HLA and Transplantation

This week's PATH3304 lecture was mainly about HLA (Human Leukocyte Antigen), also known as MHC (Major Histocompatibility Antigen). I practically spent my entire semester in Canada blogging about MHC, so most of this post will probably consist of links to previous posts that I wrote while abroad.

Understand the normal structure and function of HLA class I and II molecules
Understand the inheritance of HLA haplotypes

See previous post: The Major Histocompatibility Complex (MHC)

One thing not covered in the above post is that recombination can occur during inheritance. For example, your mum's copies of the MHC genes may cross over, resulting in some mixing of MHC alleles. The same could happen with your dad's copies of those genes. However, your mum's genes won't cross over with your dad's genes, so you won't receive a chromosome that has a mix of genes from both of your parents. (Hope that made sense.)

Define the following terms: allele vs. locus, polymorphism, linkage disequilibrium, HLA haplotype, HLA genotype

• Allele vs. locus: A locus is a region of a chromosome in which a certain gene can be found (e.g. DQ gene). An allele is the specific form of that gene that a patient might have (e.g. DQ2 or DQ3).
• Polymorphism: A term that basically means that there is more than one allele in a population (e.g. there are over 400 alleles for DQ).
• Linkage disequilibrium: Multiple loci are inherited together more frequently than you would expect by random chance alone, suggesting that they are linked together somehow.
• HLA haplotype: The combination of alleles on the same chromosome.
• HLA genotype: The combination of haplotypes found in an individual (one from the mother, one from the father).

Develop a basic understanding of HLA nomenclature

To explain HLA nomenclature, I'll give you an example: HLA-B*08:01. The HLA stands for HLA (amazing...), the B stands for the B gene, and the 08:01 refers to a specific allele. The * indicates that this particular allele was identified via DNA sequencing. Serological recognition methods (i.e. using antibodies to detect a particular allele) are also used, but they tend to be less accurate (so the same allele might only be identified as HLA-B8 using this method).

Understand that HLA matching is important in all forms of transplantation: HSCT (haematopoietic stem cell transplantation) vs. solid organ transplantation

See previous post: Immune Responses to Transplants

HLA matching is very important for many reasons. Well-matched HLA types can extend the life of the transplanted organ and reduce the need for immunosuppressive drugs, which in turn reduces the risk of infection and extends the life of the patient. HLA matching may even reduce the risk of lymphoma (which is usually associated with Epstein-Barr Virus reactivation).

HLA matching is particularly important in HSCT (haematopoietic stem cell transplantation), which includes things like bone marrow transplants. That is because the graft, which contains leukocytes, can attack and really damage the host (Graft vs. Host Disease). As such, more loci are considered in matching for HSCT compared to other solid organ transplants (in which usually only HLA-A, HLA-B and HLA-DR are considered). In order to find a perfect match, HSCT transplants are matched with donors all over the world, whereas solid organ transplants may only be matched nation-wide.

Describe some common HLA disease associations (non-transplant related)

Certain HLA alleles have also been implicated in drug sensitivity reactions and in autoimmune diseases. For example, the drug abacavir can interact with the binding site of some HLA types, causing a conformational change. The conformational change induced by abacavir may then allow HLA to bind to peptides that it wouldn't normally bind to, which may or may not provoke a reaction from the immune system.

Coeliac disease is an example of an autoimmune disease that may be linked to HLA type. When gluten is taken up, glutamine residues are converted into glutamic acid. The modified peptide can then bind to certain HLA types, which may or may not provoke an immune response. Other immune diseases that may be affected by HLA type include ankylosing spondylitis, rheumatoid arthritis and multiple sclerosis.

Thermal Physiology I: Heat Balance

Describe and understand the different definitions of animal thermoregulation

There are many different terms that can be used to describe how animals regulate body heat:

• Poikilothermic- Literally means "change heat." Body temperature changes according to ambient temperature.
• Homeothermic- Literally means "same heat." Body temperature remains constant despite changes in ambient temperature.
• Heterothermic- Somewhere between poikilothermic and heterothermic.
• Endothermic- Body heat is generated from metabolic processes.
• Ectothermic- The animal seeks an external source of body heat.

Describe, understand, and be able to calculate the Q10 effect of temperature on processes
Understand why changes in body temperature have profound effects on all life processes

The Q10 effect is basically a ratio of the reaction rates at a given temperature, and at another temperature that is 10 degrees higher than the given temperature. It is calculated as (reaction rate at higher temperature) / (reaction rate at given temperature). Generally, increasing temperature increases reaction rate (as the energy of the particles is brought closer to activation energy), but at high enough temperatures you might also have problems with denaturation of enzymes. As we're basically just big bags of chemical reactions, this is pretty bloody important.

Describe and understand the relationship between heat capacity and temperature

Heat capacity is the amount of energy that it takes to raise body temperature by 1 degree. For most animal tissue, heat capacity is around 3.5 joules/gram/°C. In other words, if the amount of heat energy generated (by metabolic processes etc.) in 1g of animal tissue is 3.5J higher than the amount of heat that is lost, then that 1g of animal tissue will become 1°C warmer.

Describe and understand the heat balance equation including its individual components, what they mean, and how they are affected by various environmental and biological factors
Be able to calculate the heat balance and its effect on body temperature given the relevant information

The heat balance equation is as follows:

Heat storage (S) = Metabolic heat production (M) ± Conductive heat exchange ± Convective heat exchange ± Radiative heat exchange ± Evaporative heat exchange (E) - External work (W)

which can be abbreviated to:

S = M ± Cond ± Conv ± Rad ± E - W

In order for body temperature to remain constant, S must be equal to 0. If S is not equal to 0, then body temperature will change. As mentioned above, since the heat capacity of animal tissue is 3.5 joules/gram/°C (or 3.5 kJ/kg/°C), for every 3.5J increase in S, 1g of animal tissue will increase in temperature by 1°C.

If you haven't done physics (like me :P), here are some quick definitions for the four main methods of heat exchange (conduction, convection, radiation, evaporation):

• Conduction- Transfer of thermal energy by direct contact (i.e. you transfer heat to things that you touch, and things that you touch transfer heat to you).
• Convection- Transfer of thermal energy to a moving fluid. This can be natural, like the buoyancy effect (heat rising), or forced (e.g. wind).
• Radiation- Transfer of thermal energy by electromagnetic radiation. The amount of radiation emitted depends on their surface temperature. The temperature and maximum wavelength are related according to Wien's law: λ_max = 2.898 * 10⁶ / T (°K).
• Evaporation- Transfer of thermal energy as latent heat of evaporation (2400J/g of water).

Describe and understand body temperature classifications of hypo- and hyperthermia

Hypothermia: Body temperature less than around 36°C.
Hyperthermia: Body temperature greater than around 38°C.

Describe and understand the basic components of the thermoregulatory control system in homeothermic animals

Just a warning from here on out: I wasn't able to attend this lecture in person, and the lecture recorded without sound. A lot of the other points on this post were not covered in the replacement lectures, so I'm basically just gonna wing the rest of this post by looking at the lecture slides.

Just like in other control systems, the thermoregulatory control system needs certain components. These components are: the variable to be regulated (skin temperature), receptors that measure the variable (thermosensors in the skin and hypothalamus), some kind of command centre where the information is integrated and then new information is sent out (presumably the CNS???), and the effectors that regulate the variable (skin blood flow, sweating, shivering and behavioural changes).

Describe and understand how peripheral and central temperatures are integrated by the control system to stimulate effector mechanisms that result in the regulation of body temperature

Both the skin and hypothalamus have cold and warm thermosensors. In the skin, the cold receptors are called A delta, and the warm receptors are C fibres. (Not sure if they have different names in the hypothalamus.) Warm sensors increase firing when they are warm, and cool sensors increase firing when they are cool.

Describe and understand the four main effector systems in thermoregulation and how they are controlled 

The four main effector systems are control of skin blood flow, sweating, shivering and behaviour changes.

Skin blood flow

At a comfortable temperature, our skin blood flow is around 25 mL/min.100g. This can decrease to 1 mL/min.100g in the cold, and increase to 150mL/min.100g in heat. The skin of the hands, feet, ears and nose have arteriovenous anastomoses (AVAs) which, when open, shunt blood directly to the veins (and not to the skin). AVAs are opened when warm, and closed when cold, thus directing blood flow to meet our needs for warmth. Indeed, changing skin temperature can adjust heat loss by a factor of 8.

Sweating

As you may (or may not) remember from second year, nerves that innervate sweat glands are part of the sympathetic nervous system, but they secrete acetylcholine rather than noradrenaline. These nerves are also called sudomotor nerves. There are two types of sweat gland: eccrine glands, which cover most of the body surface and are mainly responsible for thermoregulation, and apocrine glands, which are pretty much only found on the armpit and pubis. Sweating increases pretty much linearly once head temperature is higher than a set point. (Skin temperature also helps to determine the set point.)

