FOM: week 6 Flashcards
What is a morula?
A morula is an early embryo that has approximately 16 spherical cells (blastomeres) and divides into inner and outer cells
Where do the first few cell divisions of the embryo occur?
The first cell divisions occur in the oviduct as the embryo travels to the uterus (usually takes 6-7 days for the zygote to travel to uterus)
What is the significance of the inner and outer cells of the morula?
After compaction, the morula divides into two layers of cells: inner gives rise to embryo, and outer gives rise to extraembryonic tissues (including the placenta)
What is compaction?
Compaction occurs when the blastomeres within the morula flatten into a tight ball and become polarized. Fluid fills the space where the non-condensed blastomeres used to be. The embryo is now called a blastocyst.
When and where does implanation occur?
Implanation involves the attachment and embedding of a zygote into the uterine lining. It usually occurs roughly 6-7 days after fertilization and is initiated by hatching. Implantation typically occurs in the posterior wall of the uterine cavity.
What is ectopic pregnancy?
Ectopic pregnancy is the implantation of an embryo in an atypical location.
What are two common sites of ectopic pregnancy?
- ampulla of oviduct 2. close to cervix – results in placenta previa which causes hemorrhaging and threatens the survival of the mother and fetus.
What is hatching and what does it do to the zona pellucida?
Hatching occurs about 6-7 days after fertilization and involves the degradation of the zona pellucida by hydrolytic enzymes released from the trophoblast cells of the embryo. Degrading the zona allows the syncytiotrophoblast cells to penetrate the uterine lining and implant themselves.
How does the bilaminar disc form?
The bilaminar disc forms from the inner cell mass of the blastocyst. Fluid appears between the inner cells and separates them from the trophoblast into two regions: epiblast and hypoblast.
What are epiblasts and what do they eventually become?
Epiblasts are tall columnar epithelia that hold the amniotic fluid. These eventually give rise to the embryo proper and some extraembryonic structures.
What are hypoblasts and what do they eventually become?
Hypoblasts are cuboidal epithelia that become the yolk sac. The yolk sac eventually gives rise to extraembryonic structures like the connecting stalk which becomes the umbilical cord.
What is gastrulation and when does it occur?
Gastrulation is termed as the migration of epiblast cells to form three distinct layers: ectoderm, mesoderm, and endoderm. This process occurs in the third week of development.
What is the primitive streak?
The primitive streak is a result of epiblast cells that are oriented rostral to caudal. Epiblast cells migrate ventrally through the primitive streak and form embryonic endoderm (most ventral cells) and mesoderm (cells that reside between epiblast and hypoblast cells).
What is the primitive node and what structure does it form?
The primitive node forms at the rostral end of the primitive streak and has epiblast cells that descend through it to form a thick cord of cells called the notochord.
What are the two main functions of the notochord?
- Lends mechanical support to embryo 2. serves as a powerful inductive force for subsequent cell differentiation (organizing center)
What major body structures result from ectoderm?
Epidermis, hair, nails, cutaneous and mammary glands; central and peripheral nervous system
What major body structures result from mesoderm?
Paraxial: form somites that become muscles of head, trunk, limbs, axial skeleton, dermis, connective tissue; Intermediate: Urogenital system, including gonads; Lateral: Serous membranes of pleura, pericardium, and peritoneum, connective tissue and muscle of viscera, heart, blood cells
What major body structures result from endoderm?
Epithelium of lung, bladder and gastrointestinal tract; glands associated with G.I. tract, including liver and pancreas
What is involved in the transformation of trophoblast cells?
At the time of attachment, the some trophoblast cells fuse and become syncytiotrophoblast cells while the cells that don’t fuse are termed cytotrophoblasts.
What are some functions of the syncytiotrphoblasts?
Syncytiotrophoblasts are involved in perforating uterine vasculature and glands to eventually form the placenta. They also secrete human chorionic gonadotropin (HCG) which signals to the ovaries to keep producing estrogen and progesterone which maintains the uterine lining for pregnancy. Syncytiotrophoblasts are also involved in making decidual cells that form a distinct layer around the fetus to protect it from the immune system of the mother.
Where does chorion fluid come from?
Chorion fluid is produced from the trophoblast cells and is the fluid that surround the amnion and yolk sacs.
How does the neural plate form and from what cells does it arise from?
The neural plate is formed from the epiblast cells directly above the notochord. The notochord secretes the hormone sonic hedgehog to stimulate epiblast cell growth to form the thickened neural plate.
How does the neural tube form?
The neural plate begins to buckle and pucker into neural folds. These folds eventually fuse and form the neural tube.
What are neural crest cells and what do they become eventually?
Neural crest cells are the cells at the top of the neural folds and become detached from the lateral border of the folds and migrate within the embryo. The neural crest cells eventually become spinal and autonomic ganglia, Schwann cells, meninges, adrenal medulla, and melanocytes.
What is an epithelial-to-mesenchymal transition (EMT)?
This is the process by which differentiated epithelial cells become undifferentiated mesenchymal cells (“stem cells”) and is one way cancer can result.
What is are mesenchymal-to-epithelial transitions?
These are the transitions where undifferentiated cells become differentiated cells. This is typical in development.
What are some molecular tests used for DNA?
Southern blotting, restriction enzyme digestion, conventional sequencing, PCR
Go review these DNA tests..
:)
What are some molecular tests used for mRNA?
Q-RT-PCR, Northern blotting, microarray analysis
go review these mRNA tests…
super
What are some molecular tests used for protein?
ELISA, Western blotting
Please review these protein tests as well.
Nice
What are some emerging tests in the molecular biology field?
Deep sequencing – aka “next generation”
Therapeutic antibodies
Interference RNA (RNAi, microRNA, siRNA)
go review these tests too
What is regulative development?
Regulative development involves blastomeres that initially have similar developmental potencies each capable of giving rise to a complete embryo. Differentiation results from environmental signals which adjust to various perturbations.
