Contemporary issues in medical science

Theodor Escherich

(discovered e-coli)

Claimed meconium was sterile and intestinal colonisation was attributed to infants early environment (milk)

Necrosis

Accidental and unregulated, results from damage to cells and loss of ionic homeostasis, not energy dependent

Necrosis mechanism

1. Damage to lysosomal membrane leads to spillage of hydrolytic enzymes into cytoplasm which digests cellular contents.

2. Cell content leaks through damaged plasma membrane into extracellular space and elicit inflammation

3. nuclear changes all due to non-specific breakdown of DNA (karyolysis, pyknosis, karyorrhexis)

4. Leads to ischaemia, infections, trauma and exposure to toxins

Mitochondrial damage results in:

Decreased ATP giving multiple downstream effects

Increased ROS so damage to lipids, proteins and DNA

DNA damage effect

Activation of pro-apoptotic proteins

Membrane damage effect

Plasma: loss of cellular components

Lysosomal: Enzymatic digestion of cellular components

Homeostasis definition

The maintenance of a constant internal environment. Purpose of physiological regulation is to clamp internal parameter at a 'setpoint'

Allostasis definition

Stability through change. Parameters are not constant and variations are designed to reduce error

Aetiology

Cause of disease

Pathogenesis

Mechanism of disease

Inheritance of mitochondrial DNA (mtDNA)

Normally inherited exclusively from the mother

The mitochondria in mammalian sperm are usually destroyed after fertilisation

Structure of mitochondria DNA (mtDNA)

Compact, circular, double stranded. One mitochondria has dozens of copies of mito genome and each cell has numerous mitochondria

mtDNA defects

Proportion if mtDNA pathogenic variants must exceed a threshold before abnormality is expressed (threshold effect)

Heteroplasmy

Mitodisorders: mix of mutated and wild type mtDNA

Homoplasmy

At birth: all mitochondria identical

Mitochondrial disorders

Inherited, reduced ATP production.

E.g. Leigh syndrome, Pearson syndrome

Clinical presentation of mitochondrial disorders

Organs and tissues with high energy demands (brain, nerve, eye, cardiac/skeletal muscles susceptible)

Symptoms diverse: developmental delay, seizures, myopathy, retinopathy

Mitochondrial replacement Therapy

Ccombine the nuclear DNA of a woman carrying a mtDNA mutation with DNA of a sperm and the egg of a healthy donor, allowing her to give birth to a child free from mitochondrial disease.

Mitochondrial replacement Therapy techniques

Pronuclear transfer

Maternal spindle transfer

Pronuclear transfer technique

1. Patient couple and donor egg both fertilised making a pro nuclear zygote

2. Discard the nucleus of donor egg

3. Insert nucleus of patient couple egg into donor egg

Maternal spindle technique

1. Remove karyoplast (containing meiotic spindle) from patient couple and donor egg.

2. Discard karyoplast of donor egg

3. Insert patient couple karyoplast into donor egg

4. Fertilise egg resulting in pronuclear zygote

Phosphatidylinositol

On the inner membrane leaflet,

Can be phosphorylated, serving as an electrostatic scaffold for intracellular proteins,

(poly)phosphoinositides can be hydrolyzed by PLC (phospholipase C) to generate intracellular second signals (diacylglycerol and IP3.)

Phosphatidylserine

Found on inner leaflet but flips to extracellular in cells during apoptosis,

Can act as a cofactor in blood clotting

Structural changes in response to alterations in physiological states and some pathological stimuli

Hypertrophy, hyperplasia, atrophy, metaplasia

Hypertrophy

Increases sizes of cells/ affected organ (e.g. myocardial hypertrophy)

Hyperplasia

Increase in number of cells

Atrophy

Reduction in size of tissue/organ due to decrease in cell number (can be resulted from reduced protein synthesis and increased protein degradation)

Can be proteasomal or autophagic

(E.g Cerebrovascular diseases, Alzheimers)

Metaplasia

Reversible change where one differentiated cell type is replaced by another cell type (e.g. in smokers: columnar to squamous in respiratory tract)

