Final Flashcards
What is immunology?
Study of hosts reactions to foreign substances that are introduced into the body
What is an antigen?
A substance that reacts with an antibodies or sensitized cells but may or may not be able to elicit an immune response
What is immunity?
Condition of being resistant to infection
Who is the father of immunology?
- Louise Pasteur
- he created the first attenuated vaccine
What is attenuation?
- makes pathogens
- takes place through heat, aging and chemicals
What is phagocytosis?
Cells that eat cells
What is humoral immunity?
- Protection from disease resulting from substances in the serum/plasma (antibodies and acute phase reactants)
What are the two types of the adaptive immune response?
- cellular
- humoral
What is the function of humoral immunity?
Involves production of antibodies by B cells and plasma cells
What are antibodies?
- serum proteins used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses
What is innate immunity?
An individual ability to resist infection by means of normally present body functions
What are characteristics of the innate immunity?
- nonspecific
- no memory
- immediate exposure
- same response for all pathogens
- influenced by nutrition, age, fatigue, stress, and genetic determinants
What is adaptive immunity?
Resistance that is characterized by specificity for each individuals pathogen
What are characteristics of adaptive immunity?
- delayed response
- stronger response
- specific
- has memory
What is the function of WBCs?
Defend against invasion by bacteria, virus, fungi, and other foreign substances
What are the 5 types of leukocytes?
- monocytes
- lymphocytes
- basophil
- eosinophil
- neutrophils
What WBC is part of the adaptive immunity?
Lymphocytes
What are the main cells found in tissues?
- dendritic cells
- macrophages
- mast cells
What are hematopoietic stem cells (HSC)?
All blood cell types arise from this cell
Where are WBC produced? How much?
- bone marrow
- one and a half billion daily
What two distinct types of precursors does HSC give rise to?
- common myeloid precursors (CMP)
- common lymphoid precursors (CLP)
What do CMPs do?
- give rise to WBC that participate in phagocytosis
- also known as myeloid line
What are characteristics of Neutrophils?
- life span in several days
- nucleus has 2-5 lobes (has lots of granules)
- 50-70% of WBC
- use wright stain
What is the main function of neutrophils?
Phagocytosis
What is diapedesis?
Movement through blood vessel wall
What are chemotaxins?
Chemical messengers that cause cells to migrate in particular direction
What are characteristics of eosinophils?
- 1-4% of WBC
- eosin stain
- bilobed/eccentrically located nucleus
What are the functions of eosinophils?
1) phagocytosis (not as efficient as neutrophils)
2) neutralize basophils and mast cells
3) kills parasites using cationic proteins
4) MOST IMPORTANT: regulation of adaptive immune response through cytokine release
What are characteristics of basophils?
- less than 1% of WBC
- smallest
- nucleus is bilobed, and has deep purple-blue granules (hard to see)
- life span is a few hours
What happens to basophils in the spleen?
Removed and destroyed by marcrophages
What are the 3 main functions of basophils?
1) induce and maintain allergic reactions
2) regulate some Th cell response
3) stimulate B-cells to produce IgE antibody
What is the function of histamine?
Contracts smooth muscle
What is the function of heparin?
Anticoagulant
What are characteristics of monocytes?
- Largest
- irregular horseshoe or folded nucleus (occupies half or more of cell)
- 2-10% of WBC
- 30 hour lifespan
- after 30 hours, travels to tissue and becomes macrophage
Why are neutrophils more efficient at phagocytosis than macrophages?
Macrophages are slower
Why are neutrophils more efficient at phagocytosis than eosinophils?
- less eosinophils
- eosinophils lack digestive enzymes
What is the lifespan of macrophages?
A few months
What are the functions of innate immunity?
1) phagocytosis
2) microbial killing
3) anti-tumor activity
4) intracellular parasite eradication
5) secretion of cell membrane
How do macrophages play an important role in the adaptive system?
By presenting phagocytosized antigens to T cells
Where are mast cells found?
- located in variety of tissues (resembles a basophil)
What are the functions of mast cells?
1)act to increase vascular permeability and increase blood flow to affected area
2) role in allergic reactions
3) APC
Describe the appearance of dendritic cells
Covered with long membraneous extensions that resemble nerve cell dendrites
What are dendritic cells?
- APC that links innate and adaptive immunity and are critical for the induction of immune response
- classified according to location
What is the most potent phagocytic cell?
Dendritic cells
What are characteristics of lymphocytes?
- 20-40% of WBC
- large, rounded and indented nucleus
- similar size to RBC
- has narrow ring
- chromatin stains deep blue
- sparse cytoplasm
Why are lymphocyte granules unique?
They arise from HSC
What are the 3 types of lymphocytes?
- T cells
- B cells
- innate lymphoid cells
What is the most prominent innate lymphoid cells?
NK cells
What % of lymphocytes are B cells?
10-20%
What % of lymphocytes are T cells?
61-80%
What % of lymphocytes are NK cells?
10-15%
What is cluster of differentiation?
Protein found of cells surfaces that can be used to identify specific cell types and stages of differentiation
What are the primary lymphoid organs
Bone marrow
Thymus
What is the largest tissue in the body?
Bone marrow
What is bone marrow the main source of?
HSC
What can HSC develop into?
- RBCs
- lymphocytes
- monocytes
- granulocytes
- platelets
What occurs when lymphocytes remain in the bone marrow?
Mature and become NK cells or B cells
What occurs when the lymphocytes travel to the thymus?
Mature and become T cells
Describe the thymus
Filled with epithelial cells that play central role in differentiation process
How long does it take T cells to mature?
3 weeks
What are mature T cells released from?
Medulla thymus
What are the secondary lymphoid organs?
- spleen
- lymph nodes
- CALT
- MALT
Where does main contact of foreign antigens occur?
- secondary lymphoid organs
- T cells and B cells meet here as well
Where do lymphocytes spend most of their time?
The tissues
How do lymphocytes travel to blood stream?
Via. Thoracic duct
What is the largest lymphatic vessel?
Thoracic duct
What is lymphopoiesis?
Multiplication of lymphocytes
Where is the spleen located?
- upper left quadrant
- below diaphragm
- largest secondary organ
What is the function of the spleen?
Removes old/damaged cells and foreign antigens from the blood
What is the function of red pulp?
- to destroy old RBCs, platelets, and pathogens
What is rich in red pulp?
Macrophages
What is % of red pulp found in the spleen?
50%
What % of white pulp is found in the spleen?
20%
What is the main function of lymph nodes?
- Filtration of interstitial fluid from around cells in tissues
- important because it allows contact between lymphocytes and foreign antigens that have penetrated tissue
What are the layers of lymph node tissues?
- outer cortex
- para cortex
- inner medulla
What is lymph fluid?
A filtrate of blood and arises from the passage of water and low molecular weight solutes out of blood vessels walls and into the interstitial spaces between cells
What cells could be found in a primary follicle of the lymph nodes?
- stimulated B cells
- macrophages
- follicular dendritic
What cells can be found in a secondary follicle of lymph nodes?
Antigen stimulated proliferating B cells
What is at the center of a secondary follicle of the lymph nodes called?
Germinal center
What happens when B cells come in contact with antigens?
Plasma cells and memory cells form
Where can T cells be found in the lymph nodes?
Paracortex
What is the main function of plasma cells?
Actively secrete antibodies
What is the main function of memory cells?
Can quickly change into plasma cells
Where can MALT be found?
- appendix
- GI tract
- ileum
- peyers patch
- respiratory tract
- tonsils
- urogenital tract
Why are macrophages and lymphocytes and localized in MALT?
Mucosal surface are main ports of entry for a foreign antigen
What cells cab be found in CALT?
- dendritic cells
- macrophages
- monocytes
What are innate phagocytic cells?
- dendritic cells
- macrophages
- monocytes
- neutrophils
Describe NK cells
- can kill virally infected or cancerous target cells without previous exposure to them
- first line of defense against virally infected cells
- no specificity
- enhanced by exposure to cytokines: IL-12, INF-alpha, and INF-beta
How are T cells different from NK cells?
- only NK cells are able to kill target cells without prior exposure to them
What is the most potent phagocytic cell?
Dendritic cells
What is the main function of T-cells?
- produce cytokines that regulate both innate and adaptive immunity
What blood cell kills parasite?
Eosinophils
What are the 2 systems of the innate immunity?
- external defense system
- internal defense system
Describe the external defense system of innate immunity
- contains chemical, physical, and biological factors that work together to prevent most infections agents from entering the body
Describe the internal defense system of the innate immunity
- triggered within minutes and clears invaders as quickly as possible
Describe the outer layer of the external defense barrier
- outer layer of skin is the epidermis and contains several layers of tightly packed epithelial cells
- proteins coated cell called keratin
What is the role of keratin in the epidermis?
Makes skin impermeable to most infectious agents
What is the layer under the epidermis?
Dermis
Describe dermis
- thicker than epidermis
- connective tissue with blood vessels, hair follicles, sebaceous glands, and WBC
What cells are found in the dermis?
- dendritic cells
- macrophages
- mast cells
Why does skin have several secretions?
Discourage growth of microorganisms
What is secreted on the skin to maintain pH balance? What is the pH?
- fatty acids and lactic acid
- they prevent growth of microorganisms
- ~5.6
what is psoriasis?
- produced by skin
- has antibacterial effects, especially for gram negative bacteria
What is the respiratory tracts external defense system?
- mucous secretion block adherence
- coughing and sneezing clear out pathogens
What is the genitourinary tracts external defense systems?
- flushing out urine
- acidity helps to remove many potential pathogens
- lactic acid of vagina maintains pH of 5
What is the external defense for the digestive tract?
-hydrochloric acid keeps pH as low as 1 to kill pathogens brought in by food and drink
What are the lysosomes role in the external defense system?
- attack cell wall of microorganisms, especially gram positive bacteria
What is microbiota?
A mix of bacteria that are normally found at species body sites and do not typically cause disease
Explain the internal defense system of innate immunity
- composed of both cells
- soluble factors that have specific and essential functions
What is the function of phagocytes?
- they engulf and destroy most foreign cells or particles that enter the body
What is the % of dendritic cells and macrophages found in tissues?
10-15%
What are the most important cells in pathogen recognition?
- dendritic cells
- macrophages
What is the main function of patter recognition receptors (PRRs)?
- able to distinguish pathogens from normally present molecules in the body by means of receptors
What happens when PRRs binds to pathogen?
1) phagocytic cells activate and are better able to engulf and kill organisms
2) activated cells secrete proinflammatory cytokines and chemokines
3) cytokines and chemokines also trigger adaptive immune response
Describe pathogen associated molecular patterns (PAMPs)?
Substances that allows PRRs to have ability to distinguish self from non-self
What TLRs are found in cytoplasm?
- TLR1
- TLR2
- TLR4
- TLR5
- TLR6
What TLRs are found in the endosomal compartment of cells?
- TLR3
- TLR7
- TLR8
- TLR9
What are c-lectin receptors (CLRs)?
- plasma membrane receptors that bind to Mannan and beta-glucans found in fungal cell walls
Describe serum amyloid A
- major protein who concentration can increase X1000 in response to infection/injury
- apolipoprotein synthesized in liver
- acts like cytokines, chemical messenger (activates monocytes and macrophages
Why would serum amyloid A be increased?
- chronic inflammation
- atherosclerosis
- cancer
Describe complement
- series of serum proteins that are normally present and contribute to inflammation
What are the functions of complement?
1) opsonization
2) chemotaxis
3) lysis of cells
What is elastase?
- an enzyme secreted by neutrophils during inflammation that can degrade elastin and collagen
What are the pro inflammatory cytokines?
- TNF- alpha
- interleukin-1beta
- interleukin-6
Describe haptoglobin
- alpha1-globulin
- binds irreversibly to free hemoglobin
- acts as antioxidant to protect against oxidative damage from free hemoglobin
Describe fibrinogen
- acute phase protein involved in coagulation pathway
- small portion cleaned by thrombin to form fibrils that create a fibrils clot
What do fibrin clots do?
- increases strength with wound and stimulate endothelial cell adhesion and proliferation
- creates barrier of microorganisms further into the body
Describe ceruloplasmin
- consists of a single polypeptide chain
- copper transporting protein in human plasma
- acts as enzyme, converting the toxic ferrous ion (Fe^2+) to non toxic ferric form ( Fe^3+)
What disease is characterized by depletion of ceruloplasmin?
Wilsons disease
What is inflammation?
Body’s overall reaction to injury or invasion by an infectious agent
What are the 4 cardinal signs of inflammation?
- erythema (redness)
- edema (swelling)
- heat
- pain
What is the acute inflammatory response?
- acts it combat the early stages of infection and also begins a process that repairs tissues damage
What cells are most active in phagocytosis?
- dendritic cells
- macrophages
- monocytes
- neutrophils
What are selectins?
- adhesion molecules on endothelial cells lining blood vessels
what are integrins?
- adhesion molecules on endothelial cell wall
What is chemotaxis?
- cells are attracted to site of inflammation by chemical substances such as soluble bacteria factors and acute phase reactants
What enhances the binding process during phagocytosis?
Opsonins
what are opsonins?
Serum proteins that attach to a foreign cell or pathogen and make it more susceptible to phagocytosis
What are phagosomes?
Pseudopodia fuse to completely enclose pathogen
What are phagolysosome?
Lysosomal granule fused to phagosome
What are the two processes that eliminate pathogens?
- oxygen-dependent pathway
- oxygen-independent pathway
Describe oxygen-dependent pathway
- increase in oxygen consumption (oxidative burst) occurs in cell as the pseudopodia enclose particles within vacuole
- generates considerable energy through oxidative metabolism
What is an important bacterial agent in oxygen dependent process?
Hydrogen peroxide
Describe oxygen-independent pathway
- defensins kill gram-negative bacteria, many fungi, and some viruses
What are defensins?
Small cationic proteins that cleave segments without benefits of oxygen
What is cytotoxicity?
- the degree to which a substance can cause damage to a cell
What are two main binding receptors for NK cells?
1) inhibitory receptors
2) activating receptors
What do inhibitory receptors do?
Deliver inhibitor signals
What do activating receptors do?
Delivers signals to activate cytotoxic mechanism
What are perforins?
Proteins that form channels in target cells
What are granzymes?
Packets of enzymes tat may enter through channels and mediate cell lysis
Describe antibody-dependent cellular cytotxocity
- NK cells recognize and lysis antibody-coated target cells
- binding occurs through surface receptors; CD16 and CD32, which bind to Fc portion of immunoglobulins
- destruction occurs outside of NK cells
- important contributor to anti-tumor activity
Describe innate lymphoid cells
- growing family of immune cells that develop from the common lymphoid progenitor but does not express markers of lymphocytes lineage
- primarily found in mucosal sites
What can measurements of CRP levels be used for?
- tracking progress of organ transplant
- monitoring drug therapy with anti-inflammatory agents
- determining active phases of RA
What are the functions of C-reactive protein?
1) opsonization
2) complement activation
What is the function of serum amyloid A?
Activates monocytes and macrophages
What is the function of alpha1-antitrypsin?
Proteases inhibitor
What is the function of fibrinogen?
Clot formation
What is the function of haptoglobin?
Binds hemoglobin
What is the function of ceruloplasmin?
Binds copper and oxidizes iron
What are the functions of complement C3?
1) opsonization
2) lysis
What causes increased vasodilation and vasopermability?
Inflammation
Are all immunogens antigens?
Yes
Are all antigens immunogens?
No
What is a distinct difference between immunogens and antigens?
Immunogens successfully stimulate an immune response
What is immunogenicity?
The ability of an antigen to stimulate an immune response
What factors affect the strength of immunogenicity?
1) macromolecule size
2) foreignness
3) ability to be processed and presented with MHC molecules
4) chemical composition
5) molecular complexity
What are the most effective immunogens?
- proteins (strongest)
- polysaccharides
Describe the structure of a proteins primary structure?
- made up of subunits known as amino acids that are covalently linked together in polypeptide chains of various links
describe the structure of proteins secondary structure
Interactions between amino acids within primary structure causes chain to band/kink/loop creating 3D shapes
What are the two common secondary structures found in proteins?
- alpha-helices
- beta-pleated sheets
Describe the alpha helices of the secondary structures of protein
- peptide chains that twist to form a signal
Describe the beta-pleated sheet secondary structure of protein
Chains of undulating zig zags that have a planar shape
Describe the tertiary stucture of proteins
Secondary folds upon themselves once again, bringing distant regions of Amino acid chains together and embodying the 3D orientation of the entire molecule
Describe the quaternary structure of proteins
Two or ore polypeptide chains come together, forming a multimeric unit
What are polysaccharides less immunogenic than proteins?
- their small size
- T cells do not recognize carbohydrates
What are not immunogenic by themselves?
- pure nucleic acids
- lipids
What is required for a substance to elicit an immune response?
- must be subject to antigen processing
What is antigen processing?
- involves enzymatic digestion to create small peptides that can be completed by MHC molecules for presentation to responsiveness of an specific antigen
What is a hapten?
- a substance that isn’t immunogenic by itself but is able to form a new antigenic determinant when combined with larger carrier molecule
Are haptens considered an antigen or an immunogen?
Antigen
What is an epitope?
- part of antigen that hosts immune system recognizes, eliciting the immune response to an invading pathogen
- specifically binds to the corresponding antigen receptor on the immune cell
What are the two types of epitopes that B cells recognize?
1) linear epitopes
2) conformation epitopes
describe linear epitopes
Consist of sequential amino acids on a single polypeptide chain
Describe conformational epitopes
- results from the folding of one or more polypeptide chains, bringing together amino acids that may be distant from each other so that they are recognized together
What is an autoantigen?
- antigens that belong to host
- does not evoke an immune response under normal circumstances
What happens if an immune response does occur to autoantigens?
May result in autoimmune disease
What is an alloantigen?
From other members of the hosts species and are capable of eliciting an immune response
When are alloantigens important to consider?
- tissue transplant
- blood transfusion
What is a heteroantigen?
From other speciesm such as other animals, plants or microorganisms
What is a heterophile antigen?
- heteroantigens that exist in unrelated plants or animals but are either identical or closely related in structures so that an antibody against either antigen will cross- react with each other
Describe current rapid screening tests for IM
- detect heterophil antibodies present in the serum of infected patients that cross react with horse or bovine RBCs antigens
where are MHC molecules found?
On all nucleated cells
What do MHC molecules play a pivotal role in?
- development both humoral and cell-mediated immunity
What is the main immune function of MHC molecules?
- bring antigens to cell surface for recognition by the T cells
- only when the antigens is combined with the MHC molecules does the T cell activation occur
What is B cell recognition?
Surface receptors bind to antigen directly
What is T cell recognition?
Requires antigens to be cradled within MHC molecules
Where are class I MHC molecule genes found?
3 loci’s: A, B, C
Where are class II MHC molecule genes found?
- D region: DR, DQ, and DP
Where are class III MHC molecules found?
Lies between the class I and class II regions on chromosome 6
describe class III MHC molecules
- are secreted protein that have an immune function, but they are not expressed on cell surfaces
What is codominant?
All alleles that an individual inherits code for products that are expressed on cells
What is a haplotype?
- group of alleles in an organism that are inherited together from a single parent
- one haplotype is inherited from each parent
Where cells are class II MHC molecules found in?
Primarily on antigen-presenting cells (APCs)
What are the most important class I MHC antigens to match for transplantation?
- HLA-A antigens
- HLA-B antigens
What are the major class II molecules?
- HLA-DP
- HLA-DQ
- HLA-DR
Describe antigen presentation
- peptide fragments derived from degraded proteins are transported to the plasma membrane, allowing recognition by T lymphocytes
What does the class I MHC molecules mainly present?