What is mosaic development?
Mosaic development involves cell fate that has already been assigned during cleavage and a strict development plan is already in place; removal of one or more cells results in an incomplete embryo
What are the three methods of monozygotic twin development?
After the two-cell stage of development the embryo can:
- separate into two cells
- remain one cell with two separate inner cell masses
- remain one cell with a semi-divided inner cell mass – this can result in conjoined twins
Timing of splitting determines which path the embryo will take.
Totipotent
can give rise to all embryonic and extra-embryonic cell types and structures
Pluripotent
can give rise to all embryonic cell types and structures
Multipotent
can give rise to multiple (but not all) cell types
Unipotent
can give rise to just one cell type
induction
Induction involves the ability of one cell (or some type of environmental signal) to influence the development of another cell.
Cells not born different but are influenced during development by environmental signals regulative development
morphogens
chemicals/signals that alter cell fate
These are what cause induction to occur…
There are many different patterns for induction signaling. What are they?
- Inductive signaling – affects one other cell
- Gradient signaling – affects closest cell the most and tapers off as cells become farther away
- Antagonist signaling – cells receive an induced signal and an inhibitory signal which mix and affect the cells differently
- Cascade signaling – inductive signal affects the closest cell which then sends out another signal to the next closest cell, so on and so forth
- Combinatorial signaling – two inductive cells send signals to the close cells – mix of signals
- Lateral signaling – cells that are next to each other signal to one another
What determines whether a cell turns into a trophoblast or a cell in the inner cell mass?
This process involves Hippo signaling:
Outer cells: Yap expressed which associates with Tead4 and acts as a transcription factor for TE-specific genes such as Cdx2
Inner cells: Hippo expressed which phosphorylates Yap blocking it from entering the nucleus, thus TE-specific genes are not transcribed
What determines whether a cell will turn into an epiblast or a hypoblast?
The inner cell mass cells maintain Hippo signaling and express Oct4, Nanog, and Gata to maintain pluripotency.
Inner cells can only express either Oct 4 and Nanor OR Gata.
Gata-positive cells segregate out to form hypoblasts.
What is involved in the determination of the dorsal-ventral axis?
The mechanism is not well understood. However, an example is involved with neural tube development:
- Notochord induces ventral floor plate by secreting sonic hedgehog (Shh)
- Overlying ectoderm (former neural plate) induces the dorsal roof plate by secreting BMP
What is involved in the determination of the left-right axis in the body?
The left-right axis is determined by the direction of ciliary movement in the primitive node (flow counterclockwise by beating in same direction). This results in expression of Nodal on left side of the embryo and not the right side. Nodal then stimulates itself and another protein, Lefty, which inhibits nodal (mainly on the right side of body). Nodal stimulates production of Pitx2 which modulates gene expression patterns associated with l-r asymmetry such as heart development on the left.
Dynein mutations found to inhibit ciliary movement which affects l-r development.
What is involved in the determination of the anterior-posterior axis in the body?
Hox genes, which encode transcription factors that make homeodomains (DNA binding motifs) are involved in ant-post axis formation. Retinoic acid (RA) has been shown to regulate Hox expression and results in cell that have a more posterior fate.
What are the five major signaling pathways involved in morphogenesis and their basic features?
- TGF pathway (includes TGF and BMP proteins)
- neuronal differentiation, osteogenesis, LR asym - Hedgehog pathway (Sonic hedgehog - Shh)
- neural fate along DV axis, AP digit identity - FGF pathway (includes many FGFs)
- eye formation - Wnt pathway (involves APC – cell cycle)
- hematopoiesis, neural crest - Notch pathway
- hematopoiesis, neural development, somitogenesis
What morphogenesis pathways are associated with EMTs?
TGF-B, FGF, Wnt, and Notch pathways
What morphogenesis pathways are associated with METs?
BMP
What are the two types of cell migration?
Collective and single cell migration
How are cadherins involved in cell migration and adhesion?
- Selective cell-cell adhesion is mediated by cadherins that are expressed in tissue-specific patterns
- Cadherins of a certain type associate to one another
What is chemotaxis?
It is the characteristic movement or orientation of an organism or cell along a chemical concentration gradient either toward or away from the chemical stimulus – important in embryonic development
ex. sperm towards egg for fertilization; migration of neurons and lymphocytes
Affinity
very tight interaction at one small region; description of selectivity and strength of interaction
Avidity
several interactions that resemble a zipper or velcro; produces high binding strength, does not lead to refined selectivity
PAMP
(Pathogen associated molecular patterns) – outside
molecular moieties that are absolutely required for pathogen survival
ex. endotoxin (triggers production of IL-1), flaggelin, dsRNA, peptidoglycan, terminal mannose (cleaved by neuraminidase) –> sialic acid
DAMP
(Damage associated molecular patterns) – inside
recognized by innate immunity, sends out a danger/damage signal
ex. heat shock proteins; uric acid crystals, heparin sulfate, hyaluronan fragments
negative selection
T-cells that recognize self MHC molecules (and peptides) with high affinity are deleted from the repertoire of cells
Occurs when double positive T cells bind to bone-marrow derived APC (macrophages and dendritic cells) expressing Class I or Class II MHC plus self peptides with a high enough affinity to receive an apoptosis signal
Happens after positive selection. Involved in T cell education.
positive selection
The positive selection process leaves only T cells that can bind MHC molecules alive and are able to mature further
Occurs when double positive T cells bind cortical epithelial cells expressing Class I or Class II MHC plus self peptides with a high enough affinity to get the survival signal.
Happens before negative selection. Involved in T cell education.
