened: *proposed that recognition of microbes* leading to dendritic cells stimulating T regulatory cells. This procress build up homeostasis in the maturation of T reg cells to have a better immune system -> basically microbes are "old friends" that help the immune system *iNKT cells*: invariant natural killer T cells -> 2012 study -> Germ-free mice: more prone to allergies and colitis, which can be fixed with early colonization of proper microbial exposure *LPS variations* -> different amount of LPS can stimulate different immune response -> A Type 1 diabetes study looking at participant microbiota from Finland vs Russia. -> the Finland group have *bacteroides* while the Russian group does not. -> E. coli LPS is a major stimulant for inflammation: increased immune activity and never build tolerant to other LPS exposure leading to higher rate of allergy/infection

What is sequenced when analyzing fungal communities?

The IT regions are transcribed. These regions are more informative for fungal species. Fungal species don't contain 16s genes but contain 18s genes but if you try to sequence these, you will miss huge percentages of the fungal population.

What is the evidence for microbial involvement in IBS? Include the specific information discussed in class, along with an explanation of the how the genetic risk factors, dietary factors, and post-infection IBS also support this idea.*

There are not well defined risk factors. 1. There is an increased risk with genetics (identical/monozygotic twins have a higher rate than dizygotic twins) 2. Some people are bad at absorbing simple carbs. Rather than being absorbed, they may go to the colon. Some people won't eat specific foods that will irritate their IBS which will lead to symptom relief 3. Gastroenteritis is a trigger for some (6x - 7x increased risk) If you develop some type of infectious enteritis from bacteria or viruses, there is a significantly increased risk of developing IBS after the infection. If got salmonella infection when little, increases lifetime likelihood that develops later in life. Meta-analysis type studies. 4. Stress and menstrual cycles seems to impact the flareups and with stress may impact individual development as well 5. Additional risk factors: being female and being a young adult.