CD8 T cells (cytotoxic)
What does the class II MHC molecules mainly present?
CD4 Th cell
What is the function of class I MHC molecule?
Watchdogs of viral, tumor and intracellular bodies
What is the function of class II MHC molecule?
- help to mount an immune response to bacterial infections or other pathogens usually found outside of cells
Describe modern transplant HLA testing
- involves the use of molecular techniques to determine the MHC types of both donor and recipient
What could occur when an individual inherits certain HLA types?
May predispose individuals to the development of autoimmunity
What is rheumatoid arthritis?
Inflammation of multiple joints
Describe type I diabetes
Increase in blood glucose because of destruction of insulin-producing cells
What are two transporters associated with antigen processing?
TAP1
TAP2
What is immunogen?
A substance that reacts with an antibody or sensitized cell but always triggers an immune response
What is an adjuvant?
- substances delivered simultaneously with an antigen to enhance the immune response; used in vaccines
- also prevents antigen from diffusing more immune cells to the injection site of inoculation
What is an allele?
Alernate forms of a gene that code for a slightly different variety of the same product
Where are class II MHC molecules synthesized?
Rough endoplasmic reticulum
Describe helper T cells
- recognize antigen along with class II MHC protein
- orchestrate the adaptive immune response, influencing activities of other immune cells
- express CD4 receptors
What is the key portion of an antigen?
Epitope
How do adjuvants work?
Acts by activating innate immune cells
What is the most polymorphic gene system in humans?
HLA systems
What is the function of cytotoxic T cells?
To kill cells infected with intracellular pathogens, such as viruses, bacteria and cancerous cells
What is the function of Th cells?
- control the immune response through the secretion of cytokines or signaling molecules that allow communication between immune cells
What are the roles of antibodies in immunity?
1) labeling targets for ingestion by phagocytes
2) rendering viruses and toxins inert through neutralization
3) blocking adhesion of microbes to body tissues
Describe clonal expansion when infection occurs
Only lymphocytes responsive to epitopes found on/in the invading pathogens are activated and proliferate
What are effector cells?
Either T cells that secrete cytokines or cytotoxic OR B cells that produce antibodies
Describe memory cells
- enter quiescent state and become long-lived
- lie in wait for reinfection with same microbe
- when they encounter their specific epitope, they activate rapidly, resulting in faster response of greater magnitude that the primary response
Where do thymocytes enter the thymus?
Cortico-medullary junction
What are the stages of T cell differentiation?
- double negative stage
- double positive stage
- mature T cells
What are the 3 pairs that occur in CD3 complex?
- delta-epsilon
- gamma-epsilon
- tau-tau
What type of antigens is not recognized alone by T cell receptors?
Peptide antigens
Where does negative selection occur?
In the corticomedullary and medulla region of the thymus
When is a T cell considered mature?
When it exits the thymus
What is a naive T cells?
- mature T cells that have not yet encounter d the specific peptide epitope recognized by their TCR
What are all B cells derived from?
Hematopoietic stem cells
What is antigen-independent phase?
- first phase of B cell development in bone marrow that results in mature B-cells that have not been exposed to antigens
What are the subpopulations of antigen independent phase?
- pro-B cells
- pre-B cells
- immature B cells
- mature B cells
Describe the antigen dependent phase?
If B cells are stimulated by antigen, it undergoes transformation to a blast stage that forms memory cells and plasma cells
Describe Pro B cells
- stage in B-cell development in which rearrangement of the genes that code for the heavy-chain region of an antibody occurs
- B-cell progenitors receive signals from bone marrow stromal cell to cell contact as well as soluble cytokines
What is the most important event of Pro B cell phase?
Rearrangement of the B-cell receptors (BCR) genes
What do BCR and TCRs have in common?
1) composed of 2 different chains
2) have variable regions which determine their specificity
3) have constant regions, which allow for intracellular signaling activation of the lymphocytes expressing them
4) have a similar gene regions (V, D, J)
5) use similar mechanisms for gene rearrangement
What are the two chains in BCRs?
Light and heavy chains
What genes in BCR contain multiple V, D, and J segements?
Heavy chain genes
What needs to occur for a Pro B cell to progress to the next phase of differentiation?
At least one heavy chain gene must undergo successful rearrangement
Describe the Pre-B cell phase
- the stage of B cell development where the heavy chain part of the antibody molecule is present
What are the two types of light chains possessed by humans?
Kappa
Lambda
What is the first immunoglobulin produced?
IgM
What signifies the Pre-B cells entry into the next phase, immature B cells?
The appearance of a functional BCR on the B cell surface
What determines the antigen specificity of immature B cell and its IgM BCR?
The immunoglobulin variable region, found on both the light and heavy chains
What happens to prevent BCRs from responding to self-antigens?
Negative selection
What occurs when B cells reach maturity?
B-cells respond to binding of antigen to the BCR by activation, proliferation, and antibody production
What is the reaction of immature B cells when signaled?
Halting their development and undergoing apoptosis
Describe central tolerance
- elimination of B cells that bear self-reactive receptors
- it is estimated that more than 90% of B cells die in this manner
What surface markers begin to make an appearance during the immature B cell phase?
- IgM
- CD21
- CD40
- class II MHC
When is a B cell considered mature?
Expresses a functional IgM BCR, survives selection by not reacting to self antigens and begins to display certain B cell markers
What occurs once a B cell is considered mature?
- exit bone marrow and are carried in the blood to the spleen for the next stage of development
What are the two types of mature B cells that develop in the spleen?
- follicular B cells (majority)
- marginal-zone cells
Where can follicular cells be found?
Constantly recirculate between blood and secondary lymphoid organs in search of their specific antigens
Where can marginal-zone B cells be found?
Remain in spleen to respond quickly to blood-borne pathogens
Describe lymphoid follicles
Represent dense clusters of naiver B cells awaiting exposure to their specific antigens
Where are marginal-zone B cells located in the spleen?
Marginal sinus
What does a the presen se of both IgM and IgD on the cell membrane signify?
A mature B cell
What are plasma cells role?
A differentiated B cell that actively secretes antibodies
What is a common resident of bone marrow and the germinal centers of peripheral lymphoid organs?
Plasma cells`
What occurs when an infection occurs in the body tissues?
- macrophages and dendritic cells are among the first immune cells to respond
- APCs engulf pathogens at these distal sites of infection and carry associated antigen to local lymph nodes
What transforms a naive T cell to an activated T cells?
Combination of signals that arises when the TCR recognizes its specific peptide and CD28 is ligated
What are Th cells considered the most important cells of the adaptive response?
- driving activities of other immune cells that act directly fight infection
What are the most prominent subsets of Th cells?
Th1
Th2
Th17
What influences the type of Th cell subset that is produced after differentiation?
Influenced by the cytokines present during activation
Describe role of Th1
-produce IFN-gamma, IL-2, and TNF-alpha which protect cells against intracellular pathogens by activating cytotoxic lymphocytes and macrophages
Describe role of Th2
-essential role is to help B cells produce antibodies against extracellular pathogens and to generally regulate B cell activity
- produces: IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13
- also may play role in allergies
Describe the role of Th17
Produce cytokines IL-17 and IL-22 which lead to the recruitment of granulocytes in response to an extracellular bacterial infection damage
Describe T regulatory cells (Treg cells)?
- subset of T cell
- posses the CD4 and CD25 antigens
- play important role in suppressing the immune response to self-antigens and harmless antigens
How do Treg cells suppress the immune response to self and harmless antigens?
They proliferate of other T cell populations by secreting inhibitory cytokines
What are the two main distinct population created after cell division of Th cells?
1) most Th cells begin to secrete to cytokines and may travel to infected tissues where their activities are most needed
2) a small percentage of Th cells generated after activation will differentiate into memory cells
What are the two toxic substances found in cytotoxic T cell granules?
- perforins
- granzymes
How do T-dependent antigens get their name?
The follicular B cell response depends heavily on the activity of Tfh cells to promote an effective antibody response, antigens that provoke this type of response
How did T-independent antigens get their name?
Marginal-zone B cells don’t require the help of Tfh cells
T-dependent antigens are most likely what?
Proteins, only type of antigen that can stimulate a T cell response
What are the two possible fates of daughter B cells that are produced during proliferative phase of T dependent response?
1) remain in contact with T cells and differentiate into IgM secreting plasma cells
2) others form germinal centers within follicles and participate in a series of processes that enhance the antibody response through time
What are the three overlapping processes of the germinal center formation require?
1) immunoglobulin isotope switching
2) activity maturation
3) memory cell generation
Describe IgG
- predominant form of antibody found in blood
- found in intestines and the bodies secretion
- associated with allergies
What is isotope switching?
- under the direction of T cells, germinal center B cells can change which class of antibody they express
- also determines the class of antibody secreted once the B cell differentiates into a plasma cell
What is affinity maturation?
- immunoglobulin binds antigen with increasing strength (affinity) through the course of an immune response, resulting in the production of even more effective antibodies
- this is accomplished through somatic hypermutation
What is somatic hypermutations?
Appearance of mutations in immunoglobulin gene variable regions
What causes daughter cells to be produced with slightly different antigen-binding abilities
Somatic hypermutation
For each generation of daughter cells produced within the germinal center, there are 3 basic populations of cells formed. What are these cells?
1) plasma cells
2) memory cells
3) B cells hat remain in the germinal center to continue the process of affinity maturation
What occurs once a memory B cell is re-exposed to an antigen?
It can rapidly respond with the production of high affinity, class switched antibody
What are the Pro-B cells key CD markers?
CD10 and CD19
What are the Pre-B cell key CD markers>?
CD10
CD19
CD20
What are the immature B cell key CD markers?
CD10
CD19
CD20
CD21
CD40
What are the mature B cell key CD markers?
CD19
CD20
CD21
CD40
What are the Pro-B cell receptors?
None
What is the Pre-B cell receptor
Pre-BCR: immunoglobulin heavy chain and surrogate light chain
What is the immature B cell receptors?
Functional BCR: IgM heavy chains and kappa or lambda light chains
What is the mature B cell receptor?
Functional BCR: IgD or IgM gravy chains and kappa or lambda light chains
What is the double negative key CD marker?
CD3
What is the double negative key CD marker?
CD3
What is the double negative T cell receptors?
None
What are the double positive key CD markers?
CD3
CD4
CD8
What are the double positive T cell receptors?
TCR alpha
TCR beta
What are the single positive key CD markers?
CD3
CD4
CD8
What are the single positive T cell receptors?
TCR alpha
TCR beta
What is a characteristic of immune response to a T-independent antigen?
- antigen are often polysaccharides
How does the humoral immunity produce antibodies?
By plasma cells
What is negative selection?>
- process that takes place among surviving DP T cells in the corticomedullary region and the medulla of the thymus
- strong reactions with self-peptides other than MHC antigens triggers apoptosis
What are thymocytes?
Lymphocyte precursors in the thymus that are committed to becoming T cells
Describe immature B cells
A phase in the growth of B cells characterized by the appearance of complete IgM antibody molecules on the cell surface
What are variables?
- contained by both alpha and beta chains on TCR
- recognizes specific antigens
What is MHC restriction?
Selection of thymocytes that will only interact with the MHC antigens found on host cells
What is a surrogate light chain?
- consists of two short polypeptide chains that are noncovalently associated, along with two shorter chains, Ig-alpha and Ig-beta
What is allelic exclusions?
Selection of an allele on one chromosome only
What is clonal deletion?
Process of elimination of clones of cells that would be capable of an autoimmune responses
Describe Flow cytometry
Automated system for identifying cells based on the scattering of light as cells flow single file in a stream of fluid by a laser beam
What is positive selection?
allows only DP cells with functional TCR receptors to survive
What is a distinguishing feature of Pre-B cells?
Mu chains in the cytoplasm
What are the 3 characteristics of acute phase reactants?
1) rapid increase following infection
2) enhancement of phagocytosis
3) nonspecific indicators of inflammation
What are 3 characteristics of an immunogen?
1) large molecular weight
2) internal complexity
3) presence of numerous epitopes
Where are class II MHC ANTIGENS found
B cells
macrophage
What is an immunoglobulin?
Globular proteins (glycoproteins) that play a role in immunity
What is the composition of immunoglobulins?
- 86-98% polypeptides
- 2-14% carbohydrates
What percentage of plasma proteins are immunoglobulins?
20%
Describe serum protein electrophoresis procedure
1) serum is placed on a agarose gel
2) electric current applied to separate the proteins
At what pH does electrophoresis separates most serum proteins by size and change?
8.6
What do the results of electrophoresis appear as?
Five bands
What are the slowest moving protein?
Immunoglobulins
What band of electrophoresis contains the most antibody activity’?
Gamma band
What are the 5 classes of immunoglobulins?
IgG
IgM
IgD
IgE
IgA
Describe the two dimensional forms of IgG
- two large peptide chains (heavy chains, unique)
- two small peptide chains (light chains, kappa or lambda)
How do the kappa and lambda light chains differ?
- differ by just a few amino acid substitutions along their length
- no functional difference
What are heavy and light chains held together by?
- Disulfide bonds (s-s)
- hydrogen bonds
- hydrophobic forces
- electrostatic attractions
What is the structural unit of all the immunoglobulin classes?
Tetrapeptide structure
Where is the variable region of an immunoglobulin located?
The first 110 amino acids of the molecule at the amino terminal
Describe the variable region of an immunoglobulin
- unique to each other antibody
- allows the molecule to bind specifically to a particular antigen
Where is the constant region of the immunoglobulin located?
From amino acid 111 to the carboxyl terminal
Describe the constant region of an immunoglobulin
- are the same in each immunoglobulin class/subclass
- responsible for the biological functions that play a role in immune defense against an antigen
What are the three constant regions of an immunoglobulin?
CH1
CH2
CH3
What are CH2 and CH3 responsible for?
For binding to complement and Fc receptors on phagocytic cells
What are the prototypic enzymes?
Pepsin and papain
Describe Papain
- cleaves the IgG molecule below the set of the disulfide bonds that holds the two heavy chains together, resulting in formation of 3 fragments: 2 Fab fragments and Fc fragment
Describe Fab fragments
- each have one light chain and one half of a heavy chain
- located at amino-terminal end o the molecule
- both are identical
- have antigen-binding capability
Describe the Fc fragment
- consists of carboxyl-terminal halves of two heavy chain
- spontaneously crystallizes at 4 C
- does not bind antigen
- contain constant regions CH2 and CH3
Describe pepsin
- cleaves IgG at the carboxyl-terminal side of the inter chain disulfide bonds
- 2 fragments: F(ab’)2 fragment and Fc’
Describe the F(ab’)2 fragment
- contains all antigen binding ability in pepsin
Describe Fc’ (fragment of pepsin)
- in carboxyl-terminal portion of the molecule which is disintergrated into several smaller pieces
- has no biological activity
Describe the hinge region of an immunoglobulin
- segment of heavy chain located between the CH1 and CH2
- rich in hydrophobic residues
- high proline content that allows for flexibility
What is flexibility important in the hinge region of immunoglobulin?
- let’s two antigen binding sites operate independently and engage in angular motion relative to each other and to the Fc stem
- initiation of complement cascade
- binding to cells with specific receptors for the Fc portion of molecule
What immunoglobulins have a hinge region?
IgG
IgD
IgA
Why do IgM and IgE gave flexibility even though they do not have a hinge region?
CH2 are paired in such a way as to confer flexibility to the Fab arms
What do immunoglobulins contain>?
4 polypeptide chains
Carbohydrate portion
Where is the carbohydrate portion of immunoglobulins located?
Between the CH2 domains of the two heavy chains
What are the functions of the carbohydate portion of immunoglobulins?
1) increase solubility of immunoglobulin
2) provide protection against degradation of molecule
3) enhancing functional activity of Fc domain (most important)
What are the three antigenic determinants?
Isotype
Allotype
Idiotype
describe isotype
- unique amino acid sequences that are common to all immunoglobulins of given class/subclass
- identical in all individuals or given species and differ from one species to another
How can antibodies to human isotypes be prepared?
By immunizing animals with human serum
What are allotypes?
- minor variations of amino acid sequences that are present in some individuals of the same species but not others
What classes do allotypes occur in?
- four Ig classes
- one IgA subclass
- lambda light chain
Where are allotypes located in an immunoglobulin?
- found in the constant region
What are idiotypes?
- the variable portion of each immunoglobulin chain are unique to a specific-antibody molecule
- essential to the formation of the antigen binding site
Where are idiotypes located in the immunoglobulin?
At the amino-terminal ends of both heavy chains and light chains
What is the predominant immunoglobulin in humans?
IgG
What % of immunoglobulins are IgG?
70-75%
What immunoglobulin has the longest half life?
IgG
Describe structure of IgG
- monomer
- consists of one tetrapeptide unit
What are the 4 subclasses of IgG? What is their compositions?
IgG1: 66%
IgG2: 23%
IgG3: 7%
IgG4: 4%
What are IgG1 and IgG3 associated with?
Protein antigens
What are IgG2 and IgG4 associated with?
Polysaccharide antigens
What immunoglobulins can cross the placenta?
IgG (except IgG2)
What are the functions of IgG?
1) CH2 region is able to bind to complement
2) important mediator of opsonization
3) participates in antibody-dependent cellular cytotoxicity
4) ability to bind to bacterial toxins and viruses and neutralizes their activity
5) can cross placenta
6) agglutination reactions and precipitation takes place in vitro
What does activation of complement result in?
An enhanced inflammatory response and destruction of foreign cells such as bacteria
What is the most efficient subclass of IgG to bind completeIgM? Why?
IgG3: it has the largest hinge region and the largest number of interchain disulfide bonds
What is the 2nd most efficient subclass of IgG to bind complement
?
IgG1
What is opsonization?
The coating of a foreign antigen that leads to enhanced phagocytosis
Why are IgG1 and IgG3 particularly good opsonins?
They bind most strongly to Fc receptors
Why is passive transfer of maternal IgG to the fetus important?
Providing immunity to the newborn during its first few months of life, when its immune system is immature
Is IgG better at agglutination or precipitation? Why?
Precipitation because it involves small soluble particles, which are brought together more easily by the relatively small IgG molecule
What else is IgM called? Where does it get this name?
Macroglobulin because it is the largest of all the immunoglobulin classes
Describe the structure of IgM
- pentameter of five monomers held together by J chain (joining)
Describe J chain
- glycoprotein that holds together monomers
- cysteine residues form disulfide bonds that link the carboxyl-terminal ends of adjacent monomers in IgM and IgA
What does each monomer of IgM contain?
- Mu heavy chains and either kappa or lambda chains
- Mu heavy chains possess one more constant domain (CH4) adding to it large size
What % of immunoglobulins are IgM?
5-10%
What does IgM perform things more efficiently than IgG?
Because of IgMs multiple binding sites
What immunoglobulin is most efficient at triggering the classical pathway?
IgM because a single molecule can initiate the reaction when complement binds to two adjacent CH region
Where are IGN mainly found?
In the intravascular pool and not in other body fluids or tissues because of its large size
What immunoglobulin is the first to appear after antigenic stimulation?
IgM
What is the first immunoglobulin that appears in maturing infants?
IgM
How many binding sites does IgM have?
10
What % of immunoglobulins are IgA?
10-15%
Where does IgA migrate in electrophoresis?
Between the beta and gamma bands
What immunoglobulins have complement fixation?
IgG
IgM
Describe the basic structure of IgA
- alpha heavy chain
- contains one variable regions
- contains three constant regions
- dimer, two monomers held together by J chain
What are the two subclasses of IgA?
IgA1
IgA2
How do IgA1 and IgA2 differ?
By 22 amino acids, 13 of which are located in hinge region (deleted in IgA2)
Where are IgA2 found?