MHC I
(major histocompatibility complex I) found on nearly every nucleated cell of the body; function is to display fragments of proteins from within the cell to T cells; healthy cells will be ignored, while cells containing foreign proteins will be attacked by the immune system
MHC II
(major histocompatibility complex II) MHC IIs are a family of receptor molecules normally found only on antigen-presenting cells and lymphocytes. The antigens presented by class II peptides are derived from extracellular proteins
CD1
clusters of differentiation – phenotypic protein markers on T cell membranes at different stages of differentiation in the thymus
CD1 is related to the class I MHC molecules, and are involved in the presentation of lipid antigens to T cells
CD2 and 3 are retained at all times on peripheral T cells
Primary response for antibody production
Initially there is an increase in IgM response followed by and even greater IgG response
Secondary response for antibody production
After first infection, the response of IgM occurs but is lower than first insult, the total amount of antibody is drastically higher, and the amount of IgG is also greatly increased.
Dendritic cells
main function is to process antigen material and present it on the surface to other cells of the immune system; act as antigen-presenting cells
function as messengers between innate and adaptive immunity
Eosinophils
kill antibody-coated parasites through phagocytosis and extracellular degranulation
responds to allergies and helminthic parasites
Neutrophils
phagocytose targeted cells; have Fc and complement receptors; when cells die they form pus
CD4+ helper T cell
the T cell that reads the antigen and recruits the B cell to make antibody against it
CD8+ cytotoxic T cell
the T cell that attacks and kills the foreign element that has antibodies attached
Th0, Th1, Th2, Th17, and Treg cells
Th0, Th1, Th2, Th17 = T cells that differentiate in the thymus based on differences in the cytokines they secrete
Treg = T cells that can suppress other T cells and adaptive Treg cells that are active at the end of a normal immune response
Role of IFN gamma, TGF-B, IL4, and IL2 in antibody generation
- Th1 cells produce cytokines, such as interleukin-2 (IL-2), gamma interferon (IFN-γ), tumor necrosis factor beta, and lymphotoxin
- Th2 cells secrete IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13
Complement
associated with a group of proteins that attach to a foreign element and portions of the C# proteins are cleaved forming anaphlatoxin (C3a, C4a, C5a) while the remainder of the protein is (C3b, C4b, etc) are attached to the foreign element and attract phagocytic vesicles to come and kill the foreign element
Somatic hypermutation
a cellular mechanism by which the immune system adapts to the new foreign elements that confront it
Mantoux test and delayed type hypersensitivity
The Mantoux test is a type IV delayed hypersensitivity reaction that involves injection of either purified protein derivative (PPD) or old tuberculin (OT). A positive test results when the memory Th1 cells produce cytokines that attract macrophages and cause induration (hardening of skin) and erythema (red swelling).
memory
property of adaptive immunity and produced by immunization; mechanism unknown
Innate immunity
is genetically hardwired, rapid response, limited repertoire; complement
Effector cells that:
- kill by contact = cytotoxic T cells, NK cells, and eosinophils
- phagocytosis = neutrophils, eosinophils, and macrophages
Adaptive immunity
acquired immunity creates immunological memory after an initial response to a specific pathogen, leading to an enhanced response to subsequent encounters with that same pathogen
B-cells make antibodies
Passive immunity
immunity that was given to a person such as already made antibodies
i.e. anti-venom, maternal, therapeutic monoclonal antibodies
Active immunity
introduction of an antigen that the body then makes antibodies against
i.e. vaccines, infection response
specific binding
used to label a molecule or cell for removal; criteria for both strength of interaction and selectivity
affinity vs. avidity
mast cells
release granules containing histamine, heparin, and TNF alpha – inflammatory response
basophil
considered to regulate inflammatory processes, take resident in lymph nodes; have histamine granules
What are cytokines?
They are soluble glycoprotein mediators that are synthesized on demand, direct cellular differentiation of hematopoietic cells, commonly operate in sequential patterns such as cascades
Hypertrophy
Hypertrophy refers to the increase in the size of cells, resulting in increase in the size of the organ
Results from increased production of cellular proteins. Selective hypertrophy can occur at subcellular organelle level (SER).
Hyperplasia
Hyperplasia refers to the increase in the number of cells, resulting in an increase in the size of the organ.
Occurs if organ cells can divide. Hypertrophy and hyperplasia usually occur together.
Atrophy
Atrophy refers to the decrease in cell size and number, resulting in reduced size of a tissue or organ.
Common causes include: decreased workload, loss of innervation, diminished blood supply, and inadequate nutrition.
Results from decreased protein synthesis and increased degradation within cells; increased autophagy may be involved?
Metaplasia
Metaplasia refers to the reversible change in which one differentiated type of cell is replaced by another cell type.
Results from reprogramming stem cells present in normal tissue or reprogramming of undifferentiated mesenchymal cells.
Ex. Barrett esophagus leading to adenocarcinoma (columnar to squamous epithelial change)
What are the two cell death pathways?
Necrosis and apoptosis
How is necrosis characterized?
Changes in ultrastructure include:
• Plasma membrane alterations: blebbing, blunting of microvilli
• Mitochondrial changes: swelling, condensation
• Dilation of endoplasmic reticulum
• Nuclear alterations
What are the morphological changes in necrosis?
• Morphologic appearance of necrosis is due to denaturation of intracellular proteins and enzymatic digestion of lethally injured cells
Microscopic changes include:
- increased cytoplasmic eosinophila in tissue stains (loss of nucleus)
- myelin figures
- nuclear changes: Karyolysis, pyknosis (shrunken nucleus), karyorrhexis (pyknotic nucleus undergoes fragmentation)
Cell contents leak out and can be detected in the blood.
What are some morphologic features of apoptosis?
- condensation of nuclear chromatin
- blebbing and fragmentation of cell
- cell shrinkage
- phagocytosis, usually by macrophages
Review extrinsic and intrinsic pathways of apoptosis – good review for the final!!! :)
Super
What is coagulative necrosis?
architecture of dead tissue is preserved (infarct)
ex. kidney nuclei disappear
What is liquefactive necrosis?
o Digestion of the dead tissue results in liquid viscous mass
- typically seen in focal bacterial or occasionally fungal infections - the microbes stimulate the accumulation of neutrophils which liberate tissue destroying enzymes, resulting in creamy necrotic material filled with neutrophils (pus)
What is gangrenous necrosis?
o Clinical term, not specific pattern, usually applied to necrosis of a limb undergoing coagulative ischemic necrosis
Can be wet or dry based on production, or lack thereof, of pus, respectively. Wet gangrene is more progressive and deadly than dry. Dry can become wet though…
What is caseous necrosis?
