MLSP 5511 Exam 1

Hematopoietic Stem Cell (HSC)

Precursor to ALL blood cells

Myeloid Progenitor Cells

-Erythrocytes

-Neutrophils

-Eosinophils

-Basophils

-Monocytes

-Macrophages

Lymphoid Progenitor Cells

-T lymphocytes

-B lymphocytes

-NK cells

How to collect HSC's

-Bone marrow aspiration

-Leukopheresis

-Cord blood collection

-Peripheral blood (using G-CSF)

Variolation

Exposing patients to smallpox lesions through the nostrils or under skin to induce immunity

Cross-Immunity

Jenner took cowpox, infected a child and induced immunity to smallpox

Miasma Theory

Theory that disease was caused by bad air emanating from rotting organic material

Herd Immunity

Majority of individuals in a population are vaccinated so that:

-Pathogen reservoir decreases

-Prevents spread of disease to the immunosupressed, elderly, and the young

Inoculation

Introduction of a pathogen into an individual to induce an immune response

Vaccination

Introduction of a weakened or modified pathogen into an individual to develop immunity

Innate Immunity

Immunity that is given to humans from birth

4 Types of Innate Immunity

-Anatomic (skin, mucous membranes)

-Physiologic (temp., low pH, chemical mediators)

-Phagocytic

-Inflammatory

Adaptive Immunity

Immunity to specific foreign microorganisms and molecules

4 Characteristics of Adaptive Immunity

-Antigenic specificity

-Diversity

-Immunologic memory

-Self/non-self recognition

3 Types of Adaptive Immunity

-Active (natural=response to infection ; artificial=response to vaccination)

-Passive (natural=antibody transfer across placenta ; artificial=antibody injection to help fight infection

-Adoptive (received from bone marrow transplants)

Immunogen

Substance that induces an immune response (proteins most potent, followed by polysaccharides)

Antigen

Substance that combines with final products of immune response (think ANTI-body GEN-erator)

Epitope

Immunologically active region of antigen

Immunogenicity Factors

-Molecule must be recognized as foreign

-Molecular size (most active immunogens have mass > 10,000 daltons)

-Chemical composition and heterogeneity (protein organization contribute to immunogenicity)

-Susceptibility to antigen processing & presentation (large molecules generally more immunogenic because they're easily phagocytized ; polymers of D-amino acids can't be processed)

-Immunogen dosage & route of administration (low or high dose can induce tolerance ; immunogen administered IV goes to spleen, immunogen administered subcutaneously goes to lymph nodes)

Adjuvant

Substance that is mixed with an antigen to enhance the immune response

Blood Cell Classification

Based on expression of CD markers on cell surface

T-Helper Cells Express What CD's?

CD4, CD2, CD3, CD5, CD28, CD45

T-Cytotoxic Cells Express What CD's?

CD8, CD2, CD3, CD5, CD28, CD45

Hematopoiesis

Formation of blood cells

Erythrocyte

-RBC

-Carry O2 around body

-Contain many surface molecules that act as antigens

Megakaryocyte

-Produce platelets

-Remain in bone marrow

Monocyte

-Enter the tissues & differentiate into macrophages or dendritic cells

-2-10% of circulating WBC's

Macrophage

-Inflammatory macrophages clear and process pathogen to present to T-helper cells

-Osteoclast=macrophage of the bone

-Microglial cell=macrophage of CNS

-Alveolar macrophage=macrophage of the lung

5 Phagocyte Types

-Neutrophils

-Monocytes

-Macrophages

-Dendritic Cells

-Mast Cells

4 Granulocyte Cell Types

-Neutrophil

-Eosinophil

-Basophil

-Mast cell

Neutrophil

-~50-70% of circulating WBC's

-Circulate 7-10 hours before entering tissue

-1st responders to inflammation

-Secrete antibacterial proteins from granules

-Main component of pus

-Phagocytic

Eosinophil

-1-3% of circulating WBC's

-Red/orange granules

-Bi-lobed nucleus

-Respond to parasitic organisms

-Phagocytic

Basophil

-<1% of circulating WBC's

-Dark blue/purple granules

-Granules contain histamine

-NON-phagocytic

Mast Cell

-Released from bone marrow as undifferentiated cell

-Mature inside the tissues

-Dark blue/purple granules

-Granules contain histamine & heparin

-Role in development of allergies

Dendritic Cell

-Long extensions that capture & process antigens (phagocytosis, receptor mediated, pinocytosis)

-Travel to lymph nodes, presents antigens to naïve T lymphocytes (initiates adaptive immune response)

2 Types of Lymphocytes

-B lymphocytes

-T lymphocytes

B Lymphocytes

-Mature in bone marrow

-Expresses antigen-binding receptor on membrane (aka membrane-bound antibody molecule)

-Naïve B lymphocyte encounters corresponding antigen & divides rapidly

-Differentiates into Memory B Lymphocyte or Effector B lymphocyte (aka plasma cell)

