Bio Lab: Week 1

how else do we we equate the effiency of phagocytosis?

We actively count the number of beads.

Necrosis

When cells begin to lyse, the opposite of apoptosis

Macrophages

White blood cell which engulf and digest: cellular debris, foreign substances, microbes, and cancer cells in a process called phagocytosis.

Foam cell

a macrophage with lots of LDL, suitcase filled with cholesterol

Dexamethasone

control: glucorticoid enhance efferocytosis.

What are effectors? what are they that we will study?

drugs that can be used to enhance the efficieny of efferocytosis 1. Lipoxin A-4 and derivative 2. Resolvin D1

Deffective Efferocytosis

efferocytosis breakds down leads to build up of necrotic core

Lipoxin A4

enchance efferocytosis, effector, resolution of inflammation (Made from arachidonic acid)

Rac1

enchance rac enhance apoptosis, kinases which controls actin polymerization, you want to upregulate this to enhance movement.

Resolvin D1

enhance efferocytosis, effector, resolution of inflammation (Made from omega-3 fatty acid)

How can we go about fixing defective efferocytosis?

hopefully with Lapoxin A4 or Resolvin D1 the effectors to enchance efferocytosis.

RhoA

inhibits apoptosis. by stoping movement towards what needs to be engulfed.

Apoptosis induced by

oxidant stress, cytokines, unesterified cholesterole, oxLDL, Fas Ligan (Fas death Pathway), Endoplasmic Reticulum (ER) stress.

what is the main signal? what are the main signals?

phosphotidylserine, eat me signal, flips from inside face to outside face. and apoptosome forms, and cell blebs. 1 Phosphatidylserine externalization 2 DNA fragmentation 3 Apoptosome (caspase) formation 4 cell Blebs

what induces apoptosis?

1 Oxidant stress on endoplasmic reticulum upregulates unfolded protein response, if not fixed cell undergoes apoptosis. 2 Cytokines signal from outside cell 3 Unesterified cholesterol 4 ox-LDL 5 Fas ligand - Fas death pathway 6 Endoplasmic reticulum (ER) stress

what doe we use to measure? two things

1) BioTek Plate reader 2) pick 10 cells and count the number of beads in those cells.

What happens normally? What happens during defective efferocytosis?

1. build up of LDL macrophages eat and become foam cell 2. Macrophages cant eat anymore become apoptotic 3. secrete inflmmatory signals 4. ideal macrophage are recruited before proinflmmatory signals are sent out and stops inflmmation 4. non ideal: no additional macrophages come.. there is build up leading to necrotic core, i.e. defective efferocytosis 5. fibrous cap weakens 6. thrombus formation

What cells do we use in lab?

1. macrophages. 2. LDL/apototic cells representitive of beads.

What happens?

1. recruit monocytes 2. cross barrier 3. differentiat into monocytes 4. monocytes eat LDL 5. monocytes become foam cells

Atherosclerosis

Atherosclerosis, is a disease in which plaque builds up inside your arteries. It occurs when fat, cholesterol, and other substances build up in the walls of arteries and form hard structures called plaques. Over time, these plaques can block the arteries and cause problems throughout the body.

Role of Scavangers? what do they do? what are the two we will be looking at?

Cell surface proteins, transmembrane receptors, important for recognition of dead cells!. Expressed on cell surface, important for recognizeing phosphotidylserine and bridge molecules on apoptotic cell. MERTK - tyrosine kinase receptor CD36 - cluster of differentiation 36

Apoptosis limits lesion cellularity

Characterized by changes within a cell. 1. phosphotidylserine externalization (eat me signal) 2. DNA fragmentation 3. Apoptosome (caspase) formation 4. Blebs

Cytochalasin D

Control: inhibits actin polymerization; decreases phagocytosis

Phosphotidylserine

Eat me the signal, the lipid, that is found on the inside normally, and outside when the cell needs to be eaten.

What are we studying?

Efferocytosis - apoptotic cell clearance by phagocytes -Recognition/Signaling 1. can I recognize the dead cell 2. can i signal to macrphage that this is the cell I need to eat -Actin reorganization and polymerization - can the macrophage move in the proper direction and eat the dead cell -Investigate the role of effectors -formation of phagasome

What does fluoresence measure? and represent?

Fluoresence represents the the eaten beads, and the measurement equates to the effiency of phagocytosis.

Phagocytosis

Greek, meaning "to devour", is the process by which a cell—often a phagocyte —engulfs a solid particle to form an internal vesicle known as a phagosome

Role of bridging molecules? what do they do? what are the two we will look at?

Important for bridging recognition between apoptotic cell, and cell surface scavanger receptor! GAS6 - growth arrest specific 6 TSP-1 - thrombospondin-1

Efferocytosis

Latin for 'to take to the grave', 'to bury') is the process by which dying/dead cells (e.g. apoptotic or necrotic) are removed by phagocytic cells (like macrophages). "ability of phagocytes to engulf dead cells"

LDL (low density lipoproteins)

Low-density lipoprotein is one of the five major groups of lipoproteins. Lipoproteins are complex particles composed of multiple proteins which transport all fat molecules around the body within the water outside cells

Thrombus

Or colloquially a blood clot, is the final product of the blood coagulation step in hemostasis. Note, a thrombus is a solid or semi-solid mass.

Why do we use 96 well dish?

treat small populations, do many replicates, and quantitate data and hopefully get statistical significance

Apoptosis

Programmed Cell Death cause by internally mediated chemical pathways within the cell, Apoptosis is cell suicide, and it is planned, not accidental or traumatic like (necrosis) and the cell dies without interfering on the cell's neighbors.

Coronary artery aterosclerosis

Single largest killer in the US

What do we use fluorescence for?

we use it to quantitate the amount of phagocytosis.