It is a cheeselike, yellow-white necrosis associated with necrotizing granulomas, seen with tuberculosis and fungal infections
What is fat necrosis?
o Refers to focal areas of fat destruction
What is fibrinoid necrosis?
It is a pattern of necrosis seen in immune reactions involving vessels
What are the mechanisms of cell injury?
- Depletion of ATP
- Mitochondrial damage
- Influx of calcium and loss of calcium homeostasis
- Accumulation of oxygen-derived free radicals
- Defects in membrane permeability
- Damage to DNA and proteins
What are the causes and effects of ATP depletion in cell injury?
Causes: reduced blood supply of oxygen and nutrients, mitochondrial damage, exposure to certain toxins
Effects: failure Na/K ATPase (cell swelling), altered energy metabolism, calcium pump failure, misfolded proteins, reduced protein synthesis
What are the causes and effects of mitochondrial damage in cell injury?
Causes: oxygen deprivation, oxygen free radicals, increased cytosolic calcium
Effects: defects in mitochondrial membrane which leads to failure of ATP production, escape of cytochrome c can occur which activates apoptosis
What are the causes and effects of calcium influx and loss of calcium homeostasis in cell injury?
Causes: defect plasma membranes of SER, mitochondria
Effects: failure of ATP generation, activation of lytic enzymes (phospholipases, proteases, ATPases), induces apoptosis by activating caspases
What are the causes and effects of the accumulation of oxygen-derived free radicals in cell injury?
Causes: ROS accumulation leading to oxidative stress
Effects: oxidative stress has been implicated in cancer, aging, and degenerative diseases
Antioxidants include: vitamins E, A, and C; other enzymes
What are the causes and effects of defective membrane permeability in cell injury?
Causes: ROS, decreased phospholipid synthesis, increased phospholipid breakdown, cytoskeletal abnormalities (cell stretching)
Effects: mitochondrial membrane damage (decreased ATP, apoptosis), loss of osmotic balance and cellular contents, release of lytic enzymes due to lysosomal injury
What are the causes and effects of damage to DNA and proteins in cell injury?
Causes: genetic mutations, cell injury
Effects: enzymatic degradation of misfolded protein or genetic repair of DNA (if too severe, apoptosis is initiated)
What are four mechanisms of intracellular accumulations?
o Intracellular Accumulations: - Abnormal metabolism - Defect in protein folding, transport - Lack of enzyme - Ingestion of indigestible materials o Examples: fatty change of liver, accumulation of cholesterol/cholesterol esters (xanthomas), proteins accumulate (Russell bodies, Mallory bodies, etc.), glycogen storage diseases, exogenous (tattoo) and endogenous (hemosiderin granules – slide 65, melanin, lipofuscin – slide 64) pigments
What are the two types of pathologic calcification in the body?
Dystrophic and metastatic
What are some features of dystrophic calcification?
Dystrophic calcification can occur in areas of necrosis with deposition of crystalline calcium phosphate; can have heterotrophic bone formation
Ex. atherosclerosis, damaged heart valves, fat necrosis, TB
Note: serum levels of calcium are normal!!
What are some features of metastatic calcification?
Due to elevated serum calcium, there are calcium phosphate deposits in normal tissues (lung, kidneys, heart) and can lead to heterotrophic bone formation
Ex. aortic stenosis, leads to dystrophic calcification
What are some causes of hypercalcemia?
- Increased parathyroid hormone production (PTH)
- Destruction of bone tissue
- Renal failure (phosphate retention, secondary hyperparathyroidism)
- Vitamin D related disorders
What is cellular aging?
It is a result of progressive decline in cellular function and viability caused by genetic abnormalities and accumulation of sublethal cellular and molecular damage.
Often characterized by senescence, which is a fixed number of somatic cell divisions, and is through to be determined by telomere shortening.
What are the biomarkers for necrosis in the liver?
AST (asparatate aminotransferase) – elevation not specific for the liver
ALT (alanine aminotransferase) – primarily found in liver, better indicator of liver damage
What are the biomarkers for necrosis in the bile duct?
Increased presence of alkaline phosphatase (ALP) is consistent with bone or liver disease. Measuring gamma-glutamyl transferase (GGT) can assist in determining whether rise in ALP is due to bone or liver disease. GGT– liver.
What are the biomarkers for necrosis in cardiac tissue?
Eleveated cardiac troponin I reflects cardiac damage. So do CK-MB (creatine kinase-MB) and myoglobin, but these biomarkers are not as specific to cardiac tissue as troponin I.
What are two fundamental characteristics of stem cells?
- Stem cells are not terminally differentiated – can give rise to daughter cells that become terminally differentiated
- Stem cells can self-renew
What is the general fate of a stem cell?
Stem cell divides and becomes two stem cells, one will self-renew the stem cell pool (no loss) and the other will become a committed progenitor cell. The progenitor cells undergo successive division and are stimulated by certain molecules to become differentiated cells.
Common example = hemopoietic cells (NK, T, B dendritic cells, etc.)
What mechanisms protect stem cell DNA?
- use of progenitor cells as amplifying system keeps the number of stem cells low which reduces susceptibility to genetic damage
- stem cells retain their template DNA strands upon replication, daughter cells have replicated copies – ensures that replication errors would not affect stem cell DNA
What determines the potency of stem cells?
Potency is determined by epigenetics which involves modifications of DNA/chromatin by usually silencing gene activity through methylation. To regain potency, the changes are ‘reset’ through epigenetic reprogramming.