Shivering

Shivering is essentially heat production by moving your muscles. It begins once head temperature drops below a set point. (Once again, skin temperature also plays a role in determining set point.)

Behavioural Changes

This wasn't covered at all, but I'm pretty sure that this just entails wearing more clothes when you're cold, and taking off clothes when you're hot.

Energy Nutrition: Metabolism of Carbohydrates and Fats

This will likely be a very short post as most of the content has already been covered in posts for BIOC3004. (Note: CHO = carbohydrate)

Describe the molecules from which we derive energy

The molecules from which we derive energy are proteins, carbohydrates and fats. Most of our energy is derived from carbohydrates, a smaller percentage is derived from fats, and only a very small amount is derived from proteins. If you want to know even more about these molecules, I've written about them in excruciating detail for CHEM1004.

Outline hepatic processing and interconversion of fats and CHOs

The liver can carry out a lot of processes, but we'll only focus on a few of these processes here. Processes involving carbohydrates include glycogenesis, glycogenolysis, gluconeogenesis and conversion of monosaccharides into other monosaccharides (e.g. converting fructose into glucose). (If you don't know what those terms mean, see here.) Interestingly enough, glucose 6-phosphatase, the last enzyme in glycogenolysis, is only present in the liver. That means that the liver can break down glycogen into glucose that can leave the cell and be transported elsewhere around the body, whereas other tissues (such as muscle) can only get as far as glucose-6-phosphate, which cannot leave the cell.

Liver processes involving fats include beta-oxidation (the liver is the primary site of beta-oxidation), synthesis of fats and cholesterol, conversion of excess acetyl CoA to ketone bodies, storage, formation of lipoproteins (e.g. VLDL) and conversion of cholesterol into bile salts. The liver is truly one very busy organ!

Outline the mechanisms by which we derive energy from fats and CHOs

We derive energy from fats via beta-oxidation, and from carbohydrates via glycolysis. Both beta-oxidation and glycolysis result in acetyl CoA, which can enter the TCA cycle and then oxidative phosphorylation.

Outline the processes of glycolysis, the TCA cycle, & oxidative phosphorylation

Glycolysis: Glycolysis and the Pentose Phosphate Pathway
The TCA cycle: The TCA Cycle
Oxidative phosphorylation: Electron Transport Chain and Oxidative Phosphorylation

Outline the processes of lipolysis & β–oxidation

Beta-Oxidation

Describe the roles of energy carrying intermediates in energy metabolism

The energy carrying intermediates in energy metabolism are NADH, FADH₂ and ATP, which I've written about in pretty much all of the posts linked to above. (They're also discussed here.)

Absorption of water, minerals and vitamins

More absorption stuff! Once again, this lecture was largely a recap of PHYL2001, but there's some new stuff too!

Describe the mechanisms used for water absorption by the GIT.

The gastro-intestinal tract cannot actively transport water, so water is mainly transported by following an osmotic gradient. Usually this osmotic gradient is formed when some other solute (such as sodium) is absorbed or secreted.

Describe transcellular and paracellular absorption.

Transcellular: through cells. Paracellular: next to cells. Transcellular absorption often requires that channels, pumps, etc. be present on the apical and basolateral cell membranes, as many solutes (particularly charged ions, like sodium and potassium) cannot readily pass through a cell membrane. Paracellular absorption occurs through the tight junctions, and is dependent on how "tight" the tight junctions are. The "tightest" tight junctions are in the colon, whereas the "leakiest" tight junctions are in the small intestines.

Outline the roles of tight junctions and aquaporins in regulating water absorption.

Aquaporins are basically channels that allow water (and sometimes also hydrogen peroxide) to pass through. Both tight junctions and aquaporins allow water to flow down its osmotic gradient, allowing for the absorption and secretion of water.

Outline the importance of electrolytes in water absorption.

As I mentioned earlier, water travels down its osmotic gradient. The osmotic gradient, in turn, is set up by concentrations of electrolytes. Therefore, transport of electrolytes also affects the transport of water.

Name the transporters involved in absorption of major electrolytes/minerals.

Sodium

Sodium transport mechanisms are slightly different in different areas of the GI tract. Pretty much all of these mechanisms rely on the action of the Na⁺/K⁺ ATPase to keep the Na⁺ concentration within the cell low, creating a nice concentration gradient.

• Duodenum and jejunum: Na+ is taken up by an Na+/H+ antiport. This antiport also stops the inside of the cells from getting too acidic.
• Jejunum and ileum: Na+ is taken up by secondary active transport with glucose or amino acids.
• Ileum and proximal colon: These areas have both Na+/H+ antiports and Cl-/HCO3- antiports.
• Distal colon: Passive Na+ channels.

Chloride

Eh, I think I'll split this one up in terms of mechanism, rather than location (as the areas in which these transport processes occur overlap a lot more for chloride than they do for sodium).

• Passive absorption: Occurs via paracellular and transcellular routes. Cl- often follows Na+. Occurs in the jejunum, ileum and distal colon.
• Cl-/HCO3- antiports: Ileum, proximal colon and distal colon.
• Parallel Na+/H+ and Cl-/HCO3- antiports: Ileum and proximal colon (same as for sodium, which makes sense because it's essentially the same mechanism).

There are four main types of chloride channels. These include calcium-activated chloride channels (CaCC), volume regulated anion channels (VRAC), ligand-gated anion channels (LGAC) and the CFTR channel. The CFTR channel, which is regulated by cAMP, is of particular interest as it is this channel that is non-functional in cystic fibrosis patients. cAMP activates protein kinase A, allowing it to phosphorylate the R-domain of the channel, causing it to open. As such, when cAMP levels increase, chloride secretion increases, causing water secretion to increase, and a shit-ton of diarrhoea (pun intended) results.

Potassium

• "Solvent drag": When water moves down an osmotic gradient, some potassium can be dragged along with it. This phenomenon is found in the jejunum and ileum.
• Active absorption: H+/K+ pumps in the distal colon. This is quite energy-expensive.

Calcium

Some calcium can be absorbed via the paracellular route throughout the small intestine. Calcium can also be absorbed via the transcellular route in the duodenum. Absorption happens through the CAT1 calcium channel on the apical surface. Once in the cell, calcium binds to calbindin (to stop free calcium from setting off a whole bunch of other reactions). It can then be pumped out through pumps on the basal side. Transcription of the CAT1 gene relies on vitamin D, which is why vitamin D is important for calcium absorption.

Magnesium

Magnesium is absorbed in a similar way to calcium, except that the channel on the apical surface is called TRPM6, and no binding proteins are required.

Other random notes on mineral absorption

There are several different factors that can affect how much of a given mineral is absorbed. These include pH, the oxidation state of the metal (as mentioned here, Fe³⁺ is much less soluble than Fe²⁺), and the presence of certain dietary complexes (e.g. ascorbate can increase absorption, whereas phytates can reduce absorption). Fun fact: calcium blocks lead absorption, which is useful for treating lead poisoning.

Describe the different mechanisms used for absorption of water-soluble and lipid-soluble vitamins.

Water-soluble vitamins are mostly absorbed by specific transporters (with the notable exceptions of B9 and B12, which I'll discuss in a bit). Fat-soluble vitamins (A, D, E and K) are even simpler: they're absorbed along with fats (see here for information on how fats are absorbed). The flipside of this is that when fat absorption is inhibited (by a bile duct blockage etc.), fat-soluble vitamin absorption is also inhibited.

Now back to B9 and B12! B9 absorption isn't too complicated. As I mentioned here, B9 is often joined to a bunch of glutamate residues, so those residues need to be "chopped off" first before B9 can be absorbed. Not to worry: we have enzymes on the brush border that do just that! Once folate is inside the enterocytes, it is methylated and reduced before being released into the blood.

B12 absorption is pretty complicated. In the stomach, B12 binds to haptocorrin, which is secreted by gastric glands. Intrinsic factor, as mentioned here, is also released from the stomach, but it cannot bind to B12 at such a low pH. Once the B12-haptocorrin complex enters the duodenum, proteases degrade haptocorrin, allowing B12 to bind to intrinsic factor. The B12-intrinsic factor complex can be taken up in the ileum via clathrin-coated vesicles. The vesicles then acidify, allowing B12 to be released from intrinsic factor. B12 is then packaged with trans-cobalamin II for transport around the body.

Bacterial Diagnosis

This post will likely be fairly long (even by MICR3350 standards), as it will essentially cover 2 hours worth of content. As the title states, this post will cover bacterial diagnosis: mainly the detection of pathogens and their identification. Without further ado, let's get started!

Microscopy

Microscopy involves, well, looking at stuff under a microscope. There are different types of microscopy that can be used for different purposes. Note: the stuff about microscopy here is going to be massively oversimplified as I'm not a physicist.

Bright field microscopy

Bright field microscopes are the type that you're probably more familiar with: the object appears dark compared to the background. Usually, some kind of stain, such as Gram's stain, is used to make bacteria more visible.