- secretions at mucosal surfaces
- along respiratory, urogenital, and intestinal mucosa
- appears in breast milk, saliva, tears, sweat, and colostrum
Where are IgA1 found?
In serum
What is the major role of serum IgA?
- anti-inflammatory agent
- down regulate IgG mediated phagocytosis, chemotaxis, bactericidal activity, and cytokine release
Why is the J chain essential for in IgA?
Essential for polymerization and secretion of IgA
Where are secretory IgA synthesized?
Plasma cells found mainly in mucosal-associated lymphoid tissue and is released dimeric form
Describe secretary component
- serves as specific receptors for IgA
- precursor is present on surface of epithelial cells
Where do plasma cells secret IgA>?
Subepithelial tissue
What happens once IgA bind to SC precursors?
- Transcytosis
- both of them are taken inside the cell and released at the opposite surface
What is the main function if secretory IgA?
- to patrol mucosal surfaces and act as a first line of defense by preventing farther into the body
What is the important role of IgA?
- neutralizing toxins produced by microorganisms and helps prevent bacterial and viral adherence to mucosal surfaces
What immunoglobulin complex is trapped by mucus and coughed/sneezed out to protect the respiratory system?
IgA complex
Where is IgA found in humans?
Breast milk
How does breastfeeding help to maintain the health of newborns?
Passively transferring antibodies
What cells posses specific receptors for IgA?
Macrophages
Monocytes
Neutrophils
What % of immunoglobulins are IgD?
0.001%
What is the half day of IgD?
1-3 days
What antibody migrates as fast as a gamma protein?
IgD
Describe hinge region of IgD
- unusually long
- 58 amino acids
Why is IgD more susceptible to proteolysis that any other antibody?
Because of its long hinge region, this may be the reason for its short half life
Where are most IgD found?
Surface of unstimulated B cell
What is the second antibody that appears during an immune response?
IgD
What are the roles of IgD?
- B cell maturation and differentiation
- not exactly understood
Why is IgD an ideal early responder?
High level of surface expression and its intrinsic flexibility
What is IgE best known for?
Very low concentration in serum and the fact that it has the ability to activate mast cells and basophils
What % of immunoglobulins are IgE?
0.0005%
Describe the IgE structure
- epsilon heavy chain is composed of one variables and four constant domains
- a single disulfide bond joins each epsilon chain to a light chain, and two disulfide bonds link the heavy chains to one another
Does IgE participate in complement fixation, agglutination, or opsonization?
None of them
Where can plasma cells that produce IgE be located?
Primarily in lungs and in the skin
Where can mast cells be located?
- mainly in the skin
- lining of the respiratory and alimentary tracts
What does the release of IgE mediators induce?
- type I hypersensitivity
- allergic reaction
What are typical reactions with allergic reactions?
- anaphylactic shock
- asthma
- diarrhea
- hay fever
- hives
- vomiting
Describe the role of IgE
Serve a protective role by triggering an acute inflammatory reaction that recruits neutrophils and eosinophils to the area to help destroy invading antigens that have penetrated IgA defenses
What occurs when the first time an individual is exposed to an antigen?
Primary antibody response
What are characteristics of primary antibody response?
- IgM shows up first
- lag phase between the encounter with the antigen and the production of detectable antibody
- low affinity
- amount of antibody produced are relatively low and decline during the span of a few weeks
Describe the lag phase of the primary antibody response
- lasts 4 and 7 days
-T and B cells are being activated to respond to the antigen by the T-dependent mechanisms of antibody production
What does the lag phase of the primary antibody response result in?
Results in the generation of antibody-secreting plasma cells
What do activated B cells from the primary response develop into?
- plasma cells
- expand into clones of memory cells (long lived)
Describe secondary antibody response?
If memory cells are exposed to the same antigen weeks, months, or even years later, they can rapidly differentiate into plasma cells and larger amounts of antibody are produced
What are characteristics of the secondary antibody production?
- shorter lag time: 1-2 days
- high affinity
- production of low levels of IgM that rapidly declibe
- higher levels of other immunoglobulin isotypes, mainly IgG levels decline slowy and persists for longer periods
Describe clonal selection hypothesis
- body has numerous close of lymphocytes, each possessing surface receptors with a unique specificity
- when body is exposed to antigen, the antigen selectively binds to receptors on cells to proliferate and mount antibody responses
When can a gene be transcribed and translated into functional antibody molecule?
Not until it undergoes rearrangement, assisted by special recombinase enzymes
What does gene rearrangement involve?
Cutting and splicing process that removes much of the intervening DNA, resulting in a functional gene that codes for a specific antibody
Where does rearrangement of the gene begin?
The heavy chains
What are the genes that code for variable region of the heavy chain divided into?
Divided into 3 groups;
- V genes (variable) —> 45 genes
- D genes (diversity) —> 23 genes
- J genes (joining) —> 6 genes
Where are all the V,D and J genes present?
The germ line DNA of a bone marrow stem cell
Joining of V, D, J and constant region of a heavy chain is a two step process. What are the steps?
1) In Pro-B cells, one D gene and one J gene are randomly chose and are joined by means of a recombines enzyme after the intervening DNA is deleted
2) in Pre-B cells, a V gene is joined to the DJ complex, resulting in a rearranged VDJ gene
What is allelic exclusion?
The genes on the second chromosome are not rearranged because the rearrangement of DNA on one chromosome 14 was successful the first time
What occurs is the first rearrangement of heavy chains is unsuccessful?
- rearrangement of the second set of genes on the other chromosome 14 occurs
- this mechanism maintains clonal specificity by ensuring each B cell only expresses a single antigen receptor
What happens after rearrangement of the genes of the heavy chains?
- the variable and constant regions are joined
- occurs at the RNA level, thus conserving the DNA of the constant regions
What are the markers of the Pre-B cells?
Mu heavy chains
What does the presence of both C”Mu” and C”delta” regions of heavy chains allow?
Allows for mRNA for IgD and IgM t be transcribed at the same time
When do light chains rearrangement occur?
After Mu chains appear
Describe VJ joining of light chains
- accomplished by cutting out one intervening DNA
- results in the V(k) and J(k) segments becoming permanently joined to one another on the rearranged chromosome
When does lambda chain synthesis occur in a light chain?
Only it a nonfunctioning gene product arises from Kappa rearrangement
What does the lambda locus consist of?
Approximately:
- 30 V(lambda) segments
- 4 J(lambda) segments
- 4 C(lambda) segments
If functional heavy and light chains are not produced but a B cell, what occurs?
Cell dies by apoptosis
Light chains are joined with Mu chains to form what?
- a complete IgM antibody
- first appears in immature B cells
What happens once IgM and IgD are present on the surface membrane?
The B cell is fully mature and capable of responding to antigen
What are essential for initiating the VDJ recombination during B cell maturation?
Recombinase enzymes
RAG-1
RAG-2
What is the role of RAG-1 and RAG-2?
Recognize specific recombination signal sequences in the DNA that flank all immunoglobulin gene segments
What is junctional diversity?
- V, D and J segments doesn’t always occur at a fixed position, so each sequence can vary by small number of nucleotides
- major contributor to diversity in the variable-region genes
Where do immunoglobulins get their variation diversity from?
1) junctional diversity
2) different heavy chains can combine to different light chains
3) somatic hypermutation
What is class switching?
A the immune response progresses, B cells, may become capable of producing antibody of another class
What is switch recombination?
- a portion of the constant region DNA is deleted and the remaining C(h) genes are placed adjacent to the variable to the variable region genes
- allows the same VDJ region to be coupled with a different C regions to produce-antibody of a different class but having identical specificity for antigen
What are monoclonal antibodies?
- derived from singe antibody-producing cell that has reproduced many times to form a clone
- all the cells in the clone are identical and the antibody produced is exactly the same
-directed agains a specific epitope
what is hybridoma?
- fusion of activated B cell and myeloma cell
- myeloma cell chosen lacks essential enzymes and cannot produce own antibodies
How does production of hybridomas begin?
By immunizing a mouse with the desired antigen
What is the traditional process of mouse antibody production?
1) immunize a mouse with a specific-antigen
2) harvest spleen cells from the mouse spleen
3) combine spleen cells with myeloma cells in the presence of PEG
4) select fused cells and screen for presence of desired antibody
5) grow positive cells in larger quantities
What was used before monoclonal antibodies?
- antibody reagent could only be produced by immunizing animals such as horses or goats with the desired antigen and isolating polyclonal antibodies from the animal serum
What are the primary advantages of the monoclonal antibody reagent?
- provide decreased lot-to-lot variaton
- increased specificity toward a single epitope, rather than multiple epitopes of an antigen
What is a major limitation of using mouse monoclonal antibody as therapeutic agents?
- highly immunogenic for humans, inducing the development of human anti-mouse antibodies (HAMAs) that can cause severe hypersensitivity reactions
Where are isotypes located?
Constant region
Where are allotypes located?
In the constant regions of the IgG subclasses, one IgA subclass, and light chain
Where are idiotypes located?
Amino terminal regions of heavy and light chains
Where do antigens bind on immunoglobulins?
Hypervariable regions that contain CDRs
What are complement fixation functions?
1) enhances inflammatory response
2) uses positive feedback cycle to promote phagocytosis
3) enlists more defensive elements
Explain the process of complement fixation
1) antibodies bound to cells change shape and expose complement binding sites
2) this triggers complement fixation and cell lysis
Describe neutralization of antibody action
Antibodies bind to and block specific sites on viruses or exotoxins, thus preventing these antigens from binding to receptors on tissue cells
Describe agglutination of antibody action
- makes antigen-antibody complexes that are cross-linked, causing clumping
Describe precipitation of antibody action?
- soluble molecules are cross-linked into large insoluble complexes
What enhances phagocytosis at the antigen-antibody complex?
- neutralization
- agglutionation
- precipitation
- complement
What enhances inflammation of antigen-antibody complex? co
Complement
What are cytokines?
- chemical messengers that regulate immunity
- small proteins that bin to and activate receptors located on the target cells
Describe regulations of cytokines?
- Important in determining where cytokine action will be effective or inappropriate
- massive overproduction and dysregulation of cytokines can lead to yperctokinemia
- results in hypotension, fever, edema, multi-organ failure, or death
What is pleitotropy?
Single cytokine with many different actions
What is redundancy?
- occurs when different cytokines activate some of the same pathway and genes
- can be explained by the fact many cytokines share receptors subunits
How is cytokine activity classified?
According to the distance traveled between the producing cell and its targets cells
What is autocrine?
Cytokines that bind to receptors on the same cell from which they were secreted
What is a paracrine?
Cytokines that act one cells within tissue region surrounding their cellular source
What is an endocrine?
Some cytokines diffuse into the bloodstream, allowing them to influence cells far from the cells that produce them
What is synergistic?
Interactions that complement and enhance each other
What is antagonism?
If one cytokine counteracts the action of another
What is a cytokine cascade?
Many cytokines induce the production of additional cytokines by targeting cells
What is the antagonist of IL-4?
TNF-alpha
What synergies with IFN-gamma?
TNF-alpha
What is hypercytokinemia?
- also referred to as cytokine storm
- massive overproduction and dysregulation of cytokines can result in hyper stimulation of the immune response
What are the major proinflammatory cytokines?
- TNF-alpha
- IL-1
- IL-6
- IFN-gamma
What are the major anti-inflammatory cytokines?
- TGF-beta
- IL-10
- IL-13
- IL-35
What is the most important role of IL-1, IL-6, and TNF-alpha?
Recruiting effector cells, such as neutrophils and monocytes, into inflamed tissues
What are the best characterized cytokines of the IL-1 family?
IL-1alpha
IL-1beta
IL-1RA
Describe IL-1 alpha
- retained within the cell cytoplasm
- only released after cell death
- presence of IL-1alpha helps attract inflammatory cells to areas where cell and tissues are being damaged or killed
Describe IL-1beta
-mediates most paracrine (local) and hormonal (sytstemic) activity
- before secretion, IL-1beta must be cleaved intracellularly to an active form
Why is IL-1beta referred to as endogenous pyrogens?
It’s ability to induce fever
What purpose does a fever have?
1) inhibit of many bacteria and fungi.
2) increase the microbicidial activities of macrophages and neutrophils
3) contribute to feeling of discomfort and fatigue, compelling individuals to rest
What are the role of IL-1beta?
1) activation of phagocytosis
2) induce fever
3) production of acute-phase reactant
What are TNF products of?
Macrophages and lymphocytes
Describe CD40 ligand
Essential for signaling between T and B cells
What is the most important TNF of inflammation?
TNF-alpha
What is a major inducer of TNF-alpha production?
Microbial substances such as LPS
What is LPS?
A component of the cell wall of gram-negative bacteria
The activity of IL-6 is highly ________.
Pleiotropic
What are innate roles of IL-6?
- stimulates the production of acute-phase proteins by liver hepatocytes
What are the adaptive roles of IL-6?
- increases activation of B and T cells
- causes B cells to proliferate and differentiate into plasma cells
What are chemokines?
Subgroup of cytokines that influence the motility and migration of their tartlet cells
What are the 4 families of chemokines?
1) CXC
2) CC
3) C
4) CX3C
Describe CXC chemokines
Contain single amino acid between the first and second cysteines
Describe CC chemokines
The cysteines are found together, with no intervening amino acid
Describe C chemokines
Has only a single cysteine
Describe CX3C chemokines
Have 3 amino acids between the cysteines
What is the most important effect of chemokines?
- recruitment of leukocytes from the blood into infected or damaged tissues
What are interferons?
Soluble substances produced by virally infected cells that interfere with the ability or viruses to replicate by making host cells less hospitable to viral takeover
What are the 3 groups of IFNs?
Type I
Type II
Type III
What are the most important cytokines in response to viral infections?
IFN- alpha
IFN-beta
(Both type I)
What are the functions of type I IFN?`
1) interfere with viral replication
2) activate NK cells
3) enhances expression of class I MHC proteins on target cells
What is the most important innate defense mechanism against viral infection?
Combination of IFN-alpha and IFN-beta and NK cell killing
What secretes the majority of the cytokines in the adaptive immunity?
T cells, especially T helper Cells (CD4+)
What are the 4 sub populations of T helper cells?
Th1
Th2
Th17
Treg cells
Describe differentiation of Th cells
The cells undertake a process during which they are transformed into one of the four subclasses
What causes differentiation to the Th1 lineage?
Dendritic cell production of cytokine IL-12
What drives antibody mediated immunity?
IL-4
What arises in the presence of IL-23?
Th17 cells
What are produced in response to TGF-beta?
Treg cells
what is the hallmark of Th1 cells?
High level expression of the proliferative cytokine IL-2 and IFN-gamma, a type II IFN
What are the roles of Th1 cells?
1) expand to combat infection with viruses and intracellular bacteria
2) promote cell-mediated immunity with cytotoxic T cells, and activate macrophages
3) cause antigen-activated B cells to produce IgG1 and IgG3 antibodies capable of opsonization and fixing complement
What is another name for IL-2?
T-cell growth factor
What are the roles of IL-2?
1) drives proliferation and differentiation of both T and B cells
2) enhances the lytic activity of NK cells
3) causes naive Th to differentiate into Th1 cells
What is the primary cytokine of the Th1 response?
IFN-gamma
What are the roles of IFN-gamma?
1) influences expression of more than 200 target-cell genes
2) stimulates antigen presentation by class I and class II MHC
3) potent activator of macrophages
4) involved in regulation and activation of CD4+ Th1 cells, CD8+ cytotoxic T cells and NK cells
What are the most influential Th2 cytokines?
IL-4
IL-10
What is the IL-4 activity on naive T cells?
- turns on genes that generate Th2 cells and turns off genes that promote Th1 cells
What are Th2 cells responsible for regulating?
Allergies
Autoimmune disease
Parasites
What does IL-5 do along with IgE?
Drives differentiation and activation of eosinophils in both allergic immune response and responses to parasitic infection
What does IL-4 and IL-13 have in common?
- induce worm expulsion
- favor IgE class switching
How do IL-4 and IL-13 differ?
IL-13 plays anti-inflammatory role by inhibiting activation and cytokine secretion by monocytes
What are the roles of IL-10?
1) has anti-inflammatory and suppressive effects on Th1 cells
2) inhibits antigen presentation by macrophages and dendritic cells
3) down regulates immune response by counteracting IFN-gamma
What is the antagonist of IFN-gamma?
IL-10
How can Treg cells be identified?
Expression of CD4, CD25, and FoxP3
What are Treg cells essential?
For establishing peripheral tolerance to a wide variety of self-antigens and harmless antigens
What are the roles of Treg cells?
1) establish peripheral tolerance to self-antigens and harmless antigens
2) produce suppressive cytokines: TGF-beta and IL-10
3) down regulate immune response to prevent chronic inflammation (main)
4) may prevent tumor cells from attack by inhibiting cancer-fight cells
What does Th17 secrete?
IL-17 family cytokines
What cytokines play a role in finalizing the commitment to Th17?
IL-23
What are the roles of Th17?
1) host defense against bacterial and fungal infection at mucosal surfaces
2) secretion of IL-17
3) promote recruitment of neutrophils
4) promote release of antimicrobial peptides
5) when dysregulated, may promote pathogenesis of several inflammatory dieases
What are the APC cytokines?
IL-12
IL-23
What are the primary mediators of hematopoiesis called? Why?
- IL-3
- EPO
- G-CSF
- M-CSF
- GM-CSF
Why does IL-3 act one bone marrow stem cells?
To begin differentiation cycle
What happens if M-CSF is activated?
The cells become macrophages
What is the role of M-CSF?
Increases phagocytosis, chemotaxis, and additional cytokine production in monocytes and macrophages
What happens if G-CSF is activated?
Cells become neutrophils
What are the roles of G-CSF?
1) enhances function of mature neutrophils
2) affects the survival, proliferation, and differentiation of all cell types in neutrophil lineage
3) decreases IFN-gamma production
4) increases production IL-4 in T cells
5) mobilizes multi potent stem cells from the bone marrow
What does GM-CSF do?
Acts to drive differentiation toward other WBC types
What does EPO do?
Regulate RBC production in the bone marrow
What is EPO mainly produced by?
The kidneys
What is recombinant EPO?
- administered to improve RBC counts for individual with anemia and those with cancer
What is anti-cytokine therapy?
Aims to break vicious cycle of chronic inflammation by targeting the interactions between specific cytokines cytokines and their cognate receptors
Describe newer anti-cytokine agents
Greatly improved using recombinant DNA techniques to generate humanized monoclonal DNA antibodies that are much less immunogenic
What are 3 cytokine assay formats?
1) ELISAs
2) microbead assays
3) ELISpot assays
Describe ELISAs
- can detect many pro- and anti- inflammatory cytokines in one reaction
- allow for simultaneous detection of several cytokines from serum or plasma in single a test run
- has “spots”
Describe microbead assays
- allow for the simultaneous detection of multiple cytokines in a single tube
- each bead type has own fluorescent wavelength. When combined with fluorescent secondary antibody bound to a specific cytokine, allows for detection stop to 100 different analysis in one tube
Describe ELISpot
- similar to ELISA performed on in vitro activated peripheral WBC
- allow the detection and enumeration of individual cytokine-specific capture antibodies
What is the ELISpot procedure?
1) PBMCs are added to the wells of antibody-coated micro plate and treat with synthetic protein antigen
2) incubation period: Th1 cells secrete cytokines as they rest as the rest at the bottom of the microplate well
3) PBMCs and any soluble substances (cytokines) are washed from wells
4) detection antibody antibody specific to the target cytokine, is added to wells
What is the role of ELISpot detection antibody?
To produce a signal indicating where target cytokine is bound to capture antibody
What do the “spots” represent in ELISpot?