Embryonic stem cells are isolated from _______ _____ _______/ __________ and these stem cells are _____________.
inner cell mass/epiblast; pluripotent
Adult stem cells are involved in replacing and repairing tissues in a particular organ and are either ________________ or _____________.
multipotent, unipotent
What are mesenchymal stem cells?
a type of adult stem cell found in bone marrow (and other places too) that are multipotent and are able to give rise to bone, cartilage, muscle, and fat
What are iPS cells and how are they produced?
iPS cells are induced pluripotent cells that have been made by expressing certain sets of transcription factors (like Oct4 and Nanog) to revert a differentiated cell into a pluripotent cell
They are made by stimulating a differentiated cell to become an undifferentiated cell through successive treatment with different molecules. Use of miR-302 could improve the use of iPS cells.
Primordial germ cells give rise to gametes when stimulated. What kind of potency do PGCs have?
PGCs are pluripotent.
What is SCNT and what is the process involved?
SCNT = somatic cell nuclear transfer, aka cloning
Process: take an anucleated egg and inject a different nucleus into it to form a zygote which is stimulated to divide.
What is therapeutic cloning and how can it be used in medicine?
Therapeutic cloning involves the used of an embryo produced from SCNT that is not placed in a host mother, but is grown in culture to provide pluripotent cells for regenerative medicine.
Used in tissue repair, possibly a method for infertile couples to conceive a child (form of IVF)
How are Oct4 and Nanog related to potency?
o Oct4 and Nanog are transcription factors that influence the development of stem cells by ‘reprogramming’ them to become stem cells that are pluripotent (used in iPS technology)
- Oct4 and Nanog promote miR-302 expression, constituting a positive feedback loop that promotes pluripotency
What are two points in human life that global demethylation occurs?
- Primordial germ cells remove all epigenetic markings; re-imprinting occurs during spermatogenesis/oogenesis depending on sex of embryo
- Shortly after fertilization demethylation is incomplete, some methylation remains at imprinted regions – allows zygote to attain totipotency while allowing paternally and maternally derived genes to be differentially expressed (imprinting)
What are stem cells niches and what are some examples?
- Stem cell niches refers to the environment in which the stem cells resides and they allow for controlled stem cell proliferation and differentiation of progeny
- Niche cells produce paracrine factors that regulate stem cell proliferation and prevent differentiation, but once leave this environment the cells begin to differentiate
- Ex. Wnt signaling in crypts (BMP4), Connective tissue and dermal papillae, hemopoietic tissue (Kit ligand and stromal cell interaction)
What is regenerative medicine?
Regenerative medicine involves the potential to use stem cells to repair and renew the body.
What are some applications of regenerative medicine?
- Adult stem cells – tissue repair
- Embryonic stem cells
- iPS cells
- Therapeutic cloning – medicinal treatment
Go read through the Immunology Chronology Summary document
phew… that was long! Good work :)
What are the five cardinal signs of inflammation?
- redness (rubor)
- swelling (tumor)
- heat (calor)
- pain (dolor
- loss of function (floor) lol
List and describe the key stimuli for acute inflammation.
o Infections – receptors recognize microbes, triggering signaling pathways
o Tissue necrosis – ischemia, trauma, chemical/thermal injury results in release of molecules which elicit an inflammatory response
o Foreign bodies – splinters, sutures elicit inflammation and carry microbes
o Immune reactions – own immune system damages itself/self-tissue (hypersensitivity reactions)
List and describe the three major components of the acute inflammatory response.
o Alterations in vascular caliber that increase blood flow
o Structural changes in microvasculature that allow plasma proteins and leukocytes to leave the circulation
o Emigration of leukocytes from the microcirculation, accumulation at the site of injury, and removal of the offending agent
List and describe the three key steps involved in extravasation of neutrophils.
o Margination – neutrophil is a peripheral position
o Adhesion – neutrophil binds to selectins (rolling) and integrins (firm)
o Rolling – neutrophil makes contact with epithelium, but is allowed to move
o Diapedesis – adhere by virtue of integrins and are retained at site where they are bound, this is mediated by chemokines; cross epithelia with the aid of PECAM-1 (CD31)
o Chemotaxis – neutrophil is attracted to the site of injury through binding of chemotactic agent to its surface which induces actin polymerization resulting in movement
o Morphologic hallmark of acute inflammation is neutrophil emigration.
Discuss the mechanisms by which leukocytes recognize microbes and dead tissue.
o Leukocytes recognize microbes through binding of their surface receptors which activate various signals
o Examples of receptors include:
- Toll-like receptors – found on microbes (bacteria)
- GPCRs – recognize chemokines, prostaglandins, leukotrienes, and C5a
- Opsonins – recognize complement proteins, antibodies, and lectins (molecules that coat the offending agent); Fc (for immunoglobulins) and C3 (for complement proteins)
- Cytokine receptors – Interferon-gamma (IFNγ) is the major macrophage-activating cytokine (secreted by NK cells)
Discuss the mechanisms by which leukocytes remove offending agents.
o Offending agents are removed in a two-step process:
- Phagocytosis and engulfment – mannose receptors and various opsonins (complement and antibodies) allow binding and ingestion of microbes, leading to engulfment into phagosome, which then fuses with lysosome resulting in a phagolysosome.
- Killing and degradation – killing accomplished by ROS and NO generated within the phagolysosome through an oxidative burst. In neutrophils, bactericidal bleach is made to kill offending agent. Eosinophils contain basic protein which is cytotoxic to parasites.
Explain the key cell derived mediators of the inflammatory response.