Dark field microscopy

In dark field microscopy, the light hits the specimen at an angle, resulting in a bright subject against a dark background. This is typically used for detecting spirochaetes, such as T. pallidum.

Phase contrast microscopy

Phase contrast microscopes use two beams of light, which are out of phase by a quarter of a wavelength. This setup increases visible contrast, making it easy to see your specimens without using a stain. Phase contrast microscopy tends to be used more for looking at fungi, rather than looking at bacteria.

Fluorescence microscopy

Fluorescence microscopy uses fluorescent dyes which, as their name suggests, fluoresce. Common dyes include auramine-rhodamine, which stains mycobacteria, and acridine orange, which stains nucleic acids. There are two main types of stains that can be used: fluorochroming, which stains everything, and immunofluorescence, which stains only target molecules via the use of an antibody-labelled dye.

Electron microscopy

Electron microscopy uses electrons which are focused via electromagnets. Electron microscopy can be used to view very small pathogens, such as viruses. It can also be used to visualise bacterial organelles.

Culture

Culturing pathogens can allow us to determine if pathogens are present, and purify pathogens so that we can test and identify them. Bacterial culture can also help us to perform antibiotic susceptibility testing. Most pathogens will form a colony after overnight incubation, though some are very slow-growing. The original bacterium that the colony arose from is called the "colony forming unit" (CFU).

Culture requirements

In order to culture bacteria, you need to have some idea of what is likely to be in the sample, which can be determined by the type of specimen (sputum, urine, etc.) and from symptoms of the illness. Next, optimal conditions need to be provided. Things to take into consideration include nutrients, temperature, atmosphere, pH, and osmolality:

• Nutrients: Pretty much all bacteria need sources of carbon, nitrogen, phosphorous, water and certain trace elements. Fastidious bacteria may need more than this.
• Temperature: Some organisms, known as psychrophiles, prefer cooler temperatures (15-20°C), while mesophiles prefer 30-37°C and thermophiles prefer 50-60°C.
• Aerobic: Strict aerobes only grow when oxygen is present, strict anaerobes only grow when oxygen is absent, facultative aerobes/anaerobes can grow in either condition, microaerophilic microbes like only small amounts of oxygen (2-3%), and capnophilic microbes grow better when CO₂ is present.
• pH: Most organisms prefer neutral or near-neutral pH.
• Osmolality: Some organisms (not many) require salt.

Culture phases 

There are four main phases of growth:

1. Lag phase: Little to no growth occurs as the organism is still adjusting to the medium.
2. Log or exponential phase: Exponential growth occurs.
3. Stationary phase: Cell growth equals cell death, so the overall number of cells remains constant.
4. Death/decline phase: Cells die off faster than they grow. This may be because all of the nutrients have been consumed.

Media types

There are many different types of culture media, and there are different ways in which they can be classified. Many can be prepared in solid (agar plate) or liquid (broth) form. A solid plate can detect a range of organisms and single colonies can be separated out, but it is not as sensitive as broth culture, and some specimens might be difficult to plate. Liquid broth is more sensitive (i.e. can detect very low numbers of organisms) but cannot produce pure cultures, and does not allow for quantification of how many organisms there are.

Media can also be separated according to function:

• Enriched media: Media that contains one or more compounds that stimulate growth, such as yeast or meat. May also include special compounds for culturing more fastidious organisms.
• Selective media: Media that only allow a specific organism or group of organisms to grow. For example, colistin and/or salt stop growth of Gram-negative bacteria. Salt can also be used to select for Staphylococci.
• Differential media: Media that allows multiple organisms to grow, but provides some means of separating between them. For example, mannitol salt agar not only selects for Staphylococci, but can be used to distinguish between Staphylococcus species. S. aureus ferments mannitol, turning the plate yellow, whereas S. epidermidis, which does not ferment mannitol, leaves the plate pink.

Now for some more examples of media, because why not?

Blood agar is made up of Columbia agar and 5% blood, which usually comes from a horse or sheep. Most bacteria (except for some Haemophilus species) can grow on blood agar. Blood agar can also be used to test for alpha- or beta-haemolysis, which can be used to identify species. Chocolate agar, so called because of the chocolate colour (not actual chocolate, sorry) used to be made up of heated blood agar, but is now made up of Columbia agar, haemin and isovitalex. Virtually everything grows on chocolate agar, even Haemophilus.

Chromagenic agar, or Chromagar, can turn many different colours depending on species. There are different Chromagar types for different specimens: for example, Chromagar Candida can distinguish between different Candida species, whereas Chromagar Orientation can distinguish between E. coli, Enterococcus, Klebsiella and more.

New blood culture systems have been developed in order to detect organisms in the blood. A patient's blood is taken and put into a bottle. There are different bottles for different specimens, according to culture requirements. Bottles are put into an automated machine, which signals positive when growth is detected. When growth is detected, a Gram stain and traditional culture may then be performed.

Mycobacteria Growth Indicator Tubes (MGIT) are used for detecting mycobacteria. They contain Middlebrook 7H9 broth and are put into a special machine that scans the tubes for increased fluorescence. Mycobacteria grow much faster in these tubes than they do on plate culture.

Biochemical tests

Following culture, a variety of tests may be done to identify the bacteria. Some commonly conducted tests include the catalase, coagulase and oxidase tests.

The catalase test is used to distinguish between Gram-positive cocci. The presence of catalase is detected by adding hydrogen peroxide (as mentioned here, catalase breaks hydrogen peroxide down into water and oxygen). If the test is positive, bubbles of oxygen will appear, whereas if the reaction is negative, no bubbles will appear. Gram-positive cocci that test positive include Staphylococcus species, whereas Gram-positive cocci that test negative include Streptococcus and Enterococcus. Note that there are many other pathogens that are catalase-positive (but of different shapes etc.).

The coagulase test is used to distinguish between S. aureus from most other Gram-positive, catalase-positive cocci. Coagulase is an enzyme that converts fibrinogen into fibrin (a protein involved in clots). Coagulase-positive bacteria will form clumps, whereas coagulase-negative bacteria will not form clumps. S. aureus is the main coagulase-positive bacteria- it's not the only one, but it's probably by far the most important in human specimens. S. aureus actually produces two forms of coagulase: the bound form can be detected in a slide coagulase test, whereas free coagulase can be detected with a tube coagulase test.

The oxidase test is used to differentiate Pseudomonas (oxidase-positive) from Enterobacteriaceae (an oxidase-negative group that includes Escherichia, Klebsiella, Salmonella and Shigella). It contains a reagent that turns dark-blue when oxidised, and colourless when reduced.

Of course, this is only the tip of the iceberg. As mentioned earlier, selective and differential media can be used to test for certain organisms. There are also biochemical tests in kits, such as the API system and the miniature Vitek card, which can run a lot of tests at once and have their results evaluated by machine.

Recently, even newer bacterial identification techniques, which don't rely on biochemical reactions, have been developed. One of these is MALDI-TOF, or Matrix Assisted Laser Desorption Ionisation - Time of Flight Mass Spectrometry. Once again, I'm not going to go into the physics of it, but it involves vaporisation and ionisation of the specimen, movement of ions through a flight tube, and detection by a detector at the other end of the flight tube. MALDI-TOF produces a spectra, which can be compared with other samples in a database. MALDI-TOF is reliable, relatively inexpensive and very fast, but it may be expensive at the start (due to having to buy a shiny new machine etc.).

16S RNA, which comprises part of the 30S subunit of bacterial ribosomes, can also be used for bacterial identification. 16S RNA has some highly conserved regions, making it easy to design primers, and highly variable regions, which can be used to distinguish between different species. 16S RNA identification is particularly useful for difficult-to-identify organisms.

Yet another bacterial identification technique is the use of the Fatty Acid Methyl Ester (FAME) profile, though that wasn't really covered much in the lecture (bottom right corner of one slide... whoop-de-doop).

Detection of Microbial Components

Not all microbes are easily cultured or viewed under the microscope, so being able to detect their components in other ways is crucial. Let's see how it's done!

Antigen detection 

Antigens can be detected by using monoclonal antibodies. And... that's pretty much all I have to say.

Toxin detection

Toxins can also be detected by antibodies, or measured by mass spectrometry. They can also be measured more indirectly by looking at the functional properties of the toxins.

Nucleic acid amplification

The main technique here is a nucleic acid test (NAT)/nucleic acid amplification test (NAAT), which I also mentioned here. NAAT is pretty much just PCR (see here if you don't know what PCR is). PCR can be single or multiplex (i.e. search for multiple genes at once). PCR products can be analysed by electrophoresis (as described here). Alternatively, real-time PCR, which analyses products of PCR while the reaction is in progress, can be used. Real-time PCR uses fluorescent dyes that react with the product. The amount of fluorescence can be detected and quantified.