Tiny, elliptical silhouettes of captured IFN-gamma speckling plate bottom
What is PCR product?
Made using fluorescent-labeled primer and can be hybridized to either solid or liquid phase arrays
Describe the solid-phase array of PCR
- have up to 40,000 spots containing specific single-stranded DNA (ssDNA) oligonucleotides representing individual genes
- fluorescence of a spot indicates that gene was expressed in the cell and that cell was producing the cytokine
Describe liquid arrays of PCR
- uses same beads as microbeads arrays but have oligionucleotides on surfaces instead of antibodies. Show up to 100 different complementary DNAs to be identified
- combination of bead fluorescence and fluorescence of labeled cDNA produces an emission spectrum that identifies the cytokine gene that was expressed in cell
What are 4 approaches to anti-cytokine therapy?
1) disrupting interaction between cytokines and their cognate receptors
2) using monoclonal antibodies that function as cytokines agonists
3) hybrid proteins containing cytokine receptor binding sites attacked to Ig constant regions to block cytokine activity
4) blocking IL-17 function
What are Th2 cells involved in?
Antibody-mediated responses
What are Th1 induced by?
Dendritic cell production of IL-12
What are Th2 induced by?
IL-4
What are Th17 induced by?
IL-23
What are treg cells induced by?
TGF-beta
What are the roles of chemokines?
1) enhance motility and promote migration of many types of WBC toward source of the chemokines (chemotaxis)
2) modulate adhesion of WBCs to endothelial cells lining the blood vessels, facilitating extravasation into the tissues
Describe TNF-alpha
- LPS is a major trigger of TNF-alpha production
- secreted by activated monocytes and macrophages
- cause vasodilation and increased vasopermeability
- activates T cells by inducing expression of MHC class II molecules, vascular adhesion molecules and chemokines
What are the roles of complement?
1) causes lysis of foreign cells
2) acts as opsonins
3) increase vascular permeability
4) recruit monocytes and neutrophils to the area of antigen concentration
5) trigger secretion of immunoregulatory molecules that amplify the immune response
What is complement?
A series of more than 50 soluble and cell-bound proteins that interact to enhance host defense mechanisms against foreign cells
What are the three pathways of complement system activation?
1) classical
2) alternative
3) lectin
Describe classical pathway
- involves 9 proteins that are trigger primarily by antigen-antibody complexes
- major role in natural defense system
What classes of immunoglobulins activate the classical pathway?
IgM
IgG1
IgG2
IgG3
Describe recognition unit of the classical pathway?
Formation of C1
Describe activation unit of classical pathway
Once C1 is bound, C4, C2, and C3 are activated
Describe membrane attacks complex (MAC)?
- comprises of C5 through C9
- completes lysis of foreign particles
Describe C1 of classical pathway
- consists of 3 subunits: C1q, C1r, and C1s
- subunits require calcium to maintain structure
What happens once the classical pathway is activated?
C1r cleaves a thioester bond on C1s, which in turn, activates it.
What does it mean when C1s activated?
Recognition stage ends
What marks the beginning of the activation unit of the classical pathway?
Begins when C1s cleaves C4
What marks the end of the activation unit in the classical pathway?
The production of the enzyme C3 convertase
What is the second most abundant complement protein?
C4
What is the combination of C4b and C2a called?
C3 convertase
What occurs if C3 does not bind quickly?
Cleaved into two fragments, C3a and C3b
What is the central constituent of the complement system?
C3
What is the most significant step of complement system?
Cleavage of C3 to C3b
Describe structure of C3
- consists of two polypeptide chains, alpha, beta
- alpha chain highly reactive thioster beta
What occurs after cleavage of C3 in the classical pathway?
About 200 molecules are split for every molecule of C4bC2a
C3b also serves as a powerful _______
Opsonin
What has a specific receptors for C3b? Explain
Macrophages, primed to phagocytize antigen that has bound to C3b
Where does C3b bind in classical pathway? What does this create?
- within 40 nm of the C4bC2a
- C4C2aC36 is created. Also called C5 convertase
Describe the structure of C5
- contains two polypeptide chains, alpha, and beta, which are linked by disulfide bonds to form a molecule
What are the two fragments is C5 split into?
C5a and C5b
What occurs when sC5b-9 is formed in the classical pathway?
- MAC produce a pole of 70 to 100A that allows ions pass in and out of the membrane
What diseases are associated with abnormal C1q, C1r, and C1s?
SLE, recurrent infections
What is the function of C1q?
Binds to Fc region of IgM and IgG, synaptic pruning
What is the function of C1r?
Activates C1s
What is the function of C1s?
Cleaves C4 and C2
What is the function of C4?
Part of C3 convertase (C4b)
What is the function of C2?
- binds to C4b —> forming C3 convertase
What is the function of MBL?
Binds to mannose
What is the function of MASP-1?
Unknown
What is the function of MASP-2?
Cleaves C4 and C2
What is the function of factor B?
Binds to C3b to form C3 convertase
What is the function of factor D?
Cleaves factor B
What is the function of properdin?
Stabilizes C3Bb-C3 convertase
What is the function of C3?
Key intermediate in all pathways
What is the function of C5?
Initiates MAC
What is the function of C6?
Binds to C5b in MAC
What is the function of C7?
Binds to C5bC6 in MAC
What is the function of C8?
Starts pore formation on membrane
What is the function of C9?
Polymerizes to cause cell lysis
What are the steps of the classical pathway?
(Recognition unit)
1) the zygomens C1r and C1s are converted into active enzymes as binding of C1q occurs
2) autoactivation of C1r results from change that takes place as C1q is bound
3) when activated, C1r cleaves thioster bond on C1s, which activates it.
(Activation unit)
4) C1s cleaves C4 to create C4b and C2 to make C2a
5) C3 convertase (C4b2a) is formed
6) C3b binds to C4b2a to form C5 convertase (C4b2a3b)
(MAC)
7) C5 convertase splits C5 into C5a and C5b
8) C5b attaches to the cell membrane to initiate formation of the MAC (C5b6789)
How does the lectin pathway play an important role in infancy?
Defense mechanism during the interval between the loss of maternal antibody and the acquisition of as full-fledged antibody response to pathogens
What are the 3 recognition molecules of the lectin pathway?
- Lectin
- ficolins
- collectins (CL-L1)
What is the key protein of the lectin pathway? Describe this protein
- Mannan binding lectin (MBL)
- binds to mannose or other related sugars in a calcium-dependent manner to initiate this pathway
- considered acute phase protein because produced in liver and normally present in serum
What are the steps of the lectin pathway ?
1) binds to mannose or ribose sugar to initiate pathway
2) complex associates with MASP-1, MASP-2, and MASP-3. Complex act like C1qrs
3) MASP-2 cleaves C4 and C2
4) C3 convertase (C4b2a) is formed
5) C3b binds to C4b2a to form C5 convertase (C4b2a3b)
6) C5 convertase splits C5 into C5a and C5b
7) C5b attaches to the cell membrane to initiate formation of the MAC (C5b6789)
What are the steps of alternative pathway?
1) C3 hydrolysis water to produce C3b which binds Factor B, and together they attach to target cell surface
2) B is cleaved by Factor D into the fragments Ba and Bb. Bb combines with C35 to form C3bBb,an enzyme with C3 convertase activity
3) more C3 is cleared, forming more C3bBb. This enzyme is stabilized by properdin and, it continues to cleave additional C3
4) it a molecule of C3 remains attached to the C3bBbP enzyme, the convertase now has the capability to cleave C5. The C5 convertase now has the capability to cleave C5. The C5 convertase this consists of C3bBbP3b
5) C5b attaches to the cell membrane to initiate formation of MAC (C5b6789)
What is the role of the alternative pathway?
Functions largely as an amplification loop for activation started for activation started from the classical or lectin pathways
What are some triggering substances for the alternative pathway?
- bacterial cell walls
- some parasites
- fungal cell call
- viruses/ viral infected cells
- tumor cell line
- yeasts
- these all serve as binding site complex C3bBb
What proteins are involved in the alternative pathway?
- C3
- C5-C9
- factor B
- factor D
- P (properdin)
Describe the alternative pathway
Acts as a natural defense system
What protein is not stable in plasma in the alternative pathway?
C3
What has the ability to bind to factor B?
- iC3 (C3b)
What happens when C3b binds to factor B in the alternative pathway?
- factor D with magnesium can cleave B into Ba and Bb
Describe Factor D
- plasma protein that circulates in active enzyme form
- serine protease
- only substrate is bound to factor B
What occurs once B is cleaved into Ba and Bb in the alternative pathway?
1) Ba goes off into plasma
2) Bb binds to the surface of the triggering cellular antigen
3) if it does not bind quickly, it disintegrates.
What does C1 inhibitor (C1-INH) inhibit?
- activation of the first stages of the classical and lectin pathway
What is MCP also known as?
CD46
What is DAF is also known as?
CD55
What is Factor I?
- serine protease that inactivates C3b and C4b when bound to one of these regulators
What is immune adherence?
The ability of cells to bind complement-coated particles
Where are MCPs found?
- found on the cell membrane of all epithelial and endothelial cells except erythrocytes
What is the most efficient cofactor for factor I?
MCP
Describe DAF
- a membrane glycoprotein that has wide tissue distribution
- found on peripheral blood cells, endothelial cells, fibroblasts, and numerous types of epithelial cells
What does the presence of DAF on host cells protect them from?
Bystander lysis
What is bystander lysis?
- main mechanism used in discrimination of self from nonself because foreign cells do not posses this substance
What is the principle soluble regulator of alternative pathway?
Factor H
What is S protein?
- soluble control of protein that acts at a deeper level of complement activation
- terminal pathway
What does Factor H do in the alternative pathway?
- acts by binding to C3b to prevent binding of Factor B
What is the main function of C1-INH?
Dissociates C1s and C1r from C1q
What is the main function of Factor I?
Cleaves C3b and C4b
What pathways does C1-INH participate in?
Classical
Lectin
What pathways does factor I participate in?
Classical
Lectin
What are the main functions of Factor H?
1) cofactor with factor I to inactivate C3b
2) prevents binding of B to C3b
What pathway does Factor H participate in?
Alternative
What is the main function of C4BP?
Acts as a factor with factor I to inactivate C4b
What pathways does C4BP participate in?
Classical
Lectin
what is the main function of S protein?
- prevents attachment of C5b67 complex to cell membranes
What pathways does S protein participate in?
Classical
Alternative
Lectin
What is the main function of MIRL?
Prevent insertion of C9 into cell membrane
What is the main function of MCP?
Cofactor for factor I cleavage of C3b and C4b
What is the main function of CR1?
Cofactor for factor I, mediates transport of immune complexes
What is the main function of CR2?
- B cell co-receptor for antigen with CD19
What is the main function of CR3?
Adhesion and increased activity of phagocytic cells
What is the main function of CR4?
Adhesion and increased activity of phagocytic cells
What are 4 biological manifestations of complement?
- amplifies inflammatory response
- role in uptake and presentation of antigens in adaptive immune response
- facilitates B cell activation
- role in neural development
What is present when the inflammatory response is amplified by complement?
1) anaphylatoxins (C5a, C3a) - increase vascular permeability
2) chemotaxins (C5a) - attracts phagocytic cells to enhance phagocytosis
3) opsonins (C3b, C4b, iC3b, C3dg) - coat damaged or foreign cells to enhance phagocytosis
How can complement be harmful?
1) it is activated systemically on a large scale, as in gram-negative septicemia
2) it is activated by tissue necrosis and results in obstruction of the blood supply, as in myocardial infarction
3) lysis of RBCs occurs as in cold agglutinin disease or autoimmune hemolytic anemia
Describe C3 glomerulopathy
Inflammation of the Renal glomeruli tat maybe due to autoantibodies that cause C’ dysregulation (C3 nephrotic factors; C3NeFs
When detecting complement abnormalities, what are the immunologic assays of individual components?
- automated nephelometry or immunoturbidity
- radial immunodiffusion (RID)
What are classical pathway assays?
1) hemolytic titration (CH50) assay
2) CH50 test for lysis of liposomes
3) ELISA to detect C9 epitopes exposed after binding in MAC
What are the alternative pathway assays?
1) AH50
2) ELISA to detect C3bBbP or C3bP
What is the CH50 test procedure?
1) serum to be tested is diluted serially and added to sensitized sheep RBC
2) tubes are incubated at 37 C
3) centrifuged to pellet the unlysed cells
What is CH50 defined as?
As reciprocal of the dilution that causes lysis of 50% of the cells used in the assay
Describe the complement fixation test
- destroy any complement present in patient serum by heating serum to 56 C for 30 minutes
- dilutions of serum are combined with known antigen and a measurement amount of Guinea pig complement
- if patient antibody is present it will bind with the reagent antigen and complement be bound `
What are the steps of complement fixation test?
1) add sheep RBCs that are coated with a hemolysin (will lyse if any complement is present)
2) incubate and centrifuge tubes
3) read for hemolysis
4) it hemolysis is present —> no patient antibody/negative results
If hemolysis is absent —> patient antibody to complement/positive results
What are limitations of complement fixation test?
1) results are expressed as highest dilution showing no hemolysis
2) controls must be used
3) more sensitive than agglutination and precipitation, less sensitive than labeled immunoassay
4) errors
What pathways are affected by abnormal C1q, C1r, and C1s?
Classical —> low
What pathways are affected by abnormal C4 and C2?
Classical —> low
Lectin —> low
What pathways are affected by abnormal MBL and MASP-2?
Lectin —> low
What pathways are affected by abnormal B, D, and P?
Alternative —> low
What pathways are affected b abnormal C3, and C5-C9?
All three —> low
What pathways are affected by abnormal C1-INH?
Classical —> low
Lectin —> low
What pathways are affected by abnormal Factor H and I?
Al three —> low
What are levels of C3 and C4 routinely measured by?
- automated nephlometry or immunoturbidimetry
Describe nephlometry?
Measures the concentration of an individual complement protein according to the amount of light scattered by the antigen-antibody mixture
Describe immunoturbimetry
Based on reduction in light transmission resulting from immune complex formation
What is the difference between the CH50 and AH50 assays?
- magnesium chloride is added to buffer
- ethylene glycol tetraaactetic acid added to buffer
- calcium left out
Describe the AH50 assay
- buffer chelates calcium, blocking classical pathway activation
- rabbit RBC are used as indicator
Describe ELISA testing in alternative pathway
- can detect C3bBb or C3bP complexes in very small quantities
- micrometer wells typically coated with bacterial lipopolyssachardes to trigger activation of the alternative pathways
What are the assays that test for complement activation?
- ELISA or multiplex versions of immunoassays
Th cells affect the in innate immune response by inducing production of which cytokines?
IL-2
IL-4
Describe macrophage-colony stimulating factor (M-CSF)
- if activated, the cells become macrophages
- increase phagocytosis, chemotaxis, and additional production in monocytes and macrophages
What is autocrine stimulation?
Affecting the same cell that secreted it
Describe colony stimulation factor?
Stimulates formation of colonies of calls in the bone marrow
What is integrin?
Cell adhesion molecules on leukocytes
Describe erythropoietin (EPO)
CSF that regulates RBC production in bone marrow
Describe granulocytes colony stimulating factor (G-CSF)
- if activated, the cells become neutrophils band affects survival, proliferation, and differentiation of all cells types in the neutrophil lineage
Describe endocrine activity
System in affects
Describe adaptive T regulatory 1 cells
- CD4+ T cells tha are induced from antigen activated naive T cells in presence of IL-10
Describe granulocyte-macrophages colony stimulating factor (GM-CSF)
- cytokines produced by T cells and other cell lines that stimulates an increased supply of granulocytic cells and macrophages
What is the main difference between IgE ad IgG?
IgE has one more constant region
What is the best assay to measure a specific cytokine?
ELISA assay
What is precipitation?
Combine soluble antigen and stable antibody to produce insoluble complexes
What is agglutination?
Antigen-antibody complexes cross-link, causing clumping
What is affinity?
Initial forces of attraction that exists between a single Fab site on antibody and single epitope or determinant site on corresponding antigen
What is cross-reactivity?
- the more the cross-reacting antigen resembles the original antigen, the stronger the bond will between the antigen and binding sites
- cross-reacting antigens have lower affinity
What is serology?
Study of fluid components in the blood, especially antibodies
describe serum
- liquid portion of blood minus coagulation factors
- most frequently encounter specimen in immunologic testing
- can be separated from other components of blood via centrifuge
What color tube tops are serum collected in?
Red top or tiger top
What specimens would be rejected?
- hemolyzed
- clotted specimen
- wrong tube or container
- tube not labeled
- inadequate quantity
- time mislabeled
What are the 3 phases of laboratory testing?
- preanalytical
- analytical
- postanalytical
What are the examples of preanalytical testing?
- Specimen collection
- transport
- processing
What are examples of analytical testing?
Testing
What are some examples of postanalytical phase?
- testing results
- transmission
- interpretation
- follow up
- retesting
What phases of the laboratory testing constitutes for 90% of the errors that occur?
- pre-analytical
- post-analytical
What are the 3 phases of antigen/antibody complexes?
1) primary phenomenon
2) secondary phenomenon
3) tertiary phenomenon
Describe the primary phenomenon phase of complexes
- combination of binding site on antibody with a single epitope on antigen
- reversible: occurs in milk-seconds
- not easily detectable-hard to measure
Describe the secondary phenomenon phase of antigen/antibody complexes
- precipitation, agglutination, and complement fixation
- easily detectable
- most serological tests based
Describe the tertiary phenomenon phase of antigen/antibody complexes
- inflammation, phagocytosis, depression of immune complexes, immune adherence, chemotaxis (all in vivo)
What is complement fixation?
Triggers the classical complement pathway to detect antibody or antigen in the patients serum
what is avidity?
- is the sum of the attractive forces between forces between an antigen and an antibody
- strength with which a multivalent antibody binds a multivalent antigen
What does avidity measure?
Overall stability of an antigen-antibody complex
The more bonds that form between antigen and antibody, the higher the ______
Avidity
What is the law of mass action?
Free reactants are in equilibrium with bound reactants
What is the law of mass action equation?
K=(Ag/Ab)/(Ab x Ag)
In the law of mass action, what does the K value depend on?
The strength of binding between antibody and antigen
The higher the K value…..
- the larger the amount of antigen-antibody complexes
- the more visible or easily detectable the reaction
In the law of mass action equation, what does the [Ag/Ab] represent?
Concentration of the the antigen-antibody complex (mol/L)
In the law of mass action equation, what does [Ab] represent?
Concentration of free antibody (mol/L)
In the law of mass action equation, what does [Ag] represent?
Concentration of free antigen (mol/L)
What does a precipitation require antigens and antibodies to have?
- multiple binding sites for one another
- equivalent concentration
What are the 3 groups of the precipitation curve?
- zone of equivalence
- prozone
- postpone
For optimal precipitation, reactions must be run in what phase of the precipitation curve?
Zone of equivalence
Describe the zone of equivalence of the precipitation curve
- number of multi-valent sites of antigen and antibody are approximately equal
Describe the prozone of the precipitation curve?
- antibody excess
- antigen combine with one or two antibodies
- no cross-linking
What could cause a false negative result in the prozone phase of the precipitation curve?
High antibody concentration
Describe the postzone phase of the precipitation curve
- antigen excess
- small aggregates are surrounded by excess antigen
- no lattice network formed
What could cause a false negative result in the postzone phase of precipitation curve?
Presence of small amount of antibody
What are immunoturbidimetry and nephelometry used to test for?
- complement
- C-reactive protein
- immunoglobulins
- other serum proteins
What is the principle of immunoturbidity and nephelometry?