Metaplasia from squamous to columnar cells

In respiratory tract in response to chronic irritation

Metaplasia from columnar to squamous cells

Barrett oesophagus

Mechanism of reversible cell injury

1. reduced oxidative phosphorylation leads to depletion of ATP

2. Changes of ion concentrations and water influx causes cellular swelling

3. Mitochondrial/ cytoskeleton alterations

4. Calcium ions play a central role in progression to irreversible cell death

Fundamental causes of necrotic death

Depletion of ATP

1. Reduction of plasma-membrane energy dependent sodium pump

2. Increase in anaerobic glycolysis

3. Failure of Ca ion pump, influx of Ca ions

4. Disruption of protein synthetic mechinery

5. Deprevation of oxygen/ glucose leads to protein misfolding

Influx of Ca and loss of Ca homeostasis effects

1. Accumulation of Ca2+ in mitochondria results in opening of MPTP and failure of ATP generation

2. Can cause inappropriate activation of enzymes (e.g. phospholipase, proteases, endonuclease, ATPases)

3. Direct activation of caspases

Major consequences of mitochondrial damage

1. MPTP channel opens leading to loss of mitochondrial membrane potential, resulting in failure of oxidative phosphorylation and progressive depletion of ATP leading to necrosis

2. Abnormal oxidative phosphorylation also leads to formation of reactive oxygen species (ROS)

3. Mitochondria sequester various proteins capable of inducing apoptotic pathways between outer and inner membranes

CASPase

A family of proteases that, when activated, mediates the destruction of the cell by apoptosis.

Cysteine Aspartyl Specific Protease.

Accumulation of Oxygen-Derived Free Radicals

Cell damage through membrane lipid peroxidation, protein modification and DNA breakage as they as highly reactive.

reperfusion injury.

ROS

1. Superoxide

2. Hydrogen peroxide (H2O2)

3. Hydroxyl radical (OH)

Apoptosis features

1. Cell shrinkage

2. Chromatin condenstion

3. Formation of cytoplasmic blebs and apoptotic bodies

4. phagocytosis of apoptotic cells

Mechanism of apoptosis

1. Enzymatic cleavage of inactive zymogens to synthesise and activate CASPases

2. Cleave their substrates after specific tetrapeotide motifs

3. Initiator caspase (8) and executioner caspases (3.6,7) degrade critical cellular components

B-cell lymphoma-2 (BCL-2)

Critical regualtors of apoptosis

Devided into 3 groups: anti-apoptotic, pro-apoptotic, pro-apoptotic activators.

Anti-apoptotic

BCL-2, BCL-XL and MCL1 have 4 BH (1-4) domains.

They prevent efflux of cytochromeC (and other apoptotic proteins) into cytosol

Pro-apoptotic

BH-3 only proteins sense damage and activate BAX and BAK (effectors) to form oligiomers within the outer mito membrane to induce permeabilisation/ leakage of mitochondrial proteins

Pro-apoptotic activators

BAD, BIM, BID, Puma and Noxa. Only 1 BH domain (BH-3 only proteins).

Act as sensors of cellular stress and damage, regulate balance of other groups.

Regulation of apoptosis

1. Survival signals/ growth factors stimulate synthesis of anti-apoptotic proteins

2. Deprivation of growth factors is sensed by BH3-only proteins and pro-apoptotic effectors activated

3. Cytochrome C released into cytosol binds to APAF-1 to form apoptosome, binding cascade 9 leading to autoamplification

4. Other mitochondrial proteins enter cytoplasm to neutralise IAPs (inhibitors of apoptosis)

Probiotics

Introducing known microbes back into the body

Prebiotics

Non-digestible nutrients that promote growth of friendly bacteria

Symbiotics

Contains both pre/probiotics

Father of microbiology

Anton van Leeuwenhoek

Microbiota first sighting

Anton Van Leeuwenhoek examined plaque from teeth using microscope. He saw microbes moving around. Realised that drinking coffee stopped their movement so they must be alive. Called them animalcules

Holobiont

Co-dependent relationship between humans and microbes

Where are microbes present in humans

Skin, mouth, pharynx, respiratory system, urogenital tract, stomach, intestinal tract (high density, 70%)

Microbiota

Community of micro-organisms that colonise normal healthy people

Microbiome

the collective genome of all microbes in a microbiota, can influence fitness, phenotype and health of host

Ratio of Human Cells to Bacterial Cells

1:1

Parasitism

One species benefits and the other is harmed

Commensalism

One species benefits, and the other is unaffected

Mutualism (symbiosis)

Both species benefit from the relationship

How do you acquire your microbiome

Mother is principle source, breast-feeding can provide useful bacteria (formula fed has fewer probiotic bacteria), solid foods