- Histamine – (mast cell) vasodilation and increased vessel permeability
- Serotonin – (platelets) vasodilation and increased vessel permeability
- Prostaglandins – (mast, macrophages) involved in vascular response of inflammation and mediate systemic reaction, pain and fever
- Leukotrienes – (leukocytes) chemoattractant and vascular effects, vasoconstriction an bronchospasm
- PAF – (platelets, mast cells, leukocytes) vasoconstriction, bronchospasm, platelet aggregation
- ROS – (leukocytes) killing microbes, tissue damage
- NO – (endothelium, macrophages) vascular smooth muscle relaxation, killing microbes
- Cytokines – (macrophages, mast cells) fever/pain/anorexia/hypotension, shock
- Chemokines – (leukocytes, activated macrophages) chemotaxis, leukocyte activation
- Neuropeptides (substance P) – (leukocytes) inflammation response, pain
Explain the key protein derived mediators of the inflammatory response.
o Protein derived:
- Complement system (plasma, produced in liver) vasodilation, increased vascular permeability, smooth muscle contraction, pain
- Coagulation and kinin system (plasma, produced in liver) endothelial activation, leukocyte recruitment
List and describe the three outcomes of the acute inflammatory response.
o Complete resolution: removal of cellular debris and microbes by macrophages, resorption of edema fluid
o Healing by connective tissue replacement (fibrosis): collagen deposition occurs in the area of damage or exudate, converting it into a mass of fibrous tissue
o Progression of acute inflammatory response to chronic inflammation
List and describe the types of conditions or situations that give rise to chronic inflammation.
- Persistent infections: viral, mycobacterial, fungal; may get a granulomatous reaction
- Immune-mediated inflammatory disease: autoimmune diseases, allergic diseases
- Prolonged exposure to toxic agents, exogenous or endogenous: e.g. silicosis, atherosclerosis
List and describe the cell types and key functions of the cells involved in chronic inflammation.
• Macrophages – responsible for much of the tissue injury in chronic inflammation; disappear once stimulus is eliminated
o Two different pathways; 1) pro-inflammatory, phagocytic; 2) healing and anti-inflammatory effects
o Different activation occurs due to presence of different cytokines
• Lymphocytes – T and B cells can get into extracellular space; macrophage activate T cells which secrete chemokines to activate macrophages positive feedback loop
• Plasma cells – produce antibodies
• Eosinophils – mediated by IgE and parasitic infections; release granules which is toxic to parasites and causes mammalian epi cells to lyse –> tissue damage in allergies
• Mast cells – recognize foreign body and degranulate releasing histamine and prostaglandins (can result in anaphylactic shock)
Define a granuloma.
o Granuloma = a focus of chronic inflammation consisting of a microscopic aggregation of macrophages that are transformed into epithelial-like cells
List and describe the three types of granulomas found in tissues.
- Foreign body granulomas: see foreign material within histiocytes/giant cells, sometimes called “foreign body giant cell reaction” by pathologists
- Caseating granulomas: granulomas that induce cell mediated immune response with central necrosis; these are usually associated with infection (e.g. mycobacterial (TB), fungal infections)
- Non-caseating granulomas: granulomas that induce cell mediated immune response without central necrosis (e.g. Sarcoidosis, Crohn’s disease)
Explain the rational for the use of the sedimentation rate.
- This test is a nonspecific measure of inflammation. Acute phase proteins such as fibrinogen as well as immunoglobulins can bind to red blood cells (RBCs), causing them to stack (rouleaux). Stacked red blood cells will sediment out faster in a tube of blood than non-stacked RBCs. Values are expressed as mm/hour.
Explain the rational for the use of the C-reactive protein.
- This test is a sensitive but nonspecific indicator of acute injury, bacterial infection, or inflammation. For example, elevated levels are seen in patients with sepsis, acute appendicitis, pelvic inflammatory disease, and acute myocardial infarction. Elevated CRP is also a risk factor for cardiovascular disease. Values are measured in mg/dl.
Explain the rational for the use of the WBC count and differential.
o Leukocytosis refers to an increase in the number of circulating leukocytes (white blood cells, WBCs) in the peripheral blood. The type of WBC that is increased can be a clue to the underlying pathology. For example:
- Neutrophilia: acute bacterial infections, acute inflammation associated with tissue necrosis (e.g. acute myocardial infarct)
- Eosinophilia: allergic disorders, parasitic infections, drug reactions, certain malignancies
- Basophilia: rarely seen, may indicate a myeloproliferative disorder such as chronic myeloid leukemia
- Monocytosis: chronic infections, bacterial endocarditis, malaria, collagen vascular diseases, inflammatory bowel disease
- Lymphocytosis: viral infections, Bordetella pertussis infection, disorders associated with chronic immunologic stimulation
- Performing a “WBC differential” will tell you which type(s) of WBCs are increased. Find your favorite source for list of causes!
What are the two main systemic effects of inflammation.
Fever and existence of acute phase protein
Describe the systemic effect of fever in regards to inflammation.
Fever is produced in response to pyrogenes which stimulate leukocytes to release cytokines such as IL-1 and TNF. This results in increased activity of cyclooxygenases which convert AA to prostaglandins. In the hypothalamus, the prostaglandins stimulate neurotransmitter production which reset the temperature set point to a higher level. NSAIDs and aspirin lower temperature by inhibiting prostaglandin synthesis.
Describe the systemic effect of acute phase proteins in regards to inflammation.
Acute phase proteins = C-reactive protein, fibrinogen, and serum amyloid A (SAA) protein
Systemic effect: increased synthesis of these proteins in the liver results from the release of cytokines as part of inflammatory response. Prolonged production of SAA protein is associated with chronic inflammation.
There are other systemic effects of acute phase proteins. Describe these effects.