Serology

There weren't actually any slides on serology, but it did keep coming up on the "Types of laboratory tests" slide, so I might as well give a quick note here. Apparently serology is the detection of specific antibodies to a microorganism in the serum (so I guess this would be something like looking for anti-HIV antibodies or whatever).

Absorption of Carbohydrates, Protein and Fat

This is pretty much a recap of PHYL2001, but oh well.

Actually, you know what? This IS a recap of PHYL2001. More specifically, it's a recap of Gastrointestinal Function part 3. So re-read that post, and you should be set.

FUN FACTS NOT IN THE ABOVE POST:

• The middle of the intestinal lumen is the "stirred layer," and it's where mixing occurs. The area closer to the cells is the "unstirred layer." In the unstirred layer, food is trapped by the glycocalyx on the brush border.
• Carbohydrates must be broken down completely for absorption, as only monosaccharides can be absorbed.
• SGLT1 (the transporter that absorbs galactose and glucose) requires secondary active transport with Na+. Na+ concentrations are kept low via the Na+/K+ pump.
• Glucoamylase, one of the brush border enzymes that breaks down carbohydrates, is also known as maltase.
• Unlike carbohydrates, very short chains of amino acids (di- and tripeptides) can be absorbed. They are absorbed via the PepT1 transporter, which uses secondary active transport with H+.
• The H+ gradient for PepT1 is maintained via a Na+/H+ antiport.
• Amino acids are all taken up by specific transporters. I'm pretty sure these also undergo secondary active transport with Na+, but the slide says Na+ independent, so I'm really confused now...

Exercise Physiology III: The Urge to Breathe

This post is going to be kind of incomplete as we ran out of time (the first half of this lecture was basically the back end of Exercise Physiology II). As such, I'll just write about all of the stuff that we have learned about, and when we learn the rest, I'll update this post. Updated now :)

Describe and understand normal chemoreceptor control of ventilation

See previous post: Control of Ventilation

Describe and understand what happens to blood gasses during exercise

During exercise, venous PCO2 increases, but arterial PCO2 remains fairly steady. If anything, arterial PCO2 decreases a bit. This indicates that all excess CO2 produced during exercise is removed in the first pass through the lungs. The other consequence of this is that it suggests that chemoreceptors are probably not the main drivers of an increase in ventilation during exercise, as chemoreceptors are located in the arterial system, not the venous system.

Describe and understand what happens to blood gasses during exercise in the absence of normal chemoreceptor input

To study whether or not CO2 sensitivity is actually important during exercise, children with central congenital hypoventilation syndrome (CCHS) have been studied. Children with CCHS are insensitive to CO2, and while they breathe normally when awake, they stop breathing when asleep. Despite this, children with CCHS still have increased ventilation during exercise. The increase in ventilation is increased to a greater extent in fast than in slow exercise (matched for work rate), suggesting that mechanoreceptors detecting limb movements might be responsible in exercise.

Describe and understand alternative ventilator stimuli during exercise

Ventilation increases to a fairly large extent at the beginning of exercise. It has been suggested that this may be due to central command, as well as mechanoreceptors. The initial rise in Ve is larger in trained than in untrained individuals. Ventilation then gradually increases during exercise, which may be due to metaboreceptors, which are chemoreceptors in the muscle.

Describe and understand the alveolar gas equation and how ventilation and chemoreceptor input are causes and effects of each other 

The alveolar gas equation discussed in this lecture was different to the equation discussed in other units. Why not make things simple when you can make them confusing, right?

Anyway, the equation discussed in this lecture was as follows:

P_aCO₂ = K (V_CO2/V_A)
where P_aCO₂ is the arterial partial pressure of CO₂, and I think V_CO2 and V_A are the ventilation rates for carbon dioxide and for alveolar air, respectively.

As discussed previously, P_aCO₂ affects ventilation via the action of chemoreceptors. Conversely, ventilation can affect P_aCO₂, as higher ventilation rates result in lower partial pressures of CO₂, and vice versa. P_aCO₂ vs. ventilation and ventilation vs. P_aCO₂ can be graphed simultaneously (sort of like the cardiac and vascular function curves), and the equilibrium point is where the two curves intersect.

Describe and understand how work intensity and muscle fibre type recruitment affects the relationship between VE and VCO2

As mentioned here, type I fibres are activated at all intensity levels. As intensity increases, type IIa and IIb fibres are also activated. Type II fibres, especially IIb fibres, rely a lot on anaerobic respiration (e.g. glycolysis) to produce energy. One of the main downsides of glycolysis production is that lactic acid is produced. We do have a buffering system to reduce lactate levels, but this produces carbon dioxide, increasing ventilation:

Lactic acid + Carbonic acid <--> Water + Carbon Dioxide

Because of this buffering system, respiration increases more rapidly following the Onset of Blood Lactate Accumulation (OBLA), which occurs when blood lactate levels are around 4mM. Eventually, this buffering system is pushed to its limit, and the pH starts to decrease. The decrease in pH (increase in H+ ions) drives ventilation further, causing an even steeper increase in ventilation rate.

Describe and understand some experiments designed to investigate the phenomenon of ‘central command’ in ventilator control 

In the first experiment, researchers attached a vibrator to the bicep muscle tendon. This stimulated reflex contraction of the bicep via the muscle tendon reflex. While the muscle tendon reflex was stimulating the bicep, not as much input from the brain was required to lift a weight (as compared to participants who didn't have the vibrator). When central command required was reduced, ventilation also decreased.

The second experiment had a similar setup to the first experiment, but in the second experiment, participants were asked to use their tricep muscle to pull something down to lift a weight via a pulley system, rather than use their bicep to lift something up. As the vibrator was still attached to the bicep muscle, the muscle reflex actually made it harder to contract the tricep (as the bicep and tricep are antagonistic muscles). Therefore, in this setup, participants with the vibrator needed more input from the brain in order to lift the weight. When central command required was increased, ventilation also increased.

Pancreatic and biliary secretion

Define and describe the physiological functions of the pancreas & gall bladder.

See previous posts:

• Digestion and Absorption of Food- part 2
• Gastrointestinal function part 2

In this post, we will be focusing on the exocrine pancreas, which is the part that secretes digestive juices. (The endocrine pancreas secretes hormones such as insulin and glucagon.)

Describe the production of alkaline secretions by the pancreas.

Production of alkaline secretions in the pancreas is actually quite similar to production of acid in the stomach. HCO₃^- is produced from the reaction of CO₂ and H₂O, which is catalysed by carbonic anhydrase. HCO₃^- is then pumped out of duct cells, and Na⁺ and H₂O follow. To get rid of the H⁺ also produced during the carbonic anhydrase reaction, there is a H⁺/Na⁺ antiport on the side of the cell facing the blood.

List the major pancreatic enzymes and describe their secretion, activation, & function.

The major pancreatic enzymes include proteases, such as trypsin, chymotrypsin, carboxypeptidase, and elastase; nucleases; carbohydrases, such as pancreatic amylase; and lipases, such as pancreatic lipase, cholesterol esterase, and phospholipase. Proteases are all synthesised in inactive form so that the pancreas doesn't digest itself. For example, trypsin is released as trypsinogen, which is the inactive form of trypsin. The acini also have trypsin inhibitors as an added layer of protection.

Once in the small intestine, proteases become activated. The small intestine has an enzyme called enterokinase, which is held in place by glycosaminoglycans. Enterokinase activates trypsinogen to form trypsin, which can then go around and activate the other proteases.

Inactive proteases are constantly synthesised and stored in secretory vesicles known as zymogen granules. There is a very low amount of constitutive secretion. Secretion of enzymes is increased when CCK (cholecystokinin) and ACh (acetylcholine) bind to their receptors.

Describe the control mechanisms regulating pancreatic secretion.

The most important factors involved in pancreatic secretion are acetylcholine, secretin, CCK, and somatostatin. ACh, released from the vagus nerve, stimulates both bicarbonate and enzyme release. Secretin, secreted from duodenal cells in response to low pH, stimulates bicarbonate production. CCK, secreted in response to duodenal fat and protein, stimulates enzyme production and relaxes the sphincter of Oddi. Somatostatin pretty much just inhibits everything.

In the cephalic phase of secretion, the vagus nerve stimulates some pancreatic secretions, via ACh. (See here if you can't remember the different phases of digestion.) These secretions only account for 10-20% of total secretion. In the gastric phase, gastrin binds loosely to CCK receptors, stimulating a further 5-10% of pancreatic secretions. Finally, in the intestinal phase, large amounts of CCK, secretin and ACh stimulate the bulk of pancreatic secretions.

Describe the role of bile and its production and metabolism

See previous post: Gastrointestinal function part 3

Bile acids can be classified as primary or secondary bile acids. Primary bile acids are those synthesised by hepatocytes, and secondary bile acids are produced when primary bile acids are converted by bacteria in the intestinal lumen.

Bile is formed by hepatocytes secreting stuff into the bile ducts. Some substances, such as bile salts, bilirubin, cholesterol and drugs are actively secreted by specific transporters. Other substances, such as water and glucose, may enter via diffusion through tight junctions.