Benefits of intestinal microbes

Digestion,

Microbial antagonism,

Vitamins (K and B12),

Development,

Immune system

Microbial antagonism

Microbes take up space and secrete anti-microbial agents, preventing harmful pathogens taking hold

Obligate aerobes

Can only survive in presence of oxygen

Facultative anaerobes

Can use and grow faster with oxygen but doesn't require it to grow

Aerotolerant anaerobes

Don't use oxygen but can tolerate it

Microaerophilic

requires only a small amount of oxygen

Obligate anaerobes

Die in the presence of oxygen

Major phyla in gut microbiota

Bacteroidetes,

Firmicutes,

Actinobacteria,

Proteobacteria

Gram positive bacteria

Firmicutes, Acrinobacteria,

1 phospholipid bilayer and thick outer layer of peptidoglycan

Gram negative bacteria

Bacteroidetes, Proteobacteria,

2 phospholipid bilayers with a thin peptidoglycan layer in-between

Syntrophy

some bacteria live together and supply each other with essential nutrients

Metagenomics

the study of genetic material recovered directly from environmental samples

GALT (gut associated lymphoid tissue)

Learns to tolerate microbes and rejects new-comers, meaning we maintain stable gut microbiota composition

The best way of determining species diversity in human faecal microbiota is to use:

Metagenomics

3 multiple choice options

Two 16S rRNA genes are considered to be from different species if they have less than ... % similarity

97%

3 multiple choice options

Limitations of 16S approach

Highly dependent on choice of primers, some pairs may not amplify 16S DNA from whole classes of bacteria.

Some bacteria are harder to get DNA out of than others.

Dysbiosis

Microbial imbalance, found in autoimmune diseases (e.g. obesity, cardio metabolic)

Metabolic syndrome

A cluster of interrelated metabolic abnormalities: (obesity, dyslipidemia, hyperglycemias and hypertension) that enhance the risk for CV disease and type 2 diabetes

Pathogenic factors for metabolic syndrome

Insulin resistance and visceral obesity

Insulin resistance

Defect in insulin action but normal production by beta cells of pancreas, caused by abundance of circulating fatty acids

How do fatty acids reduce insulin sensitivity

Inhibit insulin-mediated glucose uptake, blocking transport of glucose into cells. This increases circulating levels of glucose which elevates pancreatic insulin secretion causing hyperinsulinaemia

Adipose tissue specialised for:

Storage and mobilisation of lipids, also an endocrine organ that releases cytokines: (FFAs, TNFa, C-reactive protein, interleukin-6, adiponectin and leptin)

Adiponectin

Produced by adipose cells that inhibits inflammation and protects against insulin resistance, type 2 diabetes, and cardiovascular disease

Leptin

Increased during obesity, decline with weight loss.

Elevated levels do not suppress appetite and increases BP through activation of sympathetic nervous system

Leptin resistance

Fundamental pathology in obesity

Insulin sensitive phenotype

normal body weight, no abdominal/visceral obesity, moderately active, diet low in saturated fats

Insulin resistance pathophysiology

1. pancreatic beta cells secrete more insulin to overcome hyperglycemia

2. overexpression of insulin activity in normally sensitive tissues

3. exaggeration of insulin action and resistance to other actions lead to clinical signs of MetS (metabolic syndrome)

4. Over time pancreas beta cells are unable to produce sufficient insulin leading to hyperglycemias and type 2 diabetes

Insulin receptor

Ligand-activated tyrosine kinase

Insulin signalling

Binding of insulin receptor (tyrosine kinase) results in tyrosine phosphorylation of downstream targets and activation of two pathways: (PI3K and MAPK pathway)

Obesity definition

Accumulation of adipose tissue that is of sufficient magnitude to impair health. BMI over 30kg/m*2

Hunger and satiety

controlled by complex neuroendocrine system- bidirectional crosstalk between feeding centres in brain and periphery

Short term regulators of food intake

Ghrelin, CCK (cholescystokinin), PYY (peptide tyrosine tyrosine), GLP-1 (glucagon-like peptide), GIP (glucose-dependent insulinotropic polypeptide), OXM (oxyntomodulin), glucagon, GFG21

Long term regulators of food intake

Leptin, insulin, amylin

How is obesity related to cancer

(Proposed)

Altered levels of adipokines, disrupted insulin signalling, local and systemic effects of inflammation, modifications of microbiome

Why is diabetes a major healthcare problem

How does necrosis happen?

- Damage to lysosomal membrane leads to spillage of hydrolytic enzymes into cytoplasm and digest cellular components