- Leukocytosis - inflammatory reactions, especially those produced by bacterial infections, can lead to elevation of the white blood cell count
- Other: decreased blood pressure with increased pulse rate, rigors, chills, anorexia, somnolence, malaise, profound life threatening hypotension, septic shock
Regulation
proliferation of cells and tissues following injury, which replaces lost structures. Occurs in tissues with high proliferative activity (have stem cells)
Repair
consists of the combination of regeneration and fibrosis which is a typical response following injury. Fibrosis refers to the deposition of collagen and is the predominant healing process that occurs when the ECM is damaged
Continuous dividing (labile tissues)
cells proliferate throughout life, replacing those that are destroyed; most of these tissues mature cells are derived from adult stem cells
- Ex. skin, vagina, oral cavity
Quiescent (stable tissues)
cells have low level of replication, but can undergo rapid division in response to stimuli; have capability of reconstituting tissue of origin
- Ex. liver, kidney, pancreas, smooth muscle
Nondividing (permanent tissues)
cells have left the cell cycle and cannot undergo mitotic division
- Ex. neurons, skeletal muscle, cardiac muscle
What is the goal of stem cell therapy?
o The goal of stem cell therapy is to repopulate damaged organs of a patient or to correct a genetic defect, using the cells of the same patient to avoid immunologic rejection.
What two main mechanisms stimulate cells to replicate?
- growth factors
2. ECM components
What are some features of growth factors?
- All growth factors function as ligands which bind to receptors on the target cell.
- These growth factors can have restricted or multiple cell targets, and may also promote cell survival, locomotion, contractility, differentiation, and angiogenesis.
- Derangements in the growth factor, growth factor receptor, and signaling pathways are associated with certain malignancies.
- Ex. EGF, TGF-a, HGF, VEGF, FGF, TGF-b, cytokines (TNF-a, IL-2)
What are some features of ECM components?
- The ECM regulates the growth, proliferation, movement, and differentiation of the cells that live within it, and is continuously being remodeled.
- ECM provides: mechanical support, control of cell growth, maintenance of cell differentiation, scaffolding for tissue renewal, establishment of microenvironment (basement membrane), storage/presentation of regulatory molecules
- Ex. fibrous proteins (collagen, elastic fibers), adhesive glycoproteins (cadherins, integrins, selectins), proteoglycans (GAGs, hyaluronan)
What are the three modes of signaling mechanisms in cell growth?
o Paracrine – cell secretes signal that acts on adjacent cells
o Autocrine – cell secretes a signal that acts on itself
o Endocrine – cell secretes a signal that goes into the blood stream and has its effect further downstream
What are the four types of receptors involved in signaling induction pathways?
- receptors with intrinsic tyrosine kinase activity
- receptors without intrinsic tyrosine kinase activity
- GPCRs
- Steroid receptors
What are some features of receptors with intrinsic tyrosine kinase activity?
- Growth factors: EGF, TGF-a, HGF, PDGF, VEGF, FGF, c-kit ligand, insulin
- Binding of ligand induces dimerization and autophosphorylation which activates transcription factors that stimulate cascades of growth factors, growth factor receptors, and proteins that control cell cycle entry
What are some features of receptors without intrinsic tyrosine kinase activity
- Ligands: cytokines (such as IL-2, IL-3), INF-a, etc.
- Transmit signal to nucleus by activating members of JAK family leading towards gene transcription cascades
What are some features of GPCRs?
- Ligands: chemokines, vasopressin, serotonin, histamine, epinephrine, glucagon, etc.
- Involves a G protein (a,b,g subunits) that transduce the signal once ligand is bound
What are some features of steroid receptors?
- Ligand: steroid hormones; thyroid hormone, vitamin D, etc.
- Receptor generally located inside the nucleus
- Activation causes binding to specific DNA sequences or binding to other transcription factors –> modify DNA transcription
What is the most important growth factor for fibrosis?
TGF-beta
What is the most important growth factor for angiogenesis?
VEGF
What are the functions of ECM?
- Mechanical support.
- Control of cell growth.
- Maintenance of cell differentiation.
- Scaffolding for tissue renewal: regeneration of a tissue will result in restitution of the normal structure only if the ECM is not damaged; disruption of the ECM leads to collagen deposition and scar formation.
- Establishment of tissue microenvironments (e.g. basement membranes)
- Storage and presentation of regulatory molecules
What are the two basic forms of ECM?
- Interstitial matrix: found in spaces between epithelial, endothelial, and smooth muscle cells, as well as in connective tissue; consists mostly of fibrillar and nonfibrillar collagen, elastin, fibronectin, proteoglycans, and hyaluronan.
- Basement membranes: associated with cell surfaces, and consist of nonfibrillar collagen (mostly type IV), laminin, heparin sulfate, and proteoglycans.
What is fibrosis?
deposition of collagen
What features are involved in repair of tissue through deposition of connective tissue (fibrosis)?
- Inflammation
- Angiogenesis
- Migration and proliferation of fibroblasts
- Fibrosis (scar formation)
- Connective tissue remodeling
In most healing processes, a combination of _________ and ____________ occurs.
repair (fibrosis), regeneration
The relative contributions of repair (fibrosis) and regeneration are influenced by?
1) proliferative capacity of the cells in the tissue,
2) integrity of the ECM
3) Resolution or chronicity of the injury and inflammation
What are the three stages of wound healing?
- inflammation
- proliferation
- maturation
What are some characteristics of inflammation?
- Alterations in vascular caliber that increase blood flow
- Structural changes in microvasculature that allow plasma proteins and leukocytes to leave the circulation
- Emigration of leukocytes from the microcirculation, accumulation at the site of injury, and removal of the offending agent
What are some characteristics of proliferation?
- Macrophages are key cellular component to tissue repair, clearing extracellular debris, fibrin, and other foreign material. They also promote angiogenesis and ECM deposition.
- Macrophages signal for fibroblasts to enter by releasing cytokines and various growth factors
- Collagen fibrils become more abundant, with TGF-b serving as the most important fibrogenic agent
What are some characteristics of maturation?
- Scar formation: leukocyte infiltrate, edema, and increased vascularity largely disappear, connective tissue scar is present, devoid of inflammatory infiltrates, and covered by an intact epidermis
- Wound contraction: mediated by myofibroblasts located at the edge of the wound that express smooth muscle alpha-actin and vimentin; helps to close the wound
- Connective tissue remodeling: replacement of granulation tissue by fibrous tissue (scar) involves ECM deposition
- Recovery of tensile strength: wound strength of an incisional wound is only 10% that of normal skin; after 3 months or so, wound strength reaches a plateau of about 70-80% of the tensile strength of unwounded skin
What are some systemic factors that influence wound healing?