-Responsible for "humoral" immunity

Memory B Lymphocyte

-Longer life span than effector B lymphocyte

-Expresses membrane-bound antibody molecule

Effector B Lymphocyte

-Produce secreted antibody (major effector of humoral immunity)

-Only lives 1-2 weeks

-Produces 2,000 antibodies per second

T Lymphocyte

-Derived from bone marrow, but mature in thymus

-Expresses antigen-binding molecule on membrane (aka T-cell receptor) which recognize antigens bound to MHC compounds

-Responsible for "cell-mediated" immunity

NK Cell

-Similar to B & T lymphocytes

-Do NOT possess antigen-specific receptors

-Involved in innate immunity

-Kills tumor & virus-infected cells

Primary Lymphoid Organs

-Bone marrow

-Thymus

Secondary Lymphoid Organs

-Spleen

-Lymph nodes

-MALT

Tertiary Lymphoid Tissues

Sites of inflammation

Thymus

-Maturation site of T cells (binding too tightly to MHC molecules or not binding at all to MHC molecules will cause apoptosis)

-Immature T cells (aka thymocytes) and "nurse cells" found in cortex

-Mature T cells and Hassell's corpuscles found in medulla

Lymphatic System Circulation

-Lymphatic vessels facilitate migration of T cells, B cells, macrophages & dendritic cells through lymph nodes

-Carries lymph from tissues back to circulation

-Majority of lymph fluid drains into thoracic duct

High Endothelial Venules (HEV's)

-Naïve lymphocytes extravasate from blood into tissues through HEV's

-5 x 10^4 lymphocytes move through a single lymph node's HEV per second

Cell Adhesion Molecules required for extravasation

-Addressins on HEV

-L-selectin (CD62L) on lymphocyte

Extravasation process

-Rolling along endothelial cell (binding between leukocyte and endothelial cell)

-Activation (chemokines bind to receptors on leukocyte)

-Arrest/Adhesion (leukocyte integrins undergo conformational changes, allow for strong adhesion to Ig supergene family on endothelial cell ; endothelieum secretes autotaxin)

-Diapedesis (leukocyte squeezes through HEV into the tissue

Lymph Nodes

-Connected to lymph & blood vessels

-Contains stromal cells, lymphocytes, macrophages & dendritic cells

3 Regions of Lymph Nodes

-Outer cortex [composed of germinal centers/follicles (contains B cells, macrophages & dendritic cells)]

-Middle paracortex (contains T cells and dendritic cells)

-Medulla (contains macrophages, plasma cells & reticular cells)

Germinal Centers/Follicles

-Often found within lymph nodes

-Site of intense B cell proliferation (rapid reproduction) following interaction with specific antigen

-Ig class switching occurs

-Contain Effector B cell precursors and memory B cells (memory B cells require repeated stimulation from macrophages & dendritic cell in the node)

Spleen

-Lymphocytes in spleen respond to blood-borne antigens

-Divided into red & white pulp (red=specialized fibroblasts, reticular cells, reticular fibers, macrophages & senescent (damaged) RBC's ; white=follicular B cells & T cells in periarteriolar lymphoid sheaths (PALS)

MALT

-Mucosa associated lymphoid tissue

-Made up of Peyer's patches, tonsils and adenoids

-Also BALT, GALT & NALT (bronchus-associated, gut-associated & nasal-associated)

Microfold Cells (M Cells)

-Epithelial cells overlying Peyer's patches in ileum

-Small breaks in basement membrane of M cells allow lymphocytes, dendritic cells and macrophages access to antigens that have been endocytosed by the M cells

-Transport antigen to lymphocyte in GALT

4 Immunologically Privileged Sites

-Brain

-Eye

-Testis

-Uterus

Inflammation

-Innate response

-Primary process for how the body repairs tissue and defends against infection

-Caused by trauma, tissue death, infection or immune response

5 Cardinal Signs of Inflammation

-Redness (rubor)

-Swelling (tumor)

-Heat (calor)

-Pain (dolor)

-Function loss (functio laesa)

Cytokine

Proteins that relay signals between different cells and cell types

Interleukin

Low molecular-weight proteins secreted by immune cells to act on blood cells

Chemokine

Low molecular-weight protein that promotes chemotaxis (directs cells to inflammation site)

4 Initial Steps to Tissue Damage

-Damage releases cytokines, increase blood flow and permeability

-Influx of fluid and cells to the tissue

-Neutrophils & Other phagocytes migrate to inflammation site

-Phagocytes destroy bacteria & damage tissue

Inflammation Initiation Process

-Injured tissue releases inflammatory chemical signals (DAMP's, PAMP's or MAMP's) that are expressed on the membrane of injured tissue cell

-Initial vasoconstriction to prevent blood loss

-Vasodilation and increased permeability (caused by histamine) leads to edema of tissue

-Capillary endothelial cells express E-selectin that binds to SLe^x on neutrophil (slows neutrophil to roll along endothelial cell)

-LFA-1 & Mac-1 (integrins) are activated on neutrophil from IL-8, CXCL-1 & CXCL-2 (chemokines)

-Neutrophil binds to endothelial cell via ICAM

-Diapedesis through endothelial cell junctions

Histamine

-Released from mast cells of damaged tissue

-Causes vasodilation (resulting in neutrophils entering the tissue, redness and swelling)

Chemokines (e.g. IL-8, CXCL-1, CXCL-2, etc.)