Light that is scattered at an angle is measured, indications the amount of antigen or antibody present
What is the radial immunodiffusion used to test for?
- complement
- immunoglobulins
What is the principe of radial immunodiffusion?
Antigen diffuses out into gel that is infused with antibody. Measurement of the radius indicates concentration of antigen
What is Ouchterlony double diffusion used to test for ?
Complex antigens, such as fungal antigens
What is the principle of Ouchterlony double diffusion?
Both antigen and antibody diffuse out from wells in a gel. The lines of precipitate formed indicate he relationship of antigens
What does immunoelectrophoresis test for?
Differentiation of serum proteins
What is the principle of immunoelectrophoresis?
Electrophoresis of serum is followed by diffusion of antibody from the wells
What is the immunofixation electrophoresis used to test for?
Over or under production of antibody
What is the principle of immunofixation electrophoresis?q
- electrophoresis of serum is followed by direct application of antibody to the gel
What machines could be used for immunoturbidity?
- spectrophotometer
- automated clinical chemistry analyzer
What machines is used for nephelometry?
Nephelotometer
Describe nephelotometer
- contains photodector located between 30-90 degrees from light source
- amount of light scatter increases as the number of immune complexes increases and is in index of the concentration of antigen or antibody in solution
What is passive immunodiffusion?
When no electric current is used to speed up the migration of the reactants
What can affect the rate of diffusion in passive immunodiffusion?
- size of particles
- temperature
- gel viscosity
What is the end point (Mancini) method?
Reaction goes to completion
What is the kinetic (Fahey) method?
- faster
- measurements taken before reaction is complete
What is the RID end point method results?
The square of the diameter is proportional to the antigen concentration
What are the three possible patterns of Ouchterlony diffusion?
- identity (arc)
- partial identity (one line extends)
- nonidentity (line cross X)
What is visualized when immunofixation electrophoresis (IFE) is performed?
Increased or decreased production of antibody classes and to differentiate monoclonal and polyclonal immunoglobulins
What are agglutinins?
Produced by antibodies
What are the 2 steps of agglutination?
1) sensitization
2) lattice formation
Describe sensitization step of agglutination
- initial bonding
- antigen and antibody unite through bonding of antigenic determinants sites
- fast and reversible
Describe the lattice formation of agglutination
- creation of large aggregates
- visible aggregates form as multiple antigen and antibody molecules bind to create a stable lattice
What is Coombs reagent?
- anti-human immunoglobulin
- frequently used in blood bank
- binds to Fc portion of IgG
Briefly describe immunoturbidity
- automated methods
- measures reduction in light intensity
Briefly describe nephelometry
- automated methods
- measures light scatter as immune complexes form
What are 4 types of agglutination reactions?
1) direct agglutination
2) passive agglutination
3) reverse passive agglutination
4) agglutination inhibition
Describe direct agglutination
- Uses particles with naturally occurring antigens to test for antibodies in patient serum
- detects bacteria (widal test) and RBCs
Describe Widal test
- a rapid screening test used to determine the possibility of typhoid fever
- uses salmonella O and H antigens
- direct agglutination test
What test are ran fro RBCs?
- hemagglutination
- ABO typing
(Direct agglutination)
Describe passive agglutination
- uses particles that are coated with antigens not normally found on their surfacesL erythrocytes, gelatin, latex, and silicates
What does passive agglutination test for ?
- RA
- antibodies to Group A streptococcus antigens
- antibodies to viruses such as rubella
- heterophile antibody in IM
Describe reverse passive agglutination process
1) antibody is attached to the carrier particle
2) agglutination occurs is antigens present in the patient sample
What can reverse passive agglutination test for?
-Staphylococcus aureus
- streptococcus Group A or B.
- rotavirus
- cryptococus neoformans
Describe agglutination inhibition
-based on competition between particle and soluble antigens for limited antibody-combining sites
- used to detect hapten antigens (illicit drugs)
- lack of agglutination —> positive reaction
What is an agglutination inhibition procedure?
- hemagglutination inhibition —> uses RBCs
- used to detect antibodies to certain viruses that can bind to RBCs and agglutinate them
What virus can hemagglutination inhibition be used to test for?
- rubella
- influenza
- respiratory syncytial virus
What is the principle of direct agglutination?
Antigen is naturally found on a particle
Describe results of direct agglutination
Agglutination indicates presence of patient antibody
What is the principle of passive (indirect) agglutination?
Particle are coated with antigens not normally found on their surfaces
Describe the results of passive (indirect) agglutination
Agglutination indicates the presence of patient antibody
What is the principle of reverse passive agglutination?
Particles are coated with reagent antibody
Describe results of reverse passive agglutination
Agglutination indicates the presence of patient antigen
What is the principle of agglutination inhibition?
- haptens are attached to carrier particles
- particles compete with patient antigens for a limited number of antibody sites
Describe results of agglutination inhibition
Lack of agglutination is a positive test, indication the presence of patient antigen
What is the principle of hemagglutination inhibiton?
RBCs spontaneously agglutinate in presence of certain viruses
Describe the results of hemagglutination inhibiton
Lack of agglutination is a positive test indication the presence of patient antibody
Describe monospot tests
Used to detect the heterophile antibody characteristics of IM by its ability to agglutinate horse or bovine RBCs
Describe cold agglutinins
- produced by patients with certain autoimmune disorders, malignancies, or infections, are antibodies that agglutinate human type O RBCs when incubated at cold temperatures
Describe PETINA
- “particle-enhanced turbidmetric inhibiton immunoassay”
- patient sample is incubated with latex beads coated with analytes will prevent antibody turbidity results
- measures small analytes such as therapeutic drugs
- homogenous competitive immunoassay
Describe the results of PETINA
- if low amount of analyte is present in the sample, the reagent antibody will bind to the latex beads, resulting in aggregate formation and high level of turbidity
- when concentration of analytes high, analyte will bind to the reagent antibody and prevent it from binding to the latex beads, resulting is less turbidity
How ca most cross-reactivity be avoided?
Through use of monoclonal antibody directed against an antigen determinant that is unique to antigen
What are advantages of agglutination reactions?
- RA did
- easy to perform
- no expensive equipment
- relatively sensitive
When does maximum binding at antigen and antibody occur?
When the aggregate number of multivalent sites of antigen and antibody are approximately equal
How does precipitation differ from agglutination?
Precipitation involves a soluble antigen, whereas agglutination involves a particulate antigen
Describe labeled immunoassays
- designed for antigen and antibodies that may be small in size or present in very low concentrations
- use a reactant labeled with a detection molecules to monitor the amount of specific binding that has take place
What is an analyte?
A biomarker
How are labeled immunoassays differ from unlabeled techniques?
A detection molecule (label) in the test system
What is a common technique to many labeled immunoassay?
Enzyme-mediated catalysis of a reagent substrate to generate a light signal
describe heterogenous immunoassays
- involve physical separation of bound and free components
- most commonly involve binding to a solid phase
- magnetic separation or centrifugation maybe used
Describe homogenous immunoassays
Do no require a physical separation step
Describe competitive immunoassays
- all reactants are mixed together simultaneously
- labeled and unlabeled antigen compete for a limited number of binding sites on reagent antibody
- have high specificity
What does competitive immunoassays measure?
Small antigens that are relatively pure (drugs and hormones)
What are the steps of competitive immunoassay procedure?
1) unknown concentration of analyte in patient sample competes with labeled analyte for binding sites on immobilized antibody
2) wash to remove unbound material
3) after substrate is added, a colored product (signal) is generated with an intensity proportional to the amount of enzyme-labeled analyte bound to the antibody
What is the principle of noncompetitive immunoassays
- patents analyte is allowed to bind with an excess amount of labeled reagents
- Also known as capture, sandwich or immunogenic assays
What are the steps of noncompetitive immunoassay procedure?
1) reagent antigen immobilized on solid phase binds antibody in patient sample
2) wash to remove unbound materials
3) add enzyme-labeled (detection) antibody that binds to human immunoglobulin
4) wash to remove unbound materials
5) add substrate and measure signal. Signal is directly proportional to concentration of antibody in patient sample
Describe radioimmunoassay (RIA)
- first immunoassay developed
- uses radioactive labels - 125I is most popular
- emits gamma radiation, which is detected by a gamma counter
- is extremely sensitive and precise
- measures trace amounts of analyte ( hormones, serum protein, and drugs) that are small in size
What are disadvantages of radioimmunoassay?
1) working with radioactive substance (hazardous)
2) disposal of low level radioactive waste
3) short shelf for some reagents
4) testing in clinical labs is limited
Describe enzyme immunoassays (EIAs)
- Highly sensitive assay that uses enzymes as labels, which react with suitable substates to produce breakdown products that maybe chromogenic, fluorogenic, or luminescent
- available in competitive and noncompetitive formats
Briefly describe heterogenous EIAs
Requires a step to physically separate free analyte from bound analyte
Describe competitive heterogenous EIAs
- involve enzyme-labeled antigens competing with unlabeled patient antigen for binding sites or antibody molecules
- have high specificity
What is competitive heterogenous EIAs used for?
For measuring small antigens that are relatively pure (drugs and hormones)
What are advantages of noncompetitive heterogenous EIAs
- high sensitivity
- high specificity
- simple to perform
- low cost
Describe ELISA
- “enzyme linked immunosorbent assay”
- used to measure antibody production to infectious agents that are difficult to isolate and form autoantibody testing
- easily applied to point of care and home testing
- detects HIV, Hep B, and Hep C, rubella virus
What is the principle of indirect ELISA?
Primary antibody specific for the antigen binds to the target, and a labeled secondary antibody against the host species of the primary antibody binds to the primary antibody
What are the immuoassays that detect antibodies?
Indirect ELISA
What are immunoassays that detect antigens?
Capture immunoassays
Why is it referred to as “indirect” ELISA?
Because the enzyme-labeled reagent does not participate in the initial antigen-antibody reaction
Describe capture immunoassays
- EIAs that detect antigen in patient sample
- best suited for antigens that have multiple determinants (cytokines, proteins, tumor marker, microbial antigens)
- can be used to detect immunoglobulins of a particular class
What is the principle of capture immunoassay?
Capture the antibody to an ELISA plate and allow the analyte of interest to bind to the capture antibody, then secondary antibody is added and binded
Describe the high dose hook effect
- antigen interference
- excess patient antigen causes falsely decreased detection
- analyte concentration appears to below or normal when it is actually high
- also called Postpone effect
When does the high dose hook effect occur?
When there are not enough capture antibody sites for antigen binding because the majority of binding sites are filled, so the remainder of patient antigen has no place to bind and gets removed during the wash step1
What are some interferences of antibodies?
- autoantibodies —> rheumatoid factor can cause a false positive, produced in vivo
- heterophile antibodies —> usually cause false-negative results. Example: human and mouse antibodies (HAMA)
Describe homogenous EIAs
- less sensitive than heterogenous assays
- rapid and simple to perform
- include EMIT and CEDIA
- does not require washing or separation step
- when antibody binds to specific determinant sites on the antigen, the active site on the enzyme is blocked, resulting in a measurable loss of activity
What are homogenous EIAs used for?q
To determine low-molecular weight analytes in serum and urine, hormones, therapeutic drugs, and drugs of abuse q
Describe enzyme multiplied immunoassay technique (EMIT)
- original homogenous design
- enzyme active site is blocked when antibody binds to enzyme
- does not allow substrate to react with enzyme
- based on the principle of change in enzyme activity as specific antigen-antibody interaction occurs in solution
What are the steps of EMIT?
1) reagent antigen bound to an an enzyme tag commonly G6PDH
2) when reagent antibody binds to specific sites on the enzyme-antigen pair, the active site on the enzyme is blocked. Results in loss of activity
Describe cloned-enzyme donor immunoassay (CEDIA)
- produce the beta-galatosidase enzyme in two parts: acceptor and donor
- when the acceptor-donor pair combine, enzymatic activity is restored; and reagent substrate is catalyzed to generate a product signal that is measured by photometry
Describe chemiluminescent immunoassays
- highly sensitive
- heterogenous or homogenous
- automated immunoassays
- detects antibodies or antigens
- the emission of light cause by a chemical reaction, typically an oxidation reaction, producing an excited molecule that decays back to its original ground state
Describe fluorescent immunoassays
- uses Fluor chrome as the label
- these compounds absorb energy from incident light and convert to to a longer wavelength electrons return to the ground state
Describe fluorescein
Absorbs light at 490-495 nm and emits green light at 520 nm
Describe rhodamine
Absorbs light at 550 nm and emits red lights at 585 nm
Describe fluorochromes
Fluorescent compound that can absorb energy from an incident source and convert to that energy into light of a longer wavelength and lower energy as excited electrons return to ground state
What is the direct immunofluorescence assay procedure?
1) antibody with a fluorescent tag is added to tissue or cells fixed onto a slide
2) after incubation and a wash step, the slide is read using a fluorescence microscopic
Describe direct immunofluorescence assays
- used to identify pathogens in patient samples
- fluorescent-labeled antibodies bind to CD antigens
- can be used to identify lymphocytes and other cells by flow cytometry
- used for cells or tissues
What is the principle of indirect immunofluorescence assay?
1) patient serum is incubated with microscopic slide to which a known antigen is attached
2) the slide is washed, then an anti-human immunoglobulin labeled with a fluorescent tag is added
3) A sandwich is formed with the first antibody, which localizes the fluorescence
* can be used to identify patient antibody (ANAs)
What is indirect immunofluorescence assay used to detect?
- antibody ID
- syphilis testing (treponemal antibodies, viral antibodies and autoantibodies)
describe multiplex immunoassay (MIA)
- fluorescent immunoassay that allows multiple antibodies or antigens to be detected simutaneously
What is the principle of multiplex immunoassay (MIA)?
1) to detect antibodies, patient serum is incubated with polystyrene beads conjugated to different antigens
2) antibody binding is detected by addition of a fluorescent- tagged anti-human immunoglobulin
3) beads containing bound antibody are identified by flow cytometry and can be distinguished by their unique shade of red
What is a benefit of MIA?
Allows for multiple antibodies to be detected simutaneously
How can MIA be used?
- ANA testing
- Detection of antibodies in transplant patients donor antigens
Describe fluorescent polarization immunoassay (FPIA)?
- determines concentrations of therapeutic drugs and hormones
- based on charge in polarization of fluorescent light emitted from a labeled molecule when it is bound by antibody
- If the labeled molecule is bound to antibody, the molecule emits increased amount of polarized light
- homogenous
Describe is the rapid immunoassays procedure?
1) patient sample is added to the test membrane and combines with labeled antigen or antibody conjugated to colored latex or colloidal gold particles
2)immune complexes are formed that migrate across the membrane to produce a colored reaction
Describe immunochormatography
Combine any methods of immunoassays into one step
How do heterogenous immunoassays differ from homogenous immunoassays?
Heterogenous immunoassays require a separation step
What is the principle of FPIA?
Fluorescent-labeled antigen competes with patient antigen for a limited number of soluble antibody-binding sites
What do nucleic acids do?
The carry genetic information codes for protein structure
What are some primary characteristics of nucleic acids?
- Nucleic acid complementary
- melting temperature
What is an advantage to molecular testing?
Advantageous because nucleic acids can be detected earlier than antibodies during the course of an illness
Describe DNA
- Nucleic acid that carries the primary genetic information within chromosomes
- contains sugar deoxyribose
Way are the purine bases of DNA?
Adenine
Guanine
What are the pyrimidine bases of DNA?
Cytosine
Thymine
Describe RNA
- helps convert the genetic information encoded within DNA into proteins
- contains sugar ribose
What are the purine bases of RNA?
Adenine
Guanine
What are the pyrimidine bases of RNA?
Cytosine
Uracil
What are the types of RNA?
- messenger RNA (mRNA)
- transfer RNA (tRNA)
- ribosomal RNA (rRNA)
- noncoding RNAs
What are nucleotide composed of?
- deoxyribose OR ribose sugar
- nitrogen base
- a phosphate group
Describe nucleotides
Units that make up DNA and RNA
What are the two strands of the DNA double helix are held together by?
Hydrogen bonds between their nucleotide bases
What is complementary to guanine in DNA?
Cytosine
What is adenine complementary to adenine in DNA?
Thymine
How many hydrogen binds are between guanine and cytosine?
3
How many hydrogen bonds are between adenine and thymine?
2
What is the length of double stranded DNA macromolecule measured in?
Base pairs
What is the length of a single stranded RNA measured in?
Bases
What is a megabase pair/megabase?
When there is one million base pairs/bases
What is chromosome?
Double helix of DNA
How many chromosomes are in a cells nucleus?
- 46
- two copies each of the 22 autosomes or non-sex chromosomes
- plus two copies of X chromosomes in females OR one X chromosome and Y chromosome for males
What are genes?
Sequences of nucleotides in chromosomes that carry information for either a protein or non coding genes making up the diploid genome
What is genome?
Entirety of DNA in the cell
Describe DNA replication
- two DNA strands separate
- each strand is a template for synthesis of a complementary strand
- DNA polymerase is needed for synthesis
- each new strand is synthesized in the 5’ to 3’ direction as the original double strand unwinds
- two identical double-stranded molecules results
What is the “origin of replication” for DNA?
Defined sequence of nucleotides that start DNA replication
What is the cell cycle?
While cells divide, they undergo a series of events in which increase in size and duplicate their DNA
What are the four stages of the cell cycle?
G1
S
G2
M
Describe S phase of the cell cycle
DNA replication occur here
Describe the G2 phase of the cell cycles
DNA complement of the cell are doubled
Describe the M phase of the cell cycle
One complement of chromosomes is divided into each of two daughter cells
Describe the G1 phase of the cell cycle
- each of the new daughter cells are here
- movement from G1 to the S phase is strictly regulated
What is lagging strand?
Copied discontinuously toward the replication fork
Describe RNA synthesis
- can start de novo, without primer
- is catalyzed by RNA polymerase
- occurs throughout the cell cycle
- mRNA codes for the amino acid sequence of a protein
Describe the protein synthesis and translation steps
1) mRNA is transcribed from DNA using RNA polymerase
2) the mRNA delivers the information to ribosomes, where protein synthesis takes place
3) as each amino acid is added, the peptide chain continues to grow
4) this translation process is accomplished with help of tRNA, which brings in individual amino acids
What is a phenotype?
Observable properties of an organism (blue eyes, height)
What is a codon?
3 base recognition sequence
Describe mutations and variants
- changes in the nucleotide sequence of DNA
- some are associated with disease
What is the difference a mutation and variant?
- variants are inherited
- mutations are spontaneous changes in somatic cells
Describe polymorphisms
- alterations in DNA or protein sequences shared by at least 2% of population
- MHC is highly polymorphic region in the human genome
Describe electrophoresis
- involves movement of particles under the force or an electrical current
- electrophoresis of nucleic acids commonly takes place in a semisolid matrix called a gel
- nucleic acids are negatively charged and migrate toward the positive pole (anode)
- smaller nucleic acid chains migrate faster than large chains
Describe capillary electrophoresis
- a more sensitive, semi-automated method that separates particles in a gas, liquid or gel
- DNA chains carry a fluorescent label that is excited by a laser to produce peaks of fluorescence
Describe molecular analysis
Nucleic acid test designed to detect changes (mutations and polymorphism) in the DNA sequence
What are the four main approaches to nucleic acid analysis?
1) strand cleavage methods
2) hybridization methods
3) amplification methods
4) sequencing methods
What do the strand cleavage methods use to cleave DNA at specific locations?
