Lecture 7 - Our Hostile Environment: viruses to pollutants

pathogens

viruses, bacteria, prions and fungi

what prevents pathogens from taking over?

our immune system

psychosocial stress

stress that impacts immune response affecting susceptibility and outcomes of infectious diseases

what prevents us from succumbing to stress?

our mental defense systems

climate stressors

difficult terrain or weather conditions, vehicular traffic or sporting

what prevents us from succumbing to climate stressors?

technological innovations and our ability to maintain homeostasis

immunity

refers tot the body’s ability to defend itself against pathogens like bacteria, viruses, fungi and parasites

innate immunity

first line of defense against pathogens that is present from birth providing immediate non specific protection through physical barriers like skin and mucous membranes as well as chemical defenses

innate immune cells

neutrophils, macrophages and natural killer cells (responsible for detecting and destroying pathogens)

adaptive immunity

highly specific defense mechanism that develops over time in response to specific pathogens

what are the two primary components of adaptive immunity?

humoral and cellular immunity

what is humoral immunity mediated by?

by anitbodies

what is cellular immunity mediated by?

by T cells

what are the cells involved in adaptive immunity?

B lymphocytes and T lymphocytes

B lymphocytes

produce antibodies that recognize and bind to specific antigens on pathogens marking them for destruction (responsible for keeping memory of the antigens)

T lymphocytes

coordinate and regulate immune response by directly killing infected cells and activating other immune cells

spinal cord injury

involves complex pathobiological process that starts with immediate mechanical damage and progresses through multiple secondary injury mechanisms

what are the primary drivers of the secondary injury mechanisms of SCI?

trauma to the spinal cord and tissue clearance

what are the stages of SCI?

acute, subacute and chronic stages

how long does the acute phase last?

seconds to hours

what does the acute phase of SCI consist of?

mechanical damage and the secondary injury cascade

mechanical damage (acute phase SCI)

immediate disruption of axons, blood vessels and cell membranes

secondary injury cascade (acute phase SCI)

includes vascular dysfunction, edema, ischemia, excitotoxicity, electrolyte shifts and free radical production leading to further tissue damage

vascular dysfunction

blood pouring out into the injury region causing an edema

edema

swelling caused by too much fluid trapped in the body’s tissues

ischemia

blood supply disrupted above and below spinal cord

excitotoxicity

too much glutamate pouring into the system which is toxic for most neurons cause it causes Ca2+ influx killing neurons cause they can depolarize

how long does the subacute phase of SCI last?

days to weeks

inflammation and immune response (subacute phase SCI)

recruitment of peripheral immune cells, activation of microglia and astrocytes which exacerbates or mitigates damage

cell death (subacute phase SCI)

necrosis in immediate injury site and apoptosis in adjacent areas

how long does the chronic phase of SCI last?

weeks to months

cystic cavitation and glial scar formation (chronic phase SCI)

potent inhibitors of axonal regeneration extending from head to tail of initial injury site

what is the byproduct of the autoimmune response in the chronic phase of SCI?

too much clearance of the tissue by the immune system

demyelination and Wallerian degeneration (chronic phase SCI)

causes further deterioration of the neural pathways

oxidative stress

causes ischemia

inflammation

causes immune cell infiltration, resident microglial activation and too much tissue clearance causing irreversible functional loss

cell death

causes neuronal and axonal dieback and necessary clearance of injured cells

what must we understand about SCI to come up with an intervention for it?

understand its cellular and molecular mechanisms that make up disease progression

RNA sequencing

a type of next gen sequencing that enables the comprehensive study of the transcriptome, the complete set of the RNA transcripts produced by the genome at any one time so looks at whole profile

what are the advantages of RNA sequencing?

can look at a cell’s response to treatment/stress and also cellular development and maturation

how do we now the molecular atlas of the tissue of interest and how it changes after disease?

by using next generation sequencing of the RNA landscape/transcriptome by using single cell, single nucleus and spatial sequencing

proteomics

high throughput analysis of protein molecules

epigenomics

describes changes int he regulation of the activities that act without or independently of changes in gene sequences (post translational modifications

what does bulk RNA sequencing sequence?

the whole tissue

what are the pros of bulk RNA sequencing?

its inexpensive

what are the cons of bulk RNA sequencing?

its not a single cell resolution so you have no idea where the transcript is coming from, its biased by abundance and you don’t know spatial position

what does single nucleus RNA sequencing sequence?

sequences individual nuclei

what are the pros of single nucleus RNA sequencing?

you get single cell resolution and its not limited by shape

what are the cons of single nucleus RNA sequencing?

its expensive, the nuclei doesn’t have that much RNA so you’re at a higher risk of not finding the target transcript and you don’t have spatial position information

what does single cell RNA sequencing sequence?

sequences individual cells

what are the pros of single cell RNA sequencing?

single cell resolution and the cytosolic RNA is abundant so you have a good chance of finding the target transcript

what are the cons of single cell RNA sequencing?

its expensive, biased by shape and you get no spatial position information

what does spatial RNA sequencing sequence?

single cell resolution areas of a tissue slice

what are the pros of spatial RNA sequencing?

you get single cell resolution and spatial position so its regionally specific

what are the cons of spatial sequencing?

its very expensive and the tissue must be very thin for it to work

luxol fast blue

chemically stains myelin

why are dyes not good for cells?

they combine too many parts of the spinal cord so you cant see any cellular changes using dye

immunohistochemistry

technique used for the localization of specific antigens (proteins) within cells and tissues through antigen-antibody interactions

in situ hybridization

used for looking at proteins

how is IHC created?

by immunizing various animals with an antigen and purifying their response to this antigen

antigen-antibody interaction (IHC)

utilizes the specific binding between an antibody and its antigen (what we want to visualize)

detection (IHC)

the antigen-antibody complex is visualized using various labels, usually fluorophores