After leaving the liver and travelling down the bile duct, bile is stored and concentrated in the gallbladder. Concentration of bile occurs via active transport of electrolytes from the gallbladder into the bloodstream. Electrolyte movement is then followed by water movement. One of the consequences of bile concentration is that gallstones can form, mainly from concentrated bilirubin and cholesterol that precipitates out with calcium.

When fats and amino acids enter the duodenum, CCK release is triggered. CCK relaxes the sphincter of Oddi and stimulates gallbladder contraction, pushing bile out of the gallbladder and into the small intestine. ACh from the vagus nerve may also be involved in gallbladder contraction.

Discuss haem removal via the bilirubin system

Haem is broken down into bilirubin in the reticuloendothelial system, which includes organs such as the spleen. Heme oxygenase cleaves the haem ring to form biliverdin, and biliverdin reductase reduces biliverdin to form bilirubin. Bilirubin travels around the blood while bound to albumin. When bilirubin reaches the liver, it is released from albumin, and enters the liver via transporters. Once in the liver, two glucose residues are added to form a water-soluble diglucuronide. This diglucuronide is what is pumped into the bile canaliculus.

After being secreted in bile, bilirubin is subjected to one of two main fates. First of all, bilirubin can be pooped out, along with everything else in your intestines. The alternative is that gut bacteria can convert bilirubin into urobilinogens, which the small intestine is permeable to. Urobilinogens can be reabsorbed and essentially recycled.

If bilirubin secretion is not sufficient, jaundice (yellowish tint to the skin) can occur. There are two main types of jaundice: haemolytic jaundice and obstructive jaundice. In haemolytic jaundice, too many red blood cells are broken down, and in obstructive jaundice, bile is unable to leave the bile duct due to an obstruction. The two types of jaundice can be differentiated by looking for conjugated vs. unconjugated bilirubin. Haemolytic jaundice has a large amount of unconjugated bilirubin (since a lot of bilirubin hasn't reached the liver yet), whereas obstructive jaundice has a large amount of conjugated bilirubin (as this is a problem downstream of the liver).

Liver Progenitor Cells- Cell therapy to treat liver disease

THIS LECTURE HAS AN OUTCOMES SLIDE, SO I ACTUALLY KNOW WHAT I NEED TO FOCUS ON!!! A-fucking-mazing.

Liver as an organ- Function and structure

See previous post: Digestion and Absorption of Food- Part 2

The liver is made up of two lobes, which can be further subdivided into small lobules. Each lobule is made up of a central vein, sinusoids, canaliculi and hepatocytes. Blood from the portal vein and hepatic artery flow through sinusoids into the central vein, the hepatocytes produce bile, and the canaliculi take bile to the bile duct. The canal of Hering is the junction between hepatocytes and bile ducts. Other cells present include endothelial cells, which line sinusoids (remember, they're basically just giant blood vessels) and Kupffer cells (which are basically macrophages in the lumen of sinusoids).

One of the most remarkable features of the liver is its ability to regenerate itself. When the liver is damaged in some way, gut-derived factors such as LPS (lipopolysaccharide) are upregulated. LPS activates Kupffer cells, which release pro-inflammatory cytokines, such as IL-6 and TNFα. The pancreas, duodenum, salivary gland and others also release a bunch of factors, which allow the hepatocytes to continue dividing. At the same time, TGFβ, an anti-inflammatory cytokine that inhibits hepatocyte DNA synthesis, is blocked.

Liver Disease

Disease progression

In early stages of liver disease, fat can build up in your liver, resulting in non-alcoholic fatty liver disease (NAFLD). Later on, this fat can become inflamed and liver cells can be damaged, resulting in non-alcoholic steatohepatitis (NASH). NASH can then result in cirrhosis (scarring of the liver). Cirrhosis, in turn, can lead to hepatocellular carcinoma or death unless a transplant is done.

Current treatment options

In early stages of liver disease, the damaged parts can simply be removed, and the healthy liver allowed to grow back. Unfortunately, it is very difficult to detect early liver disease. If the disease isn't detected until later, then a transplant is necessary, but unfortunately demand far outstrips supply. Patients who have acquired hepatocellular carcinoma (HCC) may undergo a partial hepatectomy (removal of part of the liver), transplantation, radiotherapy, or take Sorafinib, but prognosis is still very poor.

Regenerative Medicine and Cell Therapy

ELAD

ELAD, or extracorporeal liver-assist device, has four cartridges containing human liver cells. A patient's plasma passes through these cartridges, allowing for transfer of toxins, metabolites, and so forth. ELAD is currently on a phase III clinical trial.

Stem Cells – what stem cells are

OH I FUCKING WONDER. I certainly haven't spoken about them here, or here, or here, or here, or here, or here, or here...

Liver Stem Cells / progenitor cells

We still aren't 100% sure which cells serve as liver stem cells, but oval cells seem to be good candidates. Oval cells are oval shaped (BET YOU DIDN'T SEE THAT ONE COMING!) and grow as extensions of terminal biliary ductules in the liver. They form ductular structures that communicate with the biliary system at one end and form hepatocytes at their other end. The interesting part about oval cells is that they are induced as liver damage proceeds, and appear to be able to produce both hepatic cells and cholangiocytes (epithelial cells of the bile duct).

Cholangiocytes themselves may be able to act as liver stem cells. It has been shown that cholangiocytes can differentiate to produce hepatocytes, but this may only be possible when hepatocyte differentiation is inhibited.

Using LPC to treat disease

Tyrosinemia Type I

Tyrosinemia is a genetic disorder in which the FAH gene is mutated. The FAH gene codes for FAA hydrolase, which is one of the enzymes involved in the breakdown of tyrosine. In untreated tyrosinemia, tyrosine and its byproducts build up in tissues and organs. Interestingly enough, transplanted liver progenitor cells (LPCs) (which I'm assuming are oval cells?) are able to express FAH, at least in mice.

Methylmalonyl Aciduria (MMA)

I've discussed MMA before on my blog. Essentially, it is a defect in methylmalonyl CoA mutase, which leads to the toxic build-up of methylmalonic acid in the blood. Children with MMA are low weight for their age, and mortality is high due to toxicity. MMA can be treated with a liver transplant, but LPCs might be able to help too.

Gene Correction

Gene correction is another technique that is being studied. The idea is that if we can take a patient's cells, turn them into iPS cells, "correct" the defective genes by using gene technology, and then growing and differentiating the "corrected" cells, we can treat genetic illnesses. Gene technologies that can be used include viral vectors, such as the Adeno-associated virus (AAV) vector, and the CRISPR Cas9 method. There appears to have been some success in using AAV vectors to treat haemophilia, but for most diseases we haven't gotten anywhere near that stage yet. iPS cells produced with this technique can also be exposed to different drugs in order to find an effective drug- essentially personalised medicine.

Liver Organoids

Some work has been done in growing mini livers. Two cell types are required: LPCs and endothelial cells (for the blood vessels). These cells need to be grown on a scaffold, along with a third type of cell, which is taken from fat left over from reconstructive surgery.

Liver Progenitor Cells and Cancer

Unfortunately, LPCs do come with a risk of fibrosis and cancer. LPCs have been implicated in liver hepatocellular carcinoma, which is rather unfortunate, given that liver hepatocellular carcinoma was one of the outcomes that we were trying to avoid in the first place! Oh well.

Infections of the Central Nervous System

Hopefully my last "disease infodumping" post for a while! Next week's posts will be on bacterial diagnosis.

Once again, this post started with some brief anatomy. Basically you just need to know what the meninges are, and what the central and peripheral nervous systems are. You also need to know that the blood-brain barrier allows some things to cross (particularly lipid-soluble substances like chloramphenicol), but not others (though this may change during inflammation, as inflammation compromises the blood-brain barrier). I've written more detail than you probably want to know in this earlier post.

Also, just some quick terminology. Meningitis is inflammation of the meninges, encephalitis is inflammation of the brain, and meningoencephalitis is inflammation of both meninges and brain.

Bacterial meningitis

Bacterial meningitis is pretty damn nasty. It starts off suspiciously benign, with a headache, fever and stiff neck, and sometimes some nausea and vomiting. A few patients may develop the distinctive skin rash that does not fade under pressure. Later on, patients experience convulsions and go into a coma. Even survivors of meningitis don't always get away scot-free, as complications include hearing loss, epilepsy, brain damage and learning difficulties. Risk factors for bacterial meningitis include age (infants and young adults), living in a densely-populated community (e.g. university dormitory), compromised immunity, and travelling to an endemic area.

The main causes of bacterial meningitis are S. pneumoniae, H. influenzae and N. meningitidis, though there are many others. Gram-negatives can release endotoxin, and gram-positives can release cell wall fragments, leading to inflammation and shock. Diagnosis can be done via a lumbar puncture and Gram stain. Treatment is generally via broad-spectrum third generation cephalosporins. Close contacts are often treated as well in order to prevent the spread of disease.