Released from macrophages & dendritic cells

Early Chemical Mediator of Inflammation (Within Minutes)

Histamine

Fibroblasts

Form scar tissue (if damage is extensive)

Late Chemical Mediators of Inflammation (6-12 Hours)

-Fibrin-split products

-Bradykinin

-Cytokines

-Complement

Kinins

-Become activated from tissue damage

-Cause vasodilation and increased permeability

Fibrin

Deposited to wall of injured area from the rest of the body

Acute-Phase Proteins

-Plasma proteins that increase or decrease in concentration during tissue damage

-IL-6, IL-1, TNF-α, complement, fibrinogen (concentration increases by at least 25%)

-Albumin (concentration decreases by at least 25%)

6 Phases of Inflammation

-Damaged tissue releases IL-1, TNF-α, & histamine

-Vasoconstriction, followed by vasodilation

-Increased permeability leads to edema of tissue

-Infiltration of neutrophils into tissue

-Infiltration of lymphocytes & macrophages

-Resolution (if acute) or scarring (if chronic)

Acute Inflammation

-Neutrophils mobilized within 30-60 minutes

-# of neutrophils proportional to # of chemotactic factors

-Tissue resolution

Chronic Inflammation

-Prolonged inflammation

-Macrophages & Lymphocytes are primary cells

-T lymphocytes activate macrophages

-B lymphocytes produce antibodies

-Tissue damage & loss of function

Exudate

High-protein edema fluid containing immunoglobulin

Transudate

Low-protein edema fluid that increases hydrostatic pressure and causes heart failure

Pus

Exudate containing WBC's & dead debris caused by neutrophil migration & release of enzymes

Cellulitis

Spread out inflammation in tissues

Immunoprecipitation

When antibodies react with a multivalent antigen (e.g. ovalbumin), the cross-linked product precipitates out of the solution

Protein Electrophoresis

Tested serum from rabbits that had been immunized with ovalbumin

Gerald Edelman

Used ultracentrifugation, urea & mercaptoethanol to determine IgG structure (physical properties)

-Ultracentrifugation showed IgG was ~150,000 Daltons heavy

-7M urea unfolded proteins

-Mercaptoethanol broke disulfide bonds

-Produced 2 separate proteins (1 is ~50,000 Daltons, other is ~22,000 Daltons)

-IgG has structure of H2L2 (H=Heavy chain, L=Light chain)

Rodney Porter

Used papain & ion exchange chromatography to determine IgG structure (structure & functionality)

-Treat IgG with papain enzyme, get 3 different pieces of similar weight

-2 Fab (antibody)

-1 Fc (crystallizable)

Precipitation

Molecules falling out of solution due to antibody cross-linking with a soluble antigen (e.g. ovalbumin)

-Antibodies have 2 binding sites (multivalent=can bind to more than 1 antigen)

-Antigen has multiple epitopes

-Antigen-antibody complex so large, falls out of solution

Agglutination

Clumping of cells due to antibody molecules cross-linking

Opsonization

Prepares foreign particles to be phagocytized by phagocyte

-Antibodies act as opsonins (coat foreign particle)

-Phagocytes have receptors for Fc portion of antibody

-Antibody bound to antigen; phagocyte bound to antigen (easier for phagocyte to surround particle)

Complement

Series of components that, when activated, begin series of reactions that result in the formation of an apparatus that leads to cell membrane rupture and cell lysis

Antigenic Determinant (Epitope)

part of antigen recognized by an antibody

Affinity

Binding strength between 1 antibody site and 1 antigenic epitope (expressed as Ka)

Avidity

Sum strength of antigen-antibody binding sites when multiple copies of particular epitope on an antigen interact with multiple binding sites on antibody

Immune Study with Alfred Nisonhoff (Goats Injected with Rabbit Antibodies)

-Anti-Fab antibodies could bind to both heavy and light chains

-Anti-Fc antibodies could bind to only heavy chains

-Cleared IgG with pepsin and got 3 pieces (2 Fc' and 1 Fab2)

Amino Acid Structure

H2N-CHR-COOH

Glycine

R= -H

Alanine

R= -CH3

Cysteine

R= -CH2-SH (can form disulfide bonds)

Serine

R= -CH2-OH

Aspartic Acid

R= -CH2-COOH

Lysine

R= -CH2-CH2-CH2-CH2-NH2

Tyrosine

R= -CH2-benzene-OH

Primary Protein Structure

Amino acid sequence

Secondary Protein Structure

3D structure of alpha-helix and beta-pleated sheets