Restriction endonuclease enzymes
Describe the strand cleavage methods of molecular analysis
- separate cleaved fragments by size and charge using electrophoresis, revealing potential variations
- used to investigate small genomes, such as those of microorganisms or plasmids
- detect mutations and polymorphisms
Describe CRISPR-Cas9
- newer method that uses an enzyme to alter DNA at used-defined locations identified by a small guide RNA molecule
- used for DNA analysis, gene therapy, and genome editing
- a strand cleavage method
Describe the hybridization methods of molecular analysis
- involves the binding two complements nucleic acids, most notably a template and probe
- used on large, complex genomes
What are some examples of hybridization methods of molecular analysis?
- Souther blot
- microarray
- bead array
- in situ hybridization
What are probes?
Short nucleic acids that bind to complementary sequences
What is hybridization?
Involves binding of two complementary strands of nucleic acids
Describe western blot
- detection of proteins and protein modifications
- separated by gel electrophoresis and blotted to membrane
Describe microarrays
- allow for multiple targets or sample to be analyzed simultaneously
- the test sample is labeled with a fluorescent dye and added to a glass slide containing thousands of specific unlabeled probes
- fluorescent spots appear where complementary binding occurs
What are types of microarrays?
- comparative genomic array
- RNA expression arrays
- SNP arrays
Describe comparative genomic arrays
Detect amplifications or deletion in DNA
Describe RNA expression arrays
- detect that have been actively transcribed into mRNA
Describe RNA expression arrays
- detect genes that have been actively transcribed into mRNA
Describe SNP arrays
Detect single nucleotide differences in test samples, some of which may be associated with a particular disease.
What is genomics?
Analysis of hundreds to thousands of targets or whole genomes, rather than single genes
Describe bead arrays
- used for multiplex detection of proteins and nucleic acids
- tissue typing
- respiratory virus panels
- beads may have antibodies or single- stranded oligonucleotides attached
Describe in situ hybridization
Labeled probes hybridize to tissues or cells on glass sides
What are the types of in situ hybridization?
- immunochemistry
- fluorescence in situ hybridization
Describe immunohistochemistry
Uses enzymes-labeled antibodies to identify protein targets
Describe fluorescence in situ hybridization (FISH)
- uses fluorescent labeled probes to detect specific DNA sequences
- detects chromosomes abnormalities
What are some examples of the amplification method?
- PCR
- target amplification
- transcription based amplification
- strand displacement amplification
- signal amplification
describe polymerase chain reaction (PCR) procedure
1) denaturation —> DNA is separated into single strands by heat (95 C)
2) annealing —> primer attach (60 C)
3) extension —> complementary by nucleotides are added to 3’ end (~70 C)
Describe target amplification
- amplification of PCR products, under optimal conditions, each cycle results in doubling product
- in this manner, a target sequence present that are fewer than 100 copies can be detected
- resulting DNA fragments (amplicons) are detected using various methods
briefly describe PCR
In vitro DNA replication procedure
What is key for specificity of a PCR reaction?
- oligonucleotide primer —> synthetic single stranded nucleic acids, usually 18 to 30 in length
What is the instrument used to carry out the amplification program?
Thermal cycler
- chamber-based
- block-based
What is the a common approach used to detect mutations and polymorphisms?
SSP-PCR
Describe quantitative PCR (qPCR)
- determines the amount of a specific sequence in the original sample
- both DNA and RNA target can be measured by qPCR
What are the four probe types of qPCR?
- fluoroscence resonance energy transfer (FRET)
- Taqman
- molecular beacon
- scorpion probes
What are some commonly used applications of qPCR?
- detection of microorganisms, especially viruses and other pathogens, tumor associated gene expression and tissue typing
Describe reverse-transcriptase PCR (RT-PCR)
- amplifies cDNA made from an RNA template
- HIV, HCV
Describe transcription-based amplification (TMA)
- RNA is the target as well as the primary product
- product detected by chemiluminescence with acridinium ester
- isothermal process
- useful in testing got RNA viruses
- temperature cycling not required
Describe probe amplification
- number of target nucleic acid sequences does not change
- instead, primers are extended or ligated into many copies of detectable probes
What are some examples of probe amplification?
- strand displacement amplification (SDA)
- loop-mediated isothermal amplification (LAMP)
- molecular inversion probe (MIP)
Describe strand displacement amplification (SDA)
- after initial denaturation step, the reaction proceeds at one temperature
- nick formed in strand by restriction endonucleus enzyme
Describe loop-mediated isothermal amplification (LAMP)
- high specific and sensitivity for target DNA
- PCR primer carry sequences at 5’ end that will self hybridize, forming loops that self prime in cyclic manner
- advantage —> shortened Run time
What is the LAMP used to detect?
- HIV
- cytomegalovirus
- staphylococcus aureus
- E. Coli
Describe molecular inversion probe (MIP)
- high sensitive
- probe ends bind to target sequences so that, in the presence of target, probes ends are brought together and ligated to form circles
What is MIP used to detect?
- staphylococcus aureus
- streptococcus mutans
- influenza virus
- RNA typing
Describe signal amplification
- large amounts of signals are bound to the target sequences
- example: branched DNA
Describe branched DNA (bDNA)
- has been used to detect certain viruses
- series of short, single stranded DNA probes are used to capture the target nucleic acid and to bind to multiple reporter molecules, loading target nucleic acid with signal
Describe DNA sequencing
- direct determination of the order of nucleotides in a DNA chain
- the most specific method of identifying genetic mutations or polymorphisms
- methods include Sanger sequencing, pro sequencing, and next-generation sequencing (NGS)
Describe Sanger sequencing
- uses all components of PCR plus fluorescent-labeled dideoxynucleatides (ddNTPS) to synthesize DNA complementary to the target
- when correct ddNTPs is added, DNA synthesis stops and fragments of varying sizes are produced
- results are read by gel or capillary electrophoresis
- less sensitive than other methods
Describe pyrosequencing
- based on the generation of light when nucleotides are added to a growing strand of DNA
- not as versatile as Sanger sequencing, but less labor intensive
Describe next-generation sequencing (NGS)
- can sequence large numbers of templates simultaneously (massively parallel sequencing)
- using bioinformatics, the short sequences are assembled to determine the complete sequence, which is compared to known database
- more error prone than Sanger sequencing
what are applications of next-generation sequencing (NGs)?
- ID of genetic variations in inherited diseases and tumor cells
- HLA allele typing
- analysis of mixed microbial populations
Describe ion conductance sequencing
Nucleotide order is determined through release of hydrogen ions by DNA synthesis
Describe whole exonerated sequencing
Used to identify variants responsible for inherited disease conditions
Describe whole genome sequencing
- primarily research tools
- massive parallel sequencing has the capacity to investigate all known genetic loci alterations
Describe targeted libraries (sequencing)
See==elected genes or gene regions q
What is the basis of all molecular diagnostic testing?
The high specificity of detection of nucleic acid sequences through complementary base pairing
Between promotor, cytosine, S phase, and primer which one is associated with RNA synthesis only?
Promotor
What is the function of restriction endonucleuses?
The cleave DNA at specific sites
What technique uses RNA-guided enzyme?
CRISPR
What is the purpose of an amplification control in qPCR?
Avoid false negatives
What are some hybridization techniques??
Microarray technology
Fluorescent in situ hybridization
What is the principle of rapid immunochromatographic?
- patient sample is added to test strip and migrates through the strip. Patient antigen complexes to antibody-labeled particles or competes with antigen-labeled particles across individual test lanes at unique detection zones
What is immunophenotyping?
Identifying their surface and cytoplasmic antigen expression
What is flow cytometry used to detect?
- leukemia
- lymphoma
- AIDS
- chronic granulomatous disease
- leukocyte adhesion deficiency
- fetal RBC
What is a significant advantage of flow cytometry?
Because the flow rate of cells is so rapid, thousands of events can be analyzed in seconds, allowing for accurate detection of cells that are present in very small numbers
What are the 4 parts of instrumentation of flow cytometry?
1) fluidics
2) laser light source
3) optics and photodetectors
4) computer
Describe fluidics of flow cytometry
- allows for cell transport, one at a time
- each time a cell passes in front of a laser beam, light is scattered, and the interruption of laser signal is recorded
Describe laser light source of flow cytometry
- for cell illumination and identification
- solid-state diode lasers are typically used
Describe optics and photodetectors of flow cytometry
Signal detection
Describe computers of flow cytometry
Data management
What colors are lasers?
- red
- blue
- violet
- ultraviolet
- yellow-green
What are the two types of light scatter?
1) forward scatter (FSC)
2) side scatter (SSC)
Describe forward scatter
- an indicator of size
Describe side scatter
- indicative of granularity or the intracellular complexity of the cell
What are the intrinsic parameters of the light sources of flow cytometry?
- forward scatter
- side scatter
- light scattering properties s
Describe extrinsic parameters in light source of flow cytometry?
Requires the addition of a fluorescent probe for their detection
Describe principle of hydronomically focusing in flow cytometry
- cells pass single file through the intersection of the laser light source
What is the additive in the tube that is used for whole blood collection?
EDTA heparin can also be used
Describe single-parameters histogram
- the y-axis consists of the number of events
- usually extrinsic parameters (fluorescent-labeled) antibody
- operator sets a marker to isolate the positive event
- will calculate the percentage of positive events within the designated markers
Describe dual-parameters dot plot
- both parameters (axis) are chosen by operator
- the operator then draws a “gate” or isolates the population of interest for further analysis
- gated cells are analyzed for fluorescence
- suppurated into four quadrants
Describe quadrant 1 of dual parameter dot plot
X-axis —> negative for fluorescence
Y-axis —> positive for fluorescence
Describe quadrant 2 of dual parameter dot plot
X-axis —> positive for fluorescence
Y-axis —> positive for fluorescence
Describe quadrant 3 of dual-parameter dot plot
X-axis—> negative for fluorescence
Y-axis—> negative for fluorescence
Describe quadrant 4 of dual-parameter dot plot
X-axis —> positive for fluorescence
Y-axis —> positive for fluorescence
What are some applications of flow cytometry?
1) identifies particular markers for diagnosis and monitoring of leukemia and lymphomas
2) enumerates peripheral blood. CD4+ T cells to classify stages of HIV disease and guide treatment
3) enumerates CD34+ cells in stem cells transplantation
4) determines DNA content or ploidy status of tumor cells
5) helps diagnosis inherited diseases
What is the significance of the presence of CD5+?
Indication of mantle cell lymphoma (CLL)
How is HIV is classified?
Enumeration of peripheral blood CD4+ T cells by flow cytometry
What are the advantages of immunoassay automation?
- reduce error
- is more accurate and precise
- requires fewer staff
- saves on controls, duplicates, dilutes, and repeats
- potential for better sample ID with use of bar coding
What are two types of immunoassay analyzer?
- batch
- random-access analyzer
Describe batch analyzers
- can examine multiple samples
- provide access to samples for formation of reaction mixture
- permits one type of analysis at a time
Describe random access analyzers
- measure numerous analytes from multiple samples
- can perform different tests on any one sample
What is accuracy?
Tests ability to measure what it claims to measure
What is precision?
Ability to consistently reproduce a result on repeated testing of the same sample
What is analytic specificity?
An assays ability to generate a negative result when the analyze is not present
What is analytic sensitivity?
The lowest measurable amount of an analyte
What is a reportable range?
The range of values that will generate a positive results for the specimens assayed by the test procedure
What is the reference interval?
Range of values found in healthy individuals
What is the single most important requirement for samples to be analyzed on a flow cytometry?
Cells must be in a single-suspension
The various signals generate by cells intersecting with a flow cytometry laser are captured by what?
Photomultiplier tubes
What is flow cytometry most commonly used method?
Immunophenotyping of lymphoid and myeloid
Describe hypersensitivity
- exggerated immune response to a typically harmless antigen
- results in tissue injury and disease
- 4 types
Describe the immediate reaction of hypersensitivity
- develop minutes to hours after antigen exposure
- type I, II, and III
Describe the delay reaction of hypersensitivity
- develop 24 to 48 hours after antigen exposure
- type IV
Describe type I hypersensitivity
- cell bound antibodies react with antigens to release physiologically active substances
- complement is not involved
- known as “allergies”
Describe type II hypersensitivity
- free antibody reacts with antigen associated with cell surfaces
- complement plays major role in producing tissue damage
describe type III hypersensitivity
- antibody reacts with soluble antigen to form complexes that precipitate in tissues
- complement plays major role in producing tissue damage
Describe type IV hypersensitivity
- not seen until 24-48 hours
- differs from the other three because sensitized T cells rather than antibody are responsible for symptoms
- complement not involved
What is another name for type I hypersensitivity?
Anaphylactic hypersensitivity
What is another name for type II hypersensitivity?
Antibody-mediated cytotoxic hypersensitivity
What is another name for type III hypersensitivity?
Complex-mediated hypersensitivity
What is another name for type IV hypersensitivity?
Cell-mediated hypersensitivity
What are the key components of type I hypersensitivity?
- IgE
- Mast cells
- basophils
- eosinophils
Describe sensitization phase of hypersensitivity
- APCs process allergens and present them to Th cells
- Th2 cells induce production of allergen-specific IgE
- IgEb binds to FceRI receptors on most cells and basophils
Describe the activation phase of hypersensitivity I
- allergen cross-link adjacent cell-found IgEs
- mast cells and basophils degranulate
- chemical mediators are released and bind to target organs
- allergy symptoms produced
What are the type I preformed/primary mediators?
- histamine
- heparin
- eosinophil chemomatic factor of anaphylaxis (ECF-A)
- neutrophil chemomatic factor
- proteases
What are the type I newly formed mediators?
- platelets activating factor (PAF)
- prostaglandin (PG) D2
- leukotrienes (LT) —> B2, C4, D4, and E4
- cytokines
What are some common allergens?
- pollen
- mold spores
- animal danders
- dust mites
- insect venom
- certain foods
- certain dogs
- latex
What are the clinical manifestations of type I?
- Rhinitis (hay fever)
- allergic asthma
- food allergies
- urticaria (hives) —> wheal and flare
- eczema
- systemic anaphylaxis —> potentially fatal
What are treatment for type I?
- avoid allergens
- drug therapy —> antihistamines, bronchodilators, most cell stabilizers, corticosteroids epinephrine
- monoclonal anti-IgE antibody
- allergy immunotherapy (AIT) —> administer gradually, increasing doses of allergen
Describe process of percutaneous and intradermal of type I
1) apply a panel of allergens to seperate sites on the skin
2) wait 15 to 20 minutes
* positive test = wheal and flare at the site of application
describe allergen specific IgE testing of type I?
- RAST (radioallergosorbent test) was the first type of test to be used for specific IgE
- enzyme methods are now used to detect IgE specific allergen in patient serum
- safer than skin testing
Describe the total IgE test of type I
- RIST (radioimmunosorbent test) was first type of test method for total IgE
- enzymes are now use to detect total concentration of IgE in patient serum
What are allergens?
Refers to an inherited tendency to develop classic allergic responses to naturally occurring inhaled or ingested allergens
How long does it take for type I to occur?
30-60 minutes
What is passive cutaneous anaphylaxis ?
Redness and swelling at site that was injected with serum (from patient who is allergic to allergen) and later exposed to allergen
What is atopy?
- refers to an inherited tendency to develop classic allergic responses to naturally occurring inhaled or ingested allergens
What regulates IgE production?
Th2 cells
What is predominant in people with allergens? What does this cause to be produce?
- Th2
- IL-4 and IL-13
What are IL-4 and IL-13 responsible for?
- final differentiation that occurs in B cells, initiating the transcription of the genes that codes for the epsilon heavy chain of immunoglobulin molecules belonging to the IgE
describe allergic rhinitis
- most common form a of atopy
- hay fever
- symptoms —> paroxysmal sneering , itchy eyes and nose, nasal congestion, and runny nose
What is angioedema?
Skin reactions that occur deeper in the skin
Describe allergy immunotherapy (AIT)
- goal is to induce immune tolerance to a specific allergen by administering gradually increasing doses of the allergen over time
How is allergic reaction scored?
- based on presence or absence of erythema
- diameter of the wheal
What are the key components of type II?
- IgG and IgM directed against cell surface antigen
- complement
What effects can type II have on antibodies?
- cell destruction
- inhibition of cell function
- increase in cell function
What can cause cell damage in type II hypersensitivity?
- activation of classical pathway of complement and cell lysis
- opsonization and phagocytosis of the cell
- antibody
- antibody-dependent cell-mediated cytotoxicity (ADCC)
How long does it take for type II hypersensitivity to kick in?
A few hours after exposure to antigen
What happens when sensitized mast cells and basophils are activated?
Release chemicals that induce symptoms
Describe mast cells of the sensitization phase of type I?
- have abundant cytoplasmic granules that store numerous preformed inflammatory mediators
Describe bind of IgE to mast cells or basophils
- increases half life from 2-3 days to 10 days at least
- once IgE binds, functions as an antigen receptor on mast cells and basophils
What are effector cells of type I hypersensitivity?
Mast cells
Describe hemolytic disease of the newborn (HDN)
- pregnant woman produces antibodies to Rh antigens (usually RhD) on fetal RBCs
- IgG cross placenta and destroy fetal RBCs
Describe autoimmune hemolytic anemia
- type II hypersensitivity
- direct against self-antigens because this disease forms antibodies to their own RBCs
- 2 kinds of antibodies —> warm reactive antibodies and cold agglutinins
- symptoms —> jaundice, unexplained fever, lightheadedness, weakness, and malaise
What temperature does warm reactive antibodies react at?
37 C
What temperature does cold agglutinins react to?
Below 30 C
Describe the warm autoimmune hemolytic anemia
- account for more than 70% of autoimmune anemias
- characterized by formation of IgG antibody
Describe cold agglutinins
- autoantibodies that cause RBCs to clump together at cold temperatures
- below 37 C
- belong to IgM class
What is found in those affected with cold agglutinins syndrome?
- high titer of cold agglutinins of C3d resulting in a positive direct antiglobulin test
Describe paraoxysmal cold hemoglobinuria
- can cause autoimmune hemolytic anemia
- occurs after certain infections (measles, mumps, chickenpox, IM)
- agglutinate at cold temperatures
- activates complement at 37C to produce intermittent hemolysis
Describe mycoplasma pneumoniae
- cold agglutinins found in approximately 55% of patients
- cold agglutinins appear around week 2 or 3 after on set
- reoccurring respiratory infections
- symptoms —> enlarged spleen, jaundice, pallor, hemoglobinuria
describe infectious mononucleosis
- 50% of patients with disease have anti-i present
- antibody normally detectable in vitro up to a temperature of about 25C
Describe direct antiglobulin test (DAT)
- detects RBCs coated with complement components or IgG antibody in vivo
- patient RBCs are incubated with a poly-specific anti-human Ig directed against IgG and C’
- if positive , the test is repeated with mono-specific anti-IgG, anti-C3b and anti-C3d
Describe indirect antiglobulin test (IAT)
- Coombs test
- test patient serum for antibodies to RBC antigens
- positive test —> agglutination
Describe process of indirect antiglobulin test
1) incubate reagent RBCs with patient serum at 37C, wash to remove excess
2) add anti-human globulin
What is DAT used to detect?
- Transfusion reactions
- HDFN
- autoimmune hemolytic anemia
- drug induced hemolytic anemia
- detects in vivo binding
What is IAT used for?
- cross matching of blood to prevent a transfusion reaction
- determine the presence of particular antibody in patient plasma
- type patient RBCs for specific blood group antigens
- detects in vitro binding
Describe type III hypersensitivity
- keys components are IgGand IgM directed against a soluble antigen
- small antigen- antibody complexes precipitate out and deposit in tissues
- C’ binds —> vasodilation and vasopermability increase
- macrophages and neutrophils migrate to affected areas and release lysosomal enzymes, resulting in tissue damage
How long does type III hypersensitivity take to occur?