N. meningitidis is an aerobic, gram-negative diplococcus that has a polysaccharide capsule. It infects the throat before entering the blood and causing meningitis. When it proliferates in the bloodstream (i.e. sepsis), it can destroy tissue, which may lead to limb amputation. Thankfully, vaccines have been developed against the capsular serotypes, which are A, B, C, W and Y. In Australia, there is currently a free vaccine available against A, C, W and Y for those aged between 15 and 19.

H. influenzae is a gram-negative pleomorphic rod (i.e. it can appear in slightly different shapes). It is part of our commensal flora, but some capsules (particularly type b) can be more pathogenic. There is now a vaccine against H. influenzae type b, which has reduced the mortality rate.

S. pneumoniae is a gram-positive coccus that has a capsule. It commonly lives in the nasopharynx, so a lot of people are healthy carriers. S. pneumoniae is actually the leading cause of bacterial meningitis. There is a vaccine available, but it only covers a fraction of the more than 90 serotypes of S. pneumoniae.

Listeriosis

Listeriosis is caused by Listeria monocytogenes, which is a gram-positive rod. It is excreted in animal faeces and distributed in soil and water, and is spread by contaminated food. Symptoms include fever, headache, altered mental state, neck stiffness and seizures. It is more likely to affect the immunocompromised and elderly. Infection during pregnancy is particularly problematic as Listeria can cross the placenta, which may cause stillbirth. If the foetus makes it to term, they may have brain injury or die at a young age. As such, pregnant women are often advised to avoid high-risk foods, such as soft cheeses.

Leprosy

Leprosy is caused by Mycobacterium leprae, an acid-fast rod related to M. tuberculosis. M. leprae is unique in that it grows in the peripheral nervous system, as well as in skin cells (it prefers outer, cooler areas of the body). It can survive ingestion by macrophages, invade cells of the peripheral nervous system myelin sheath, and cause damage via a cell-mediated immune response.

Tetanus

Tetanus is caused by Clostridium tetani, which is an obligately anaerobic, endospore-forming, gram-positive rod. The bacteria do not spread from the site of infection and there is no inflammation, but they can release neurotoxins upon death. These neurotoxins can spread through the peripheral nerves and blood to reach the CNS, where they block the muscle relaxation pathway. The jaw is affected first, which is why tetanus is also known as "lockjaw," but later on back spasms and so forth can result. Death is usually due to respiratory muscle spasms. Tetanus is vaccine-preventable, so get yo' tetanus shots, kids!

Viral meningitis

In contrast to bacterial meningitis, viral meningitis is more common, but thankfully much less serious (and can sometimes be mistaken for a cold!). It is usually caused by enteroviruses and herpes viruses, and normally resolves without treatment.

Poliomyelitis

Polio is best known for its ability to cause paralysis, but in fact the paralytic form is quite rare, and more likely to occur in people who contract the disease for the first time in adolescence. (That's not to say that it isn't devastating, however.) Most of the time, it causes no symptoms, or only mild symptoms, such as headache, sore throat, fever and nausea. Polio multiplies in the throat and small intestines before invading the tonsils, lymph nodes and intestines. From the lymph nodes, polio can enter the blood, causing viraemia. Often the viraemia is only transient and does not cause disease, but if it is persistent, the virus can enter the CNS and nerve cells, killing off motor neurons, resulting in paralysis. Thankfully, two vaccines (the Salk and Sabin vaccines) have mostly eradicated polio from the world.

Rabies 

Rabies can be acquired from the bite of an infected animal. It has a long incubation period, so if you are bitten, a rabies vaccination will actually help you. The virus replicates in the muscle before travelling along peripheral nerves to the CNS, causing encephalitis. Symptoms include periods of agitation as well as spasms of the mouth or pharynx (which might be set off by just the sight of water). The disease is pretty much 100% fatal in humans. Thankfully, rabies is pretty rare in Australia (I think it's only found in bats). Well, I guess that's compensation for the number of other things that can kill us here!

Arboviral encephalitis

Encephalitis can also be caused by arthropod-borne (insect-borne) viruses, or arboviruses. Symptoms include chills, headache, fever, mental confusion, coma, and death. Examples include Japanese encephalitis and West Nile Virus.

Fungal meningitis

Fungal meningitis is pretty rare, and is more common in the immunocompromised. The main cause of fungal meningitis is Cryptococcus, which is a yeast that reproduces by budding. It has large polysaccharide capsules and is found in soil that has been contaminated by pigeon and chicken droppings.

Protozoal or amoebic meningitis

Protozoal meningitis is also very rare, and is usually always fatal (partly because it is often misdiagnosed as bacterial meningitis until it is too late). One cause of protozoal meningitis is Naegleria fowleri, which causes primary amoebic meningoencephalitis (PAM). It is found in recreational fresh water, and while many people are infected by it at some point, very few people actually become sick. Those that do become sick, though, are pretty much screwed, as there are only a few known survivors.

African trypanosomiasis (sleeping sickness)

African trypanosomiasis is caused by two subspecies of flagellated protozoans, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. Both species are spread by tsetse flies. T. b. gambiense causes Gambiense disease, which causes CNS symptoms after a few weeks to months, which eventually lead to death. T. b. rhodesiense causes Rhodesiense disease, which is relatively rare in humans (usually found more in livestock) and more acute, with symptoms occurring in days and death occurring in weeks to months.

Infections of the Blood

Another week, another set of diseases! I think this is the last week that we're getting infodumped about diseases- future lectures seem to cover stuff related to diagnosis and treatment. Remember: red pathogens are found in Microbe Invader (http://microbeinvader.com).

This lecture started with a bit of anatomy about the cardiovascular system. All you really need to know is that it delivers blood around the body, and that plasma can diffuse out of the capillaries and get picked up by the lymph system. The lymph system later dumps its crap back into the venous system. If you want to know more details regarding the cardiovascular system, just use the search function on this blog. I wrote a fair bit about the anatomy in ANHB2212, and a lot about the physiology in PHYL3002.

Bacterial Infections

Sepsis and Septic Shock

Sepsis is defined as "a toxic inflammatory condition arising from the spread of bacteria or bacterial toxins from the focus of infection." Septicaemia is basically sepsis that results from bacteria in the blood. (Bacteria in the blood, with or without sepsis, is also known as bacteraemia.) Low-level bacteremia is usually okay as our host defensive cells can kill bacteria, and the low levels of iron in our blood are generally insufficient to sustain bacterial growth, but when our defences fail, or when organisms are pathogenic, we might have some problems.

Sepsis can be divided up into three main stages. In the first stage of sepsis, inflammation occurs and cytokines are released, resulting in fever, chills, accelerated breathing and faster heart rate. In the second stage, severe sepsis, at least one organ is damaged. In the third stage, septic shock, there is a significant drop in blood pressure due to inflammation. Septic shock can lead to organ failure and death.

Some of the most frequent causes of sepsis are Neisseria meningitidis, as well as Staphylococcus, Streptococcus and Enterococcus species. In fact, Enterococcus species are some of the leading causes of nosocomial (hospital-acquired) sepsis- they enter the body during invasive procedures, such as dialysis. Septic shock is more likely to be caused by gram-negatives, such as N. meningitidis, as they release endotoxins when they die. The inflammatory response leads to a drop in blood pressure. N. meningitidis septicaemia is also known as meningococcal disease, which is a pretty nasty disease that can be fatal within hours. Septic shock can also be caused by gram-positive bacteria such as Staphylococcus and Streptococcus. They produce potent exotoxins (remember, gram-positive bacteria can't produce endotoxins) called superantigens (i.e. antigens that induce a really strong immune response). This form of septic shock is also called toxic shock syndrome.

Puerperal Sepsis

Puerperal sepsis is also known as "childbirth fever," as it is an infection of the uterus following childbirth or abortion. The infection can spread from the uterus to the abdominal cavity, causing peritonitis and then sepsis. The most common cause is Streptococcus pyogenes. Puerperal sepsis is pretty rare in developed countries, but it is still a leading cause of maternal death in developing countries.

Bacterial Endocarditis

Endocarditis refers to inflammation of the endothelium, which is the innermost of the layers lining the heart (see here for details). There are two main types of endocarditis: subacute and acute.

Subacute endocarditis is usually caused by alpha-haemolytic streptococci in the oral cavity. Bacteria can lodge in pre-existing lesions in the heart, where they become trapped in clots. These clots protect the bacteria from phagocytes, while they get to work destroying the heart. Untreated subacute endocarditis is fatal within months. Risk factors for subacute endocarditis include congenital heart defects, syphilis, or rheumatic fever.

Acute endocarditis is usually caused by our good friend S. aureus. As its name suggests, it progresses more rapidly than subacute endocarditis. It destroys heart valves and, if untreated, kills within days.