STORM

stochastic optical reconstruction microscopy is a type of super resolution imaging technique that allows us to view biological structures at the nanoscale level by overcoming the diffraction issue

what is diffraction and why is it an issue in regular fluorescence microscopy?

it is the wave nature of light that limits resolution causing a blooming effect so we aren’t able to tell what the center of the image or how many molecules are actually contributing to the image

how does traditional fluorescence microscopy work?

all fluorophores are being excited at once so you cant tell at a single molecule resolution

how does STORM work?

the secondary antibodies have 2 fluorophores that are differentially excited so if you excite the red one then the blue one a nanometer apart from each other you can calculate the center between the blinks to localize at a nanometer resolution

what is expansion microscopy?

an alternative to STORM that allows for super resolution imaging on conventional microscopes by physically expanding the biological specimen

what are the steps of expansion microscopy?

sample labelling, embedding, homogenization and swelling

sample labelling + embedding (ExM)

label proteins/cells of interest with fluorophores and then the specimens are embedded in a dense cross linked polyelectrolyte gel

homogenization (ExM)

cytoskeleton of the specimen is digested with enzymes that allow for expansion

swelling (ExM)

the gel specimen composite is exposed to water, causing it to swell isotopically effectively magnifying the specimen so everything is blown up to scale but stays in the same location

why would someone use ExM over STORM?

it is cheaper but still provides the same level of resolution

ligt sheet and optical clearing

cells are labelled and digestion occurs to make the tissue transparent and imaging happens layer by layer then reconstructed so that you can sample fragile structures without having to cut into them

what is the role of pericytes?

important in maintaining the integrity and function of the spinal cord barrier

how would pericytes be studied after spinal cord injury?

they would be mobilized and activated to produce a collagen/fibronectin scar that surrounds and fills the lesion site then the scar limits lesional expansion while paradoxically blocking axonal growth and regeneration

what is the role of astrocytes?

they maintain the balance of neurotransmitters in the synapse

what happens to astrocytes after SCI?

they change their molecular signature and behavior through astrogliosis, they then become larger down regulating glutamate transporters and upregulating production of ECM molecules, this causes glutamate buildup in the synapse resulting in excessive Ca2+ influx

astrogliosis

abnormal increase in the number of astrocytes due to the destruction of nearby CNS neurons by upregulating phosphorylation of Stat3

how does glutamate build up in the synapse and excess Ca2+ influx occur?

by Ca2+Na+ permeable NDMA receptors causing neuronal death due to excitotoxicity

HIV (human immunodeficiency virus)

RNA virus that attacks the body’s immune system,

AIDS (acquired immunodeficiency syndrome)

chronic life threatening condition that occurs when HIV goes untreated

initial infection and reverse transcription (HIV)

virus enters the body infecting CD4+T cells, macrophages and dendritic cells, fuses w/host cell membrane and viral RNA is reverse transcribed into DNA using virus’ reverse transcriptase enzyme so copy of virus genome is now in the host

integration and production of viral components (HIV)

virus’ integrase enzyme so viral DNA is now integrated into the host genome and then HIV uses host’s cellular machinery (DNA pol + RNA pol) to replicate via transcription and translation to produce new viral RNA and protein, they assemble and bud off host cell into mature particles

what are the reasons why viruses aren’t alive?

they lack cell structure, they don’t metabolize so they cant generate energy to produce things, they cant reproduce independently cause they depend on host cells and machinery to do so and they lack homeostasis so they cant regulate their internal temperature

virus first hypothesis

suggests viruses predate cellular life, evolving from replicating genetic elements

cellular origin theory

proposes viruses evolved from cellular DNA/RNA fragments that gained independence

reductive evolution

implies viruses came from more complex life forms losing non essential life forms over time

co evolution theory

suggests viruses and cells evolved concurrently from early protein nucleic acid components

chimeric origin

indicates viruses formed from genetic material of different origins

how does AIDS evolve?

HIV virus infection disrupting immune function, CD4+T cell depletes weakening immunity then immune response fails leaving room for opportunistic infections to manifest that patient cant combat like tuberculosis and pneumonia

HIV diversity

HIV has insane mutation/variation ability that one patient 6 yrs after infection’s variation in the virus is equivalent to the global variation in the HA gene of seasonal influenza A H3N2 in a year

what is the zoo problem?

the fact that we have to use a different species for each antigen because for instance if you have a Mouse antigen X then a secondary antibody is made by using a goat so it only recognizes the mouse antibody by tagging with a green fluorophore if you use the same animal for antibody against antigen Y too you cant tell if you tagged antigen X or or Y and so the issue is what do we do to resolve this problem when we run out of different species to use

how do we bypass the zoo problem?

by using a directly conjugate primary antibody or a biotinylated antibody

how does a directly conjugated primary antibody solve the zoo problem?

if the primary antibody mouse anti-X is directly conjugated to a fluorophore this means that there is no need for it to bind to a secondary antibody + adding a wash step since all secondary are ligated to the primary will make sure there is no floating secondary antibody so no new interaction between secondary antibody goat-anti-mouse IgG (green) with mouse anti W since its directly conjugated to a red fluorophore and avoids the need to use another species

how does a biotinylated primary antibody solve the zoo problem?

since biotin is strongly chemically linked to avidin or streptavidin you could conjugate a fluorophore to avidin or streptavidin and use that as a secondary antibody to interact with a biotinylated Anti -W per example making this interaction strong and species independent so you get a conjugated antibody and avoids need to use another antibody

what is a directly conjugated antibody?

a primary antibody that has a fluorophore attached to it already, which is good to use when its high in abundance

what is the point of creating a secondary antibody?

to amplify the fluorescent response of a low abundance primary antibody