A few hours after exposure
Describe Arthus reaction
- skin reaction caused by type III hypersensitivity
- localized inflammation characterized by redness and edema
- peaks at 3 to 8 hours
Describe serum sickness
- generalized type III hypersensitivity reaction
- caused by passive immunization of humans with animal serum
- produces antibodies against the foreign animal proteins against the foreign animal proteins in patients
- cause immune complexes to form and deposit in tissues
- symptoms —> headache, fever, nause, joint pain, rashes, lymphadenopathy
What are the laboratory tests of type III hypersensitivity?
- testing ANAs
- fluorescent staining of tissue sections to detect deposited immune complexes
- testing for rheumatoid factor (anti-IgG)
- testing complement levels
When are complementary levels decreased in serum?
- during periods of high disease activity
What methods detect antinuclear antibodies in SLE and RA?
- indirect immunofluorescence
- ELISA
- fluorescent microsphere multiplex immunoassay
What methods can detect RF?
- latex agglutination
- nephelometry
- other immunoassays
Describe type IV hypersensitivity?
- Th1 and macrophages are involved
- APCs present antigen to naive Th cells, which differentiate into Th1 cells
- Th1 cells release cytokines that attract and activate macrophages
- macrophages induce inflammation
- cytotoxic T lymphocytes are recruited and destroyed target cells
Describe hypersensitivity pneumonitis
- allergic disease of lungs
- caused by inhalation of bacterial and fungal spores
- primarily mediated by sensitized T cells that respond to inhaled allergens
What are some examples of hypersensitivity pneumonitis?
- farmers lung disease
- birds breeders lung disease
- humidifier lung disease
Describe contact dermatitis
- low-molecular weight compounds contact the skin and act as haptens to sensitive Th1 cells
What are trigger for contact dermatitis?
- poison ivy
- poison oaks
- Nickles salts
- cosmetics
- hair dye
- latex
What are granoulomas?
- cluster of cells produced by chronic persistence of antigen
- can function to wall off the organism in contained area, preventing spread
What are the skin tests for type IV hypersensitivity?
- patch test
- skin testing for immunodeficiency
- mantoux method
Describe patch test
- antigen applied to skin surface
- test for contact dermatitis
- positive test —> redness with papules or blisters
Describe mantoux method
- antigen injected intradermally
- tests for TB exposure or T cell function
- positive test —> induration
What are types of interferon gamma release assays?
- quantification TB gold plus assay
- T-spot TB test
Describe interferon gamma release assays (IGRA)
- measures production of IFN- gamma by patient T cells stimulated with MTB antigens
Describe T-spot TB test
- patient mononuclear cells are incubated with MTB antigens and tested for IFN- gamma by ELISPOT
What is the gold standard testing for contact dermatitis?
Patch test
What could a positive result in DAT show?
- presence of IgG on RBCs
- presence of C3b or C3d on RBCs
- a transfusion reaction caused by preformed antibody
What is the difference between type I and type II?
Type II involves cellular antigens
A young woman developed red, itchy papules on her wrist 2 das after wearing a new bracelet. What is this reaction caused by?a
An inflammatory response induced by cytokines released from Th1
What are reactions to latex caused by?
- type I
- type IV
- skin irritation
What is the principle of the tuberculin test?
- PPD is injected into the forearm and stimulates a delayed type hypersensitivity response mediated by T cells in patients with prior exposure causes induration at the injection site within 48-72 hours
Describe autoimmune diseases
- humoral and cell-mediarted immune responses directed toward self-antigens
- cause tissue or organ damage
- systemic or organ-specific due to loss of self tolerance
What is self tolerance?
Ability of the immune system to accept self-antigens and not initiate an immune response against them
What does autoimmunity result from?>
- complex interactions between genetic and environmental factors
- inheritance of certain genes can make some individuals some susceptible
- hormones can influence autoimmunity
- tissue injury
- microbial infections
- environmental factor that can alter gene expression
What are the microbial that can trigger autoimmunity?
- molecular mimicry
- epitope spreading
- superantigens
Why does molecular mimicry trigger autoimmunity?
- bacteria or viruses possess antigens that are like a self antigen
Why does epitope spreading trigger autoimmunity?
- immune response to a microbe expands to activate, including self antigens
Why do superantigens trigger autoimmunity?
Can activate numerous clones of T cells, or through certain viruses, which can cause polyclonal B cells activation
What are systemic diseases of autoimmunity?q
SLE
RA
GPA
Others SARDS
What are organ specific diseases of autoimmunity?
- autoimmune thyroid disease
- type I diabetes
- celiac disease
- autoimmune liver disease
- multiple sclerosis
- myasthenia gravis
- anti-glomerular basement membrane disease
Describe immunologic tolerance
A state of immune unresponsiveness that is direct against a specific antigen
Describe central tolerance
Occurs in central or primary lymphoid organs, thymus, and bone marrow
What is anergy?
Absence of the normal immune response to a particular antigen or allergen
Describe peripheral tolerance
Lymphocytes that recognize self-antigens in the secondary lymphoid organs are rendered incapable of reacting with those antigens
What are the classification of autoimmune disease?
1) systemic
2) organ specific
What are the 2 groups of system diseases?
- systemic autoimmune rheumatic diseases (SARDs)
- anti-neutrophil cytoplasmic antibodies (ANCAs)
Describe SARDs
Involve inflammation of the joints and their associated structures
Describe ANCAs
Characterized by inflammation of the blood vessel
Describe system lupus erythematous (SLE)
- chronic sytstemic inflammatory disease that affects multiple organ systems
- pathology caused by type III, tissue damage occurs at the sites in thee body were immune complexes have deposited
- develop numerous autoantibodies
- symptoms —> joint involvement, skin rash, renal involvement, weight loss, malaise, fatigue
What happens when immune complexes form within a patient with SLE?
Trigger
- complement activation
- chemotaxis of neutrophils
- inflammation
What autoantibodies develop in patients with SLE?
- dsDNA and other nuclear components
- lymphocytes
- RBCs
- platelets
Describe the skin rash that occurs on patients with SLE
- a classic butterfly rash across the nose and cheeks
- this is where lupus comes from, meaning wolf-like
Describe the LE cell
- it is a neutrophil that have engulfed the antibody-coated nucleus of another neutrophil, mainly in vitro
What are the laboratory tests to diagnosis SLE?
- CBC
- urinalysis
- CRP/ESR
- complement quantitation
- ANAs
What test is typically done first if SLE is suspected?
- screening test for antinuclear antibodies (ANAs)
What is the most common cause of death is lupus?
- renal failure and infection
Describe ANAs
- directed against antigens in cell nuclei
- present in more than 95% of lupus patients
- includes
—> anti-dsDNA
—> anti-ssDNA
—> anti-histones and nucleosomes
—> antibodies to centromere or nuclear component
—> anti-ENA
What antibody (ANA) is most specific for SLE?
- dsDNA
What is a nucleolus?
Prominent structure within the nucleus where transcription and processing of ribosomal RNA and assembly of ribosomes take place
What testing is most widely used to detect ANAs?
Fluorescent antinuclear antibody
What is the FANA test principle?
1) incubate patient serum with hep-2 cells fixed no microscope slide
2) wash and incubate with fluorescein-labeled anti-human IgG
3) wash and view under-fluorescent microscope
Describe ENA Ouchterlony test
- immunodiffusion test detects antibodies to specific ENA
- patient serum in outer wells react with ENA antigens in center well
- anti-sm is diagnostic for SLE
- positive reactions indicated by immunoprecipation lines of serological identity
- not as sensitive
- long turn around times
What are the 3 main applications of IIF?
1) as initial test to determine the presence or absence of ANAs in patients with SARDs, aiding in diagnosis
2) to provide guidance in selection of follow-up tests based on the immunofluorescence patterns obtained
3) to determine ANA titer because moderate to high titers have been better correlated with disease
Why are HEp-2 cells used in IIF?
- they have large nucleic with high antigen expression, allowing for high sensitivity and facilitating visualization of results.
What are the 4 major nuclear patter groups?
1) homogenous
2) speckled
3) centromere
4) nucleolar
Describe homogenous nuclear pattern
- characterized by uniform staining of entire nucleus in interphase cells and of condensed chromosomal region in metaphase cells
- associated with dsDNA, his tones, and nucleosomes
When is homogenous nuclear patterns presents?
Found in patients with
- SLE
- drug induced lupus
- AIH
- juvenile idiopathic arthritis
Describe the speckled nuclear pattern
- characterized by discrete fluorescent specks throughout the nuclei of interphase cells
- 3 groups: dense fine, tiny/fine, and large/course
What are fine or coarse speckled patterns associated with?
- antibodies to ENAs
- SLE
- Sjorgen’s syndrome
- SSc
- other SARDs
What is the dense speckled pattern associated with?
- antibodies to the DFS70/LEDGF antigen
- correlated with absence of a SARD upon confirmatory test
Describe the centromere nuclear pattern
- numerous discrete speckles are seen in nuclei of interphase cells and the chromatin of divding cells
- 46 speckles, represent each chromosome
- found mainly in patients with CREST syndrome
Describe the nucleolar nuclear pattern
- prominent staining of the nucleoli within nuclei of interphase cells is seen in this pattern
- seen in pateints with SSc but also can be present in other SARDs
Describe rheumatoid arthritis (RA)
- chronic arthritis of the peripheral joints that can progress to joint deformity and disability
- 25% of patients have osteoporosis
- some patients also develop
- subcutaneous nodules
- pericarditis
- interstitial lung disease
- vasculitis
Describe pathology of RA
- inflammation destroys the bone and cartilage
- TNF-alpha plays key role in the process
- overly active osteoclasts absorb the bone
- autoantibodies combine with antigen to form immune complexes
What are some laboratory tests for RA?
- Rheumatoid factor
- anti-CCP
- ANAs
- ESRs, CRP and C’
Dscescribe rheumatoid factor
- autoantibody (usually IgM) that reacts with Fc portion of IgG
- found in approximately 80% of patients with RA, not specific for RA
Describe anti-CCP
- autoantibody directed against cyclic citrullinated peptide
- high specifc for RA
- best for early detection of RA
What is pannus?
A sheet of inflammatory granulation tissue that grows into the joint space and invades the cartilage
What is the gold standard for ANA testing?
IIF
What are symptoms of RA?
- transient joint pain in hands and feet
- muscle spasms
- limited movement
- weight loss
- fatigue
- fever
- malaise
What autoimmune disease is characterized by dry mouth and eyes?
Sjorgren syndrome
What is the most common cause for death in RA?
Cardiovascular disease
What are the criteria for Identifying RA?
- number and type of joints involved
- duration of symptoms
- serology results for RF and anti-CCP
- serum level of the acute phase reactant
- CRP
- ESR
Describe sjogren syndrome
- dry eyes and mouth
- ss-A and ss-B antibodies
- characterized by chronic inflammation of the exocrine glands, most notably —> ocular and salivary gland
- have primary and secondary categories
Describe systemic sclerosis (SSc)
- fibrosis and vasculitis affecting skin, joints, other organs
- includes CREST syndrome and many ANAs
- symptoms
—> tightening and hardening of skin
—> musculoskeletal pain
—> Raynaud’s phenomenon
—> heartburn
Describe mixed connective tissue disease
- overlap of limited cutaneous SSc and other SARDs; anti-u1-RNP
Describe inflammatory myopathies
- polymytosis and dermatomyostis
- progressive muscle weakness; many ANAs
- characterized by chronic inflammation of the skeletal muscles and progressive muscle weakness
Describe granulomatosis with polyangitis
- Wegners granulomatosis
- rare autoimmune disease involving inflammation of small-to-medium sized blood vessels and respiratory tract
- progresses to more systemic disease involving other organs
- most patients have antibodies to neutrophil cytoplasmic antigens such as proteinase 3
- neutrophil activation results in damage to vascular endothelium and Th1 response
What are symptoms of granulomatosis with polyangitis? (GPA)
- collapsed nose bridge
- hearing loss
- fever
- joint pain
- anorexia/weight loss
- persistent runny nose
- pain and arthritis of large joints
- rhinitis
- oral or nasal ulcers
Describe anti-neutrophil cytoplasmic antibodies
- produced against proteins in neutrophil granules
- strongly associated with syndromes involving vascular inflammation
- detected by IIF of ethanol-fixed leukocyte
- ELISA and chemo luminescent assay are also available
Describe ANCA detected by IIF on ethanol fixed leukocytes procedure
1) patient serum incubated with microscope slide containing ethanol-fixed leukocytes
2) wash and add FITC-labeled anti-IgG conjugated
3) wash and view fluorescence in neutrophils under fluorescence in neutrophils under fluorescent microscope
What are the 3 syndromes involving vascular inflammation associated with ANCAs?
- GPA
- microscopic polyangitis (MPA)
- eosinophil granulomatosis with polyangitis (EGPA)
What are the two patterns that can be observed with ANCAs?
- cytoplasmic (c-ANCA)
- pernicular (p-ANCA)
Describe c-ANCA pattern
- primarily caused by PR3-ANCA and appears as a diffuse, granular staining in cytoplasm of neutrophils
Describe p-ANCA pattern
- fluorescence surrounds the lobes of the nucleus, blending them together so that individual lobes cannot be distinguished
What is the autoantigen of c-ANCA?
PR3 antigen
What disease is associated with c-ANCA?
GPA
What is autoantigen of p-ANCA?
Positively charged antigens, including MPO
What are disease associated with p-ANCA?
- MPA
- EGPA
What diseases can ANCA be detected in?
- SARDs, such as SLE and RA
- autoimmune gastrointestinal disease
- liver disease
- HIV
- hepatitis C
- malignancy
Describe autoimmune thyroid diseases (AITDs)
- thyroid hormone synthesis is carefully regulated by endocrine feedback loop
- autoantibodies produced in AITDs can lead to to decreased or increased production of thyroid hormones
What diseases are AITDs?
- Hashimoto’s thyroiditis
- Graves’ disease
What is the target tissue of Graves’ disease and hasimotos disease?
Thyroid gland
Describe Hashimoto’s thyroiditis
- immune destruction of the thyroid gland produces hypothyroidism
- lab results:
—> normal or high TSH
—> low free T4
—> anti-TPO
—> anti-Tg - symptoms
—> formation of goiter
—> brittle hair
—> weight gain
—> dry skin
—> fatigue
What is hypothyroidism?
- Decreased thyroid function
- symptoms
—> puffy face with edematous eyelids
—> descreased sweating
—> brittle hair
—> pallor with yellow tinge
—> dry skin
—> weight gain
—> fatigue
Describe Graves’ disease
- AITD characterized by hyperthyroidism
- TRAbs produced
- low TSH and high FT4; anitbodies to TPO and Tg may be produced
What are autoantibodies of Hashimoto’s thyroiditis?
- anti-thyroglobulin
- anti- thyroid peroxides
What are the autoantibodies of Graves’ disease?
- TSH receptor antibodies (TRAbs)
- anti-thyroglobulin
- anti-thyroid peroxides (TPO)
What is hyperthyroidism?
- state of excess thyroid function
- associated with Graves’ disease
What are the symptoms of Graves’ disease?
- bulging eyes
- nervousness
- insomnia
- depression
- heat intolerance
- sweating
- heart palpitations
- breathlessness
- lower leg edema
- cardiac dysrhythmias
What are most commonly used to detect thyroid antibodies?
- sensitive ELISA
- chemiluminescent immunoassays
What is the best indicator for Hashimoto’s thyroiditis?
Antibodies to TPO
What is highly indicative of Graves’ disease?
TRAbs
What are the 2 tests for TRAbs?
- binding assay
- bioassay
Describe binding assays for TRAbs
- automated solid-phase ELISA
- chemiluminescent immunoassays
- labeled antibody for TSH receptor bound to solid phase
- unable to distinguish between TSI and TRAbs
Describe bioassay for TRAbs
- requires tissue culture
- difficult to perform
- specificity measure the function of TSI
Describe type I diabetes mellitus (T1D)
- endocrine disorder characterized by hyperglycemia
- type I destruction of beta cells in pancreas results in insulin deficiency
- genetic susceptibility to the disease
What are the long term effects of type I diabetes mellitus?
- cardiovascular disease
- kidney dysfunction
- nerve damage
- blindness
- infections
What would the results look like for a patient with type I diabetes mellitus?
- increased glucose blood level
- elevated HbA1
- autoantibodies to GAD and IA-2, ICA
What is hyperglycemia?
A high level of glucose in the blood
What are the 4 types of diabetes?
- type I
- type II
- gestational
- neonatal/ pancreatitis
What leads to fibrosis criteria that is used to diagnose type I diabetes mellitus?
Progressive inflammation of the islets of langerhans in pancreas
What are the 4 criteria that is used to diagnosis type I diabetes mellitus?
1) fasting glucose greater than 126 mg/dL on more than one occasion
2) a random plasma glucose level of 200 mg/dL or more with classic symptoms of diabetes
3) oral glucose tolerance test of 200 mg/dL or more in a 2 hour sample with a 75 g glucose load
4) a hemoglobin A12 value (HbA1c) greater than 6.5%
Describe celiac disease?
- affects small intestine and other organs
- triggered by gluten
- symptoms
—>diarrhea
—> abdominal pain
—> bloating
What is the diagnosis of the celiac disease based on?
- clinical symptoms
- serological test findings
- duodenal biopsy
- presence of HLA-DQ8 or HLA-DQ2 haplotype
Describe autoimmune hepatitis
- hepatocytes targeted
- AIH-1 —> positive for SMA, ANAs
- AIH-2 —> positive for LKM-1 or LC-1 antibodies
- affects small interlobular bile ducts
What are the alleles are associated with AIH-1?
HLA-DR3 and HLA-DR4
What alleles are associated with AIH-2?
HLA-DRB1 and HLA-DQB1
What is necessary to confirm AIH?
Liver biopsy
Describe PBC
- destruction of intrahepatic bile ducts; cholestasis
- inflammation of portal vein in liver
- accumulation of scar tissue that can lead to cirrhosis and liver failure
- majority of patients, produce mitochondrial Abs (AMAs)
- symptoms
—> itchy skin
—> greasy stool
—> dry mouth and eye
—> abdominal pain
—> jaundice
—> fatigue
What is the most common autoimmune liver disease?
PBC
What is cholestasis?
A condition in which flow of bile is slowed or blocked
What methods are these to detect AMAs?
- IIF
- immunoblotting with mitochondrial preparations from mammal tissues
- ELISA
- fluorescent microbead immunoassay
Describe multiple sclerosis (MS)
- involves inflammation and destruction of the central nervous system
- most patients produce antibodies against myelin basic protein
- plaques form in while matter of the brain and spinal cord, causing destruction of the myelin sheath of axons
- symptoms
—> weakness in limbs
—> facial palsy
—> visual disturbance
—> dizziness
—> sensory abnormalties
What would be the laboratory findings of a patient with MS?
- lesions on magnetic resonance imaging (MRI)
- increased immunoglobulins in spinal fluid and increased IgG index
- oligoclonal bands on protein electrophoresis of CSF
What are environmental factors that have been associated with MS?
- reduced exposure to sunlight
- vitamin D deficiency
- cigarette smoking
- infection with EBV after early childhood
Describe myasthenia gravis
- affects neuromuscular junction, resulting in weak skeletal muscles
- most patients have antibodies to acetylcholine receptors (ACHR) which block binding of ACH to receptor and transmission of nerve impulses that activate muscles
- symptoms
—> drooping eyelids
—> inability to retract corners of the mouth
—> difficulty speaking, chewing, and swallowing
—> respiratory muscle weakness
—> associated with several HLA antigen abnormalities
What other diseases are associated with presence of MG?