Rheumatic fever

See previous post: Carditis

Tularaemia

Tularaemia is caused by F. tularensis, which is a gram-negative rod. It is pretty rare and is usually passed around by infected animals, particularly rabbits and squirrels (as such, it is more common in the USA). It causes ulcers and enlarged, pus-filled lymph nodes.

Brucellosis (undulant fever)

Brucellosis is caused by Brucella species, which grows inside cells. When cultured, it requires enrichment and elevated carbon dioxide. The major species are B. melitensis, B. abortus and B. suis. B. melitensis, which usually affects goats and sheep, can cause severe disease and death in humans. B. abortus, which infects livestock, causes more mild disease, while B. suis, found in swine and cattle, causes ulcers in humans. Other symptoms of Brucella infection include chills, fever, malaise, and sweating. Brucella is transmitted via contact with diseased animals, such as via abrasions in the skin or by drinking unpasteurised milk. Treatment is with long-term tetracycline and streptomycin.

Anthrax

Anthrax is caused by Bacillus anthracis, which is a gram-positive, endospore-forming rod. It is found in soil and affects grazing animals, such as cattle and sheep. There are three main types of anthrax: cutaneous, gastrointestinal, and inhalational. However, only inhalational anthrax enters the bloodstream. When endospores enter the bloodstream, they are taken up by macrophages, where they germinate into vegetative cells. This causes the macrophage to die, releasing bacteria, which then replicate rapidly and secrete toxins. B. anthracis secretes two toxins: oedema toxin and lethal toxin. These toxins share a component called protective antigen, which binds toxins to host cells.

One of the tricky things about B. anthracis is that its capsule is made out of amino acids, not polysaccharides (like most bacterial capsules). As such, protective immunity is not stimulated, and people generally do not acquire immunity to anthrax.

Viral Infections

Infectious mononucleosis (Mono/Glandular Fever)

Infectious mononucleosis is caused by the Epstein-Barr Virus (EBV). Children are often infected by EBV without showing symptoms. However, if EBV is contracted as a young adult, disease can occur. It is usually self-limiting and non-fatal, with symptoms such as fever, fatigue, and sore throat. However, deaths can occur due to rupture of the spleen. EBV is transmitted through saliva, which is why this disease is sometimes known as the "Kissing Disease."

Cytomegalovirus (CMV) 

CMV is actually a type of herpes virus- HHV5 (human herpes virus 5). Nearly all humans are infected with CMV at some point, but it usually causes no or mild symptoms. In the immunocompromised, however, CMV can cause life-threatening pneumonia, and many AIDS patients develop CMV retinitis (blindness due to CMV infection). A foetus may also suffer damage if a pregnant mother acquires CMV for the first time during pregnancy.

CMV is a very sneaky virus. It can remain latent in our white blood cells, and move between cells that are in contact with each other, in order to escape detection by the immune system. CMV can't hide from microscopes, though- they form distinctive inclusion bodies that are known as "owl's eyes."

Yellow fever

Yellow fever is transmitted by mosquitoes, particularly in tropical areas of Africa and Central and South America. It causes fever, chills, headache, nausea, vomiting, and jaundice (which is where the disease gets its name).

Dengue fever

Dengue fever is caused by Dengue virus, which is also transmitted by mosquitoes. Once again, it is endemic in tropical areas. Its symptoms can include fever, severe chills, joint pain and rash. It is usually quite mild, though some people can experience severe pain, giving this disease the nickname of "breakbone fever." There is, however, a more dangerous form called dengue haemorrhagic fever, which is more likely to occur if you have multiple Dengue fever infections in a row.

Emerging viral haemorrhagic fevers

Yellow fever and Dengue fever are known as "classic" haemorrhagic fevers, as we've known about them for a long time. Marburg virus, Ebola virus and Lassa virus also cause haemorrhagic fevers, but as we've only found out about them in the past 50 years or so, they're considered to be "emerging" haemorrhagic fevers. Marburg virus was discovered from African monkeys that were imported into Europe, Lassa virus was found to be from rodents, and Ebola is possibly from fruit bats (though we don't know for certain).

AIDS

See previous post: Chemotherapy III: Antiviral Drugs

Burkitt's Lymphoma

Burkitt's lymphoma is a jaw tumour that seems to result from infection by EBV and malaria. It is possible that malaria infection impairs the immune response to EBV.

And that's the last post covering content for next week's test! (CNS infections are up next, but I don't think they're on the test. Weird.)

Engineering Contractile Tissues for Heart Repair

This post is kind of like a part 2 to my last PATH3304 post: Scaffolds for Biological Tissue Reconstruction. This lecture didn't have an outline, so guess I'll just have to wing it.

Repair capacity of the heart

Unfortunately, our hearts are not very good at fixing themselves up. That is because cardiac muscle cells generally don't divide. If cardiac muscle cells die, they tend to be replaced with scar tissue, and remaining cells hypertrophy. There are clusters of stem cells in the heart, but they are pretty rare. Therefore, prognosis for heart attack survivors isn't the best: their heart function often declines until it ultimately fails.

Cardiac tissue engineering

Like I said in my last post, cells, scaffold, and growth factors are collectively known as the "Tissue Engineering Triad," as they are all important factors in tissue engineering. Another important consideration is vascularisation: it's pretty much impossible to grow whole organs if not all of the cells will be able to get nutrients. Several different approaches have been trialled in cardiac tissue engineering: ring structures, cell sheets, decellularised scaffolds, and in vivo vascularised chambers.

Ring structures have been created with donor cardiomyocytes (such as rat cardiomyocytes- so far, we haven't found a willing human donor- wonder why? :P) on a collagen hydrogel. This approach is relatively simple, and allows for some modification of scaffold and cells. Since this technique doesn't take vascularisation into account, its size is limited. Furthermore, its contractile force is quite low. Ah well, back to the drawing board!

A second approach involves making cell sheets out of rat cardiomyocytes or iPS (induced pluripotent stem cells). Once again, this is a relatively simple approach, and doesn't need any kind of scaffold. Once again, however, contractile force is low, and lack of vascularisation means that size is limited.

A third approach involves a decellularised scaffold, which I mentioned here. It involves rat cardiomyocytes cultured on a decellularised donor heart. These tissues can be assembled fairly quickly, and are large 3D structures (as compared to the smaller 2D structures of the other approaches). An obvious limitation with this method is that you need donor tissue, and the procedure is quite complex. Furthermore, the contractile force is still quite low.

A fourth approach involves growing tissue in a special chamber in vivo. These chambers can have holes in them (which I think encourages the growth of blood vessels or something?). Advantages include the growth of large 3D structures, but unfortunately, the procedure is fairly complex and the contractile force of the resulting structure is still low.

Obtaining cardiomyocytes

As you may have noticed, a recurring theme with the approaches for cardiac tissue engineering is that donor cardiomyocytes are required. This is a massive problem, as it's not exactly reasonable to ask a healthy person to donate part of their heart. As such, other sources, such as adipose-derived stem cells, have been considered. Adipose stem cells are derived from first performing liposuction on a patient and then isolating the stem cells.

After the adipose stem cells (ASCs) have been obtained, the next challenge is to get them to differentiate into cardiac muscle. Trichostatin A was found to increase expression of cardiac actin, though this did not cause contraction. Co-culture of ASCs with rat cardiomyocytes, however, was more likely to induce differentiation of human cardiomyocytes.

iPS (induced pluripotent stem cells) have also been investigated. iPS cells can be derived from fibroblasts which have been specially treated in order to become pluripotent. In order to optimise differentiation, several techniques have been trialled, such as adding trichostatin A or co-culture, just like with ASCs. (I think. This might have been where I was zoning out. I blame it on the lecturer turning out the lights because I just felt sleepy for the entire second lecture. But maybe I was tired because it was that time in the afternoon when I get sleepy? I don't know.) One of the risks of using iPS cells is that there seems to be a risk of forming teratomas, which are tumours of multiple cell types.

Other stuff

The lecture finished here because it went overtime, but the lecturer said that the slides would go up and they would be "pretty self-explanatory," or something along those lines. Well, I'm looking at them now, and it seems like the main points are that MSCs (remember them?) have paracrine activity involved in angiogenesis, and thus may improve vascularisation. Also there's another slide that says that hypoxic ASCs may also stimulate angiogenesis. According to the summary slide, this may also be through paracrine mechanisms, or the ASCs themselves may differentiate into vascular wall cells.

And that's all of my posts down for the week!

Exercise Physiology II: The Cardiovascular System

Understand the distribution of cardiac output and how it is controlled in different situations

At rest, only around 20% of cardiac output goes to the muscles. Around half goes to the liver and kidneys, around 14% to the brain, and the rest goes to the heart, skin, and other organs. Distribution depends on the opening and closing of pre-capillary sphincters, and local vasodilation/vasoconstriction, as discussed here.