• Nutrition: protein deficiency, deficiency in vitamin C impair healing
• Metabolic status (e.g. diabetes associated microangiopathy impairs healing)
o Diabetics with skin ulcers – deficiencies in microcirculation, have diabetic neuropathy (don’t realize that have wound…)
• Circulatory status (e.g. impaired blood supply due to arteriosclerosis or venous abnormalities impairs healing)
• Hormones such as glucocorticoids have anti-inflammatory effects that influence the inflammatory response, as well as inhibit collagen synthesis, resulting in impaired healing in acute wounds
What are some localized factors that influence wound healing?
o Infection (most important factor), delays healing o Mechanical factors can delay healing o Foreign bodies can delay healing o Size, location, and type of wound: wounds in richly vascularized areas heal faster than those in poorly vascularized areas. Small incisional wounds heal faster and with less scar formation than large excisional wounds or wounds caused by blunt trauma.
accuracy
ability of the test to actually measure what it claims to measure correctly
precision
ability of the test to reproduce the same result when repeated
sensitivity
- the probability that an individual with the disease will test positive
- high sensitivity will not miss many patients who have the disease (low false negative rate)
- If a highly sensitive test is negative, it is unlikely that the individual has the disease in question (SNOUT = sensitive test, when negative, rules out disease)
specificity
- the probability that an individual without the disease will test negative
- high specificity will infrequently identify patients as having a disease when they do not (low false positive rate)
- If a highly specific test is positive, it is quite likely that the individual has the disease in question (SPIN = specific test, when positive, rules in disease)
In some sequential testing strategies a _________ test is used first as a screening test to detect the possibility that disease may be present, followed by a more ________ test.
sensitive, specific
positive predictive value (PPV)
refers to the probability that a positive test correctly identifies an individual who actually has the disease
negative predictive value (NPV)
refers to the probability that a negative test correctly identifies an individual who does not have the disease
prevalence
percent of individuals with the disease in the population being tested
Why is prevalence the most important factor in determining the utility of a test?
Prevalence is crucial to provide context to the positive result. If the test is positive in the tested population, then it can be considered a positive result. If the test is positive in a non-tested population, then the test could be a false positive.
Draw out the aracidonic acid pathway including prostaglandins, thromboxanes, and leukotienes. Also, draw where the respective drug targets are located on the pathway.
Lipase – hydroxycortisone, prednisone, dexamethasone
COX – aspirin, NSAIDs (ibuprofen, acetaminophen, naproxen, celebrex, vioxx)
Block CysLT1 receptor – montelukast (Singulair), zafirlukast (Accolate)
The addition of sugars to molecules can change their ____________, __________, and __________.
solubility, polarity, excretability
Lactase is a dimer of?
galactosyltransferase and a-lactalbumin
What is the molecular physiology of jaundice?
o Decyclizing heme oxygenase cleaves heme from Hb to produce biliverdin (green)
- Requires NADPH and O2
o Biliverdin is reduced to bilirubin (yellow)
- Requires NADH
o Bilirubin:
- Not soluble so is bound to albumin for transport in the blood
- Is glycosylated by the addition of two glucuronates (from UDP glucuronate) – catalyzed by glucuronosyl transferase make bilirubin more soluble, thus able to excreted in bile or urine
o If bilirubin is in excess of glucuronate and it is systemic, it can result in jaundice:
- Rate of heme degradation exceeds capacity to excrete it, bilirubin will diffuse out of blood into tissues
What are some features of neonatal jaundice?
It occurs immediately after birth due to an increase in hemolysis and an immature glucuronate conjugating system.
What are some features of hemolytic jaundice?
It can occur if there is excessive red blood cell destruction.
• G6PDH, malaria, sickle cell anemia
What are some features of hepatocellular jaundice?
It can occur if the liver is not functioning, e.g. due to damage from excessive alcohol consumption
What are some features of obstructive jaundice?
It is caused by a disturbance in bile drainage, due to a gallstones or a tumor.
What are three types of glycosylated sugars?
- cerebrosides
- globosides
- gangliosides
What is a cerebroside?
Cerebrosides are composed of ceramide + glucose/galactose + hydroxyl-methyl group
What is a globoside?
Globosides have two or more sugars
What is a ganglioside?
Gangliosides have oligosaccharides and NANA
Lysosomal storage diseases result from deficiencies in carbohydrate enzymes. What are the three enzymes involved in lysosomal storage diseases?
- Hexoaminidase: A and B isoforms
o A: trimer of 1α and 2β subunits - ganglioside
o B: homotetramer of 4β sununits - globoside - b-glucosidase
- a-galactocerebrosidase
What enzyme is defective in Tay-sachs disease? What are some features of Tay-sachs disease?
There is a defect in the α subunit, thus affects hexoaminidase A ONLY!
Features: characterized by early onset mental retardation and motor impairment, caused by ganglioside accumulation in neuronal cells
What enzymes is defective in Sandhoff disease? What are some features of Sandhoff disease?
There is a defect in the β subunit, thus effects both hexoaminidase A and B!
Features: mental retardation and motor impairment (more severe than Tay-sachs), characterized by ganglioside and globoside accumulation in neuronal cells
What enzymes is defective in Gaucher disease? What are some features of Gaucher disease?
Enzyme: β-glucosidase
Features:
o results in accumulation of glucocerebreside in the brain, liver, bone marrow, and spleen
o treated with recombinant β-glucosidase (Imiglucerase)
What enzymes is defective in Fabry disease? What are some features of Fabry disease?
Enzyme: α-galactocerebrosidase A
Features:
o characterized by progressive renal, cardiovascular, and cerebrovascular failure
o caused by accumulation of glucose-galactose-globosides
o treated with recombinant enzyme replacement therapy (Agalsidase)