Describe and understand what happens to cardiac output during exercise

Cardiac output, as I'm sure you know, increases dramatically during exercise. The distribution of cardiac output also changes: a larger proportion of cardiac output goes to the muscle, and less goes to other organs. Because cardiac output has increased so much, though, the absolute amount of blood that most organs get increases. There are two exceptions to this rule: the liver and kidneys both experience a decrease in cardiac output (both absolute and proportional).

Describe and understand the determinants of cardiac output during exercise

During exercise, sympathetic stimulation increases, causing vasoconstriction. This seems counterproductive given that vasoconstriction will reduce the flow of blood, but thankfully there's also something called "functional sympatholysis" (i.e. the "breaking off" of sympathetic stimulation). Areas of the body undergoing high levels of metabolism, such as working muscle, can produce local mediators that cause vasodilation, counteracting the vasoconstricting effects of sympathetic stimulation.

Describe the known determinants of local blood flow mediation

See previous post: Microcirculation and Blood Flow

Nitric oxide (NO) may also play a role. It has been discovered that haemoglobin releases NO when it becomes deoxygenated.

Describe and understand how the local control of blood flow conflicts with blood pressure regulation

Vasodilation of capillaries causes a decrease in blood pressure, which would be bad if unchecked. (The opposite is true during vasoconstriction.) Thankfully, total peripheral resistance isn't the only determinant of blood pressure, the other being cardiac output. An increase in cardiac output can counteract a decrease in blood pressure due to local vasodilation (within limits, of course).

Understand the relationships between work rate and cardiovascular variables

As work rate increases, so too does heart rate and stroke volume (and hence cardiac output), arterial pressure (systolic increases to a much greater extent than diastolic), oxygen consumption, and arterio-venous oxygen difference. Peripheral resistance, however, decreases due to the vasodilation of capillaries in working muscle.

Describe and understand the origin of changes in the arterio-venous oxygen difference during exercise

As you might recall from PHYL2001, the haemoglobin saturation curve shifts to the right when temperature is high and pH is low (which is what happens during exercise). A rightward shift indicates that more oxygen is released from haemoglobin at the same partial pressure of oxygen in solution. As more oxygen is being released from haemoglobin during exercise, the arterio-venous oxygen difference increases.

Describe and understand the changes in cardiovascular variables during exercise

During exercise, the baroreceptor's set point is reset to a higher blood pressure. Stimuli that reset the baroreceptors may include feedback from muscle chemosensors, muscle mechanoreceptors, or the motor cortex. Our normal blood pressure is then sensed as being too low, reducing the firing rate of baroreceptors. The nervous system then responds by increasing sympathetic stimulation and decreasing sympathetic stimulation. These changes in sympathetic and parasympathetic stimulation cause an increase in certain cardiovascular variables, such as heart rate.

Describe and understand adaptations in cardiovascular variables after repeated exercise

Our bodies can adapt to repeated exercise. Fit people will tend to have a lower resting and exercise heart rate, but a greater oxygen uptake (a.k.a. VO2 max). I'm not entirely sure what the mechanisms are, though- maybe we'll find out in the next lecture?

Describe and understand the determinants of stroke volume

See previous post: Cardiac Loads

End-diastolic volume changes more in exercise than end-systolic volume. Interestingly enough, end-systolic volume changes even less when you are exercising in a supine (lying down) position, compared to exercising while standing up.

Describe and understand Starling’s capillary fluid balance and how it is affected by exercise

See previous post: Microcirculation and Blood Flow

Remember, the switch from filtration to reabsorption depends on the pressure drop across the length of an arteriole. In exercise, this pressure drop is decreased, so the outward hydrostatic presssure is greater than the inward oncotic pressure over a longer distance. This leads to increased filtration during exercise, which in turn leads to oedema and a decrease in plasma volume during exercise.

Describe and understand cardiac drift

Cardiac drift refers to the phenomenon in which heart rate increases during exercise, even when work rate remains the same. Cardiac drift occurs because of the decrease in plasma volume during exercise (see above). A decrease in plasma volume lowers blood pressure, and heart rate increases in order to compensate (baroreceptor reflex).

Describe and understand blood pressure changes in exercise

Not really sure what to say here, other than that a higher heart rate leads to a higher diastolic pressure during exercise (my understanding was that it's because the next heart beat comes along before pressure can drop all the way down to resting diastolic pressure?). Mean arterial pressure also increases during exercise.

Describe and understand blood boosting

In blood doping (or blood boosting), athletes remove a litre of their blood around a month or so prior to a competition, and then reinfuse it. As such, their haematocrit (% of blood with red blood cells) increases. Blood doping increases VO2 max and exercise performance, but if you overdo it, the blood can become very viscous and difficult to pump around (as also mentioned here).

An alternative to blood doping is to add erythropoietin (EPO) to increase production of red blood cells. Recombinant EPO, made in the lab by microbes, can be detected as microbes glycosylate EPO differently to humans.

Scaffolds for Biological Tissue Reconstruction

In this post we'll be talking about scaffolds that can be used to grow cells and tissues for later implantation.

History

Apparently people have been using various sorts of implants for over 2000 years, but the more modern kinds of implants date back to the 1940s. In the 1940s, a British ophthalmologist found that fighter pilots whose eyes were injured by shards of canopy plastic didn't have any apparent reactions to the plastic, so he looked at the use of this plastic in eye implants. In the 1960s, materials for implants were more specifically designed and engineered.

Purpose

As I said at the beginning, scaffolds can be used to grow cells and tissues for later implantation. Bioscaffolds, cells, and growth stimulating signals are collectively known as the "Tissue Engineering Triad."

Functions

The function of a scaffold is to act as a synthetic ECM (extracellular matrix) so that cells can grow on them without any problems. Therefore, it needs to be able to perform some of the functions of ECM, including structural support, provision of bioactive cues for cell alignment, provision of growth factors, and provision of a changeable environment in which processes such as remodelling and neovascularisation (growth of new blood vessels) can take place.

Properties

In order to design an appropriate scaffold, we need to take several properties into consideration:

1. Architecture- This includes things like void volume (empty space), porosity, and biodegradability. Void volume may influence vascularisation and new tissue formation, while porosity may influence metabolite and nutrient transport. Ideally, the rate of degradation should match the rate of tissue formation.
2. Tissue compatibility- The scaffold must be non-toxic and allow cells to grow and differentiate on it.
3. Bioactivity- Ideally, the scaffold should be able to interact with cells and regulate their activities. This may be achieved through topographical features (mechanobiology, anyone?) or through the use of chemical signals.
4. Mechanical properties- The scaffold should provide some shape and stability. It may be helpful if the mechanical properties of the scaffold are similar to those of the host tissue.

Approaches to bioscaffold design

Pre-made scaffolds

One approach to bioscaffold design is simply to make a scaffold and seed cells on it later. Scaffolds can be made with natural or synthetic materials. Natural scaffolds can include substances like silk, collagen, alginates, etc., or even graft tissue. Natural scaffolds have excellent biocompatibility and cells attach to them easily, but they are relatively fragile. Synthetic scaffolds include a range of materials, such as glass, ceramics, nylon, or plexiglass. Synthetic scaffolds are not as biocompatible as natural scaffolds, but they allow for much greater control over their physical and mechanical properties.

Synthetic materials can be made using a variety of different methods, including electrospinning, casting, nanoweaving, and 3D printing. Electrospinning involves a probe tip that fires a liquid jet of polymer solution at a collector (okay, that was probably way oversimplified, but unfortunately that's all I really understood about this method). Casting involves the use of a mould. Nanoweaving is basically just weaving really small threads, and you've probably already heard of 3D printing, so I won't go into that.

Decellularised ECM

Decellularised ECM uses tissue derived from an allograft (graft from a member of the same species) or xenograft (graft from a member of a different species). The tissue is chemically treated to remove all of the cells, leaving only the ECM. Decellularised ECMs have excellent biocompatibility and are generally pretty functional, but there are also disadvantages. It is difficult to seed cells at an even distribution over a decellularised ECM, and if the ECM is not properly decellularised, there is a possibility of an immune reaction.

Cell sheets

This one is pretty much what it says on the box- cells are grown in sheets, which can be stacked on top of each other. The advantages of this method are that cells secrete their own ECM, the sheets are rapidly neovascularised (I think this means that new blood vessels supplying this area will rapidly develop) and no sutures are required during implantation. Disadvantages of this method include limited thickness (they aren't vascularised before implantation, so you're limited by diffusion), and are quite fragile.

Cell encapsulation

Cell encapsulation involves trapping cells in a solid ECM. This can be done by suspending cells in a liquid, and allowing the liquid monomers to self-assemble. This method is good for irregularly-shaped defects, but is also quite fragile.

Combination scaffolds

The above techniques can also be combined to make a combination scaffold.

Clinical Example

Our lecturer is working on developing a tympanic membrane (ear drum). His team is investigating the use of fibroin (a component of silk), which is biocompatible, fairly stiff, and able to be moulded. It is not, however, very biodegradable. The goal is to be able to culture cells on this membrane and implant it into a perforated eardrum in order to heal the perforation.