- SLE
- RA
- pernicious anemia
- thyroiditis
Describe laboratory testing for MG
- RIA is used to detect antibody, based on assays that block the binding of receptors by anti-ACH receptor (ACHR) antibody
- precipitation of receptors caused by combination with antibody is then measured
- a quick immuno stick ELISA has also been developed for testing
What is the gold standard for detecting binding AChRs?
Quantitative radioimmunoassay that have radio-labeled snake venous
Describe anti-glomerular basement membrane disease
- formlerly known as good pasture syndrome
- patients produce autoantibodies to basement membranes living the renal glomeruli and lung alveoli
- immune complexes bind to basement membranes, attract complement and cause damage by type II hypersensitivity
- antibodies to GBM found in most patients
- symptoms
—> edema
—> hypertension
—> malaise
—> fatigue
Describe testing for anti-GBM disease
- detected by IIF on frozen kidney sections or ELISA for antibody to a 3-subunits
- tissue-bound anti-GBM detected by direct immunofluorescence on kidney biopsy sections
- produces a smooth, linear, ribbon-like fluorescence along the GBM
What allele has a strong association with anti-GBM disease?
HLA-DRB1-15
What detects GBM antibodies currently?
- ELISA
- fluorescent microbead immunoassay that uses recombinant alpha-3 (V) antigen substrates
- confirmation —> western blot
What is symbiotic relationship?
- hosts and microbes live together long term
- indigenous microbiota
What is a commensalstic relationship?
- no benefits or harm to either organism
What is mutualistic relationship?
Both host and microbes benefit
What is parasitic relationships?
Microbes cause harm to host
What are three types of symbiotic relationship?
Commensalisitic
Mutualistic
Parasitic
What does the relationship within our indigenous microbiota exist through?
- co-evolution
- co-adaption
- co-dependency
What needs to happen for a microorganism to survive?
- needs to stimulate a immune response
- needs to colonize the host and acquire nutrients
What is infectivity?
- Organisms ability to establish an infection
- “contagious”
What is pathogenicity?
- ability of an organism to cause disease
- qualitative trait
Describe virulence?
- extent of damage and pathology caused by an organism when it infects a host
- quantitative trait
What does virulence factors increase an organisms pathogenicity?
1) organisms ability to establish itself on or in the host
2) invade or damage host tissue
3) evade host-immune response
Describe endotoxins
- the lipid A portion of LPS in gram-negative cell walls
- powerful stimulator of cytokine release
- complement activator, which forms anaphylatoxins C3a and C5a, which causes vasodilation and increased vasopermability
Describe exotoxins
- potent toxic proteins released from living bacteria (mostly gram-positive)
- neurotoxins, cytotoxins, enterotoxins
- known as most potent molecules to harm living organisms
- extremely immunogenic
- induce production of protective antibodies
- may act like superantigen
What is virulence factor?
Bacterial properties that determine whether an organism is pathogenic and able to cause disease
What are plasmids?
- self replicating extra-chromosomal DNA molecules that are located in the bacteria’s cytoplasm
- contain limited number of genes
What are bacterial cells classified as?
Prokaryotic cells
What are human cells classified as?
Eukaryotic cells
What are the difference between prokaryotic and eukaryotic cells?
- P—> bacterial chromosomes are found in bacterial cytoplasm. E—> enclosed in membrane-bound nucleus
- P—> bacteria cell wall is outermost feature. E—> plasma membrane is outermost feature
- P—> cell wall contains alototenymes
What is the primary component that provides shape and rigidity of bacterial cells?
Peptidoglycan
What are the two variations of bacterial cell wall?
- positive gram
- negative gram
What variations of bacteria contains lipopolysaccharide layer (LPS)?
Gram-negative
What are the 3 components of LPS?
- outer core polysaccharide
- inner core polysaccharide
- lipid A
How does the capsule of bacteria contribute to the organisms ability to resist innate and adaptive immune response?
1) block the attachment of antibodies
2) inhibit activation of complement
3) act as a decoy when capsular material is released into the surrounding host environment
What is the most important feature of of the capsule?
Blocking phagocytosis by WBCs
What cytokines are produced by endotoxins?
IL-1
IL-6
IL-8
TNF
PAF
What are two defenses against bacteria?
Innate defenses
Adaptive defenses
Describe innate defense against bacteria
- intact skin and mucosal surfaces serve as structural barriers
- antibacterial defense peptides are lysozyme, defensins, ribonuclease
- complement proteins, cytokines, acute-phase reactants
- recognition of PAMPs by PRRs such as TLRs
Whta is the function of a ribonuclease?
Destroy RNA and have antimicrobial and antiviral activities
What are the 3 classes of defensins?
Alpha
Beta
Theta
What occurs when PRR engages with appropriate PAMP?
1) triggers release of immune mediators such as cytokines and chemokines
2) boosts production for various defensins and proteins
3) initiates phagocytosis
Describe adaptive defenses for bacteria
- antibody production —> binding C’, osponization, neutralization of bacterial toxins
- cell-mediated immunity —> CD4 T cells produce cytokines that induce inflammation; cytotoxic T lymphocytes attack host cells that contain intracellular bacteria
- Uses granulosomes to prevent spread
What is the main defense against bacteria in adaptive immunity?
Antibody production
What is the binding of antibodies to invading bacteria referred to as?
Opsonization
What mechanism do bacteria do use to inhibit the innate immune response?
1) avoiding antibody
2) blocking phagocytosis
3) inactivating the complement cascade
4) blocking digestion
5) inhibiting chemotaxis
6) cleaving IgA
What mechanisms do microorganisms use to resist digestion?
- block fusion of lysosome granules with phagosomes after being engulfed by phagocyte
- production of extra cellular products after bacteria is phagocytosized
- block action of complement
What are some laboratory detection tests of bacterial infection?
1) culture of causative agent
2) microscopic examination
3) detection of bacterial antigens
4) molecular detcection of bacterial DNA or RNA
5) serology
Describe cultures of causative agent
- grow on broth or solid media
- major means of diagnosis, but may take time or may not be possible
Describe microscopic examination for bacteria
Uses gram stain or special stains
What tests are for detection of bacterial antigens?
Rapid testing or special stains
- ELISA
- LFA
- LA
Describe serology testing for bacteria
- detects antibodies to bacterial antigens
- to detect and confirm when option are not available
- to diagnosis infections with nonspecific symptoms
- current infection indicated by IgM high titer of IgG, or fourfold rise in antibody
- to determine a past exposure to an organism
- to assess reactivation or re-exposure
What do the results of a serology test look like when a past exposure to an organism is present?
IgM
IgG+
What are the disadvantages of serology testing for bacterial infection?
- delay between start of infection and production of antibodies
- low antibody production by immunosuppressed patients
What are disadvantages of culturing for causative agents?
- several bacterial pathogens that don’t have cultures available
- may take too long
- some organisms are difficult to grow
- some organisms present a danger to start it handled improperly
Describe gram stain
- gram-positive bacteria stains purple with crystal violet
- gram-negative bacteria stains pink with safranin
What is the most widely known molecular technology?
PCR
Describe streptococci structure
- are gram positive bacteria
- spherical, Ovoid, or lancet shaped organisms that are catalase negative and are often seen in pairs or chains
- divided into groups on basis of certain cell wall components, including two major proteins known as M and T proteins
Describe streptococci
- transmitted person to person
- additional virulence factors include exoantigens or exotoxins, proteins are secreted by the bacterial cells as they metabolize during the course of streptococcal infections
- detection of host antibodies exotoxins is important in the diagnosis of sequelae such as glomerulonephritis and acute rheumatic fever
What is another s. Pyogenes?
Group A streptococcus (GAS)
Describe serotyping for GAS
- used to identify a particular strain of GAS
- involves ID of the M protein antigens by precipitation with type-specific sera
What are disadvantages of serotyping?
- limited availability of type sera
- new M proteins do not react with anti-sera
- difficulty interpreting results
What is the most common and ubiquitous pathogenic bacteria?
GAS
what are virulence factors of GAS?
- the M protein
- has net-negative charge at the amino terminal end that helps to inhibit phagocytosis
- M protein limits deposition of C3 on the bacterial surface (diminishing complement activation)
What are clinical manifestations of pus forming group A streptococcal (GAS) infection?
- pharyngitis (“step throat”)
- pyoderma (impetigo or skin infection)
What are the clinical manifestations non-pus forming group A streptococcal (GAS) infection?
- scarlet fever
- toxic shock syndrome
- necrotizing fasciitis
What are the two major sites of strep A infection?
- upper respiratory tract
- the skin
What are symptoms of pharyngitis?
- tonsillar exudates
- petechial rash on the soft palate
- severe sore throat
- chills
- fever
Describe impetigo
- most common skin infection
- vesicular lesions on extremities that become pustular and crusted
- symptoms
—> lesions
—> otitis media
—> cellultiis
Describe scarlet fever
- usually associated with pharyngeal infections
- initially appears on the neck and chest then spreads over the body
- results from infection with strep A that elaborates SpeA and SpeC (can act as superantigens)
- symptoms
—> fever of 101 F or higher
—> rash
—> abdominal rash
—> vomiting
—> nausea
—> headache
Describe necrotizing fasciitis
- may occur when Strep A
- such infection invades the muscles in the extremities or the trunk
- exotoxins produced by s. Pyogenes cause rapid infection deep in fascia
Describe sequelae
- conditions that are consequence of a previous disease or injury
- results from the host response to infection
What are the two serious sequela that could be developed from Strep A?
- acute rheumatic fever
- poststreptococcal glomerulonephritis
Describe acute rheumatic fever
- develops 1-3 weeks after pharyngitis or tonsillitis in 2-3% of infected individuals
- mostly likely caused by immune responses to streptococcal antigens that cross-react with human heart tissue
- only follow upper respiratory tract infections
Describe post streptococcal glomerulonephritis
- may follow strep infection of the skin or pharynx
- damages glomeruli
- deposits of immune complexes containing streptococcal antigens in glomeruli
- characterized by damage to the glomeruli in the kidneys
- impaired because glomerular filtration rate is reduced
What are symptoms of acute rheumatic fever?
- fever
- joint pain
- inflammation of heart
What are the symptoms of post streptococcal glomerulonephritis?
- hematuria
- proteinuria
- edema
- hypertension
- malaise
- backache
- abdominal discomfort
- impairment renal function
What laboratory tests are run for suppurative strep A?
- culture on sheep blood agar
- rapid assays to detect strep A
What are rapid assays used to detect suppurative strep A?
- strep screen
- LFA
- assays are easy to perform
- allow for a single sampling testing
- more sensitive
- cultured should be ran if rapid assays are negative
Describe serological detection of strep A sequelae
- antistreptolysin O (ASO) test
- classic hemolytic method for determining the ASO tier was the first test developed to measure streptococcal antibodies
- based on the ability of patient antibodies to neutralize the hemolytic activity of streptolysin O
- an ASO titer greater than 166 Todd units is considered a positive test
What are the serological detection test of nonsupprative strep A?
- antistreptolysin O (ASO)
- anti-DNase B
- streptozyme test
Describe antistreptolysin O (ASO) test of nonsupprative strep A
- nephelomatic methods currently used that measure light scatter produced by immune complexes containing streptolysin antigen
- titer is elevated in 85% of patients with acute rheumatic fever
- automatic and rapid
Describe anti-DNase B test of nonsuppurative strep A
- produced by both rheumatic fever and impetigo patients
- tested by EIA and nephelomatic methods
- useful in patients suspected of having glomerulonephritis preceded by skin infection
- highly specific for strep A
- if anti-DNase antibodies are present, they neutralize reagent DNase B, which prevents it from depolymerizing DNA
- tubes are graded by color, 4+ indicating intensity of color unchanged and 0 being complete color loss
Describe the streptozyme test
- a slide agglutination screening test for detection of antibodies streptococcal antigens
- sheep RBCs are coated with the 5 antibodies
- hemagglutination is a positive test result
- positive in 95% of patients with acute phase post streptococcal glomerulonephritis
- detects antibodies to five streptococcal products:
—> ASO
—> anti-hyaluronidase (AHase)
—> anti-streptokinase (ASKase)
—> anti-nicotinamide-adenine dinucleotide (anti-NAD)
—> anti-DNase B
What are some disadvantages of the streptozyme test?
- the lest reproducible test for antibodies
- more false negative and false positives have been reported
Describe heliobacter pylori
- gram- negative microaeorphilic spiral bacterium with flagella
- transmission likely by fecal-oral route
- major cause of gastric and duodenal ulcers
- can survive in acidic environment of the stomach because of production of urease, which provides a buffering zone around the bacteria
- if untreated, can lead to gastric carcinoma or mucosa-associated lymphoid tumors
- resides in mucus layer, b the gastric epithelium cells
What are detection tests for heliobacter pylori?
- detect urease in stomach biopsy (CLO test)
- urea breathe test
- heliobacter pylori antigens
- heliobacter pylori antibodies
- ELISA is method of choice
- IgG serum indicates active infection
What are virulence factor of H. Pylori ?
Culture
Describe urea breath test
- patient ingests urea labeled with radioactive carbon or nonradioactice carbon broken down by the Reese’s enzyme of H. Pylori producing ammonia and bicarbonate
- excellent sensitivity and specificity
An H. Pylori infection results in production of what?
IgG (test detect this class)
IgA
IgM
What is considered a successful treatment of H. Pylori infection?
Decrease in antibody titer more than 25%
Describe mycoplasma pneumonia
- tiny bacteria that lack a cell wall
- leading cause of respiratory droplets
- belong to class mollicutes
- incubation period is 1 to 3 weeks
- infection has insidious onset
- symptoms
—>fever, headache, malaise, and cough
What are clinical manifestations of mycoplasma pneumonia?
- walking pneomonia
- Raynaud syndrome
- Stevens-Johnson syndrome
What is a mycoplasma?
- represents the smallest known free-living life forms
- have small genome
What are laboratory diagnosis procedures for M. Pneumonia?
- culture
- antibody detection
- cold agglutinins
- molecular methods
Describe a culture of M. Pneumonia
- gold standard but isn’t used anymore
- produces mulberry colonies with a “fried egg” appearance on specialized media
Describe antibody detection for M. Pneumonia
- most useful diagnostic assay
- IgM —> recent-infection
- IgG —> possible reinfection
- most widely used —> EIAs
Describe cold agglutinins testing of M. Pneumonia
- present in 50% of patients M. Pneumonia but not specific for the infection
- cause RBC agglutination at 4 C
- reversible at 37 C
- not very specific or sensitive
Describe molecular methods of M. Pneumonia
- will most-likely become gold standard
- LAMP technology
Describe rickettsial infections
- obligate intracellular gram-negative bacteria
- spotted fever group (spf) —> Rocky Mountain spotted fever
- typhus group (tg) —> epidemic typhus
- organisms transmitted by arthropods (tick, mites, lice, or fleas) through biting after feeding on an infected animal
Describe Rocky Mountain spotted fever (RMSF)
- caused by R. Rickettsii
- transmitted by 3 species of ticks
- symptoms
—> rash that starts on hands and soles of feet and proceed to trunk
—> headache
—> nausea
—> vomiting
—> diarrhea
—> death
What is RMSF diagnosed?
- clinical presentation of symptoms
- serology by IFA (gold standard)
How do RMSF organisms spread in the body?
- via lymphatic and circulatory system
- attach to and invade target cells (vascular endothelium)
- organism multiply by binary fission
What is the main pathophysiological event caused by RMSF?
Endothelial cell damage which leads to increased vascular permeability, resulting in edema, hypovolemia, hypotension, and hypoalbuminemia
What is principle of the IFA test for detection of antibodies produced in RMSF?
1) specific antibodies in serum sample attach to the antigens fixed to microscope side
2) attached antibodies are stained with fluorescein-labeled anti-human immunoglobulin and visualized with a fluorescence microscope
What is the principle of LFA?
- involves movement of a liquid sample containing labeled antibodies specific to analyte
- if antigen-antibody complex is present, it is captured by another antibody at the end of the strip, resulting in a visible line
What are spirochetes?
- long, slender, helically coiled bacteria containing axial filaments or periplasmic flagella
- wind around the bacterial cell wall and are enclosed by an outer sheath
- gram-negative
- microaerophilic
- corkscrew flexi on and motility
Describe spirochete diseases
- caused by spirochete bacteria
- localized skin infection, disseminates to numerous organs as disease progresses
- cardiac and neurological involvement if disease remains untreated
What are the two major spirochete diseases?
Lyme disease
Syphilis
Describe syphilis
- sexually transmitted disease caused by treponemal pallidum
- rapidly destroyed by heat, cold, and drying
- direct contact with open lesion needed
- transmission to fetus during pregnancy
- bloodborne transmission rare
- most common spirochete disease
- have TROMP membrane proteins
What are the pathogens in the palladium subspecies?
- T. Pallidum subspecies pertenue
- T. Pallidum subspecies endemicum
- T. Carateum
Describe T. Pallidum subspecies pertenue
- agents of yaws
- found in tropics
Describe T. Pallidum subspecies endemicum
- cause of nonvernereal endemic syphilis
- found in desert regions
Describe T. Carateum
- the agent of pints
- found in central and South America
What are the stages of syphilis?
1) primary stage
2) secondary stage
3) latent stage
4) teritary stage
What is a characteristic of the primary stage of syphilis?
Development of chancre
What are symptoms of the secondary stage of syphilis?
- rash
- generalized lymphadenopathy
- fever
- malaise
- pharyngitis
Describe the latent stage of syphilis?
- asymptomatic
- follows disappearance of secondary stage
- patients are not infectious at this time with the exception of pregnant women
What are the symptoms of the tertiary stage of syphilis?
- gummatous
- cardiovascular
- neurosyphilis
- Tabes dorsalis (shuffling gate)
Whta stages of syphilis could spontaneous healing occur?
Primary
Secondary
Describe tertiary stage of syphilis
- stage occurs most often 10-30 years following the secondary stage
What are gummas?
- localized areas of granulomatous inflammation that are most often found on bones, skin or subcutaneous tissue
- contains lymphocytes, epithelial cells and fibroblasts
- may heal spontaneously with scarring
Describe cardiovascular complications in the tertiary stage of syphilis
- involve ascending aorta
- symptoms caused by destruction of elastic tissue in aortic arch
Describe congenital syphilis
- occurs when a woman who has early syphilis or early latent syphilis transmit treponemae’s to the fetus
- most affected in second and third trimester
- causes death in 10% of cases
What are laboratory diagnostic procedures of syphilis?
- direct detection
—> demonstration of treponemal in active lesion
—> dark-field microscopy
—> fluorescent antibody staining - serological test
—> nontreponemal
—> treponemal
How can primary and secondary syphilis by diagnosed?
- presence of T. Pallidum in exudates from skin lesions
When do false negatives occur in dark-field microscopy when testing for syphilis?
1) a delay in evaluating the slides
2) insufficient specimen obtained
3) patient is pretreated with antibiotics
What testing should be performed for syphilis if no lesions are present?
Serological
Describe nontreponemal tests
- detect antibody against cardiolipin (Reagin)
- sensitive, inexpensive and simple to perform
- includes:
—> venereal disease research laboratory test (VDRL)
—> rapid plasma Reagin test (RPR)
—> both - look for flocculation
—> screen - undiluted patient serum
—> titer - test two-fold dilutions of patient serum
Describe VDRL test procedure
1) patient serum mixed on slide with cardiolipin-lecithin-cholesterol antigen suspension
2) rotated for 4 minutes at 180 RPM
3) viewed under a light microscope for flocculation
Results:
- reactive —> medium to large clumps
- weakly reactive —> small clumps
- nonreactive —> no clumps or slight roughness
What is Reagin?
- a lipid released from membranes of cells damaged as a result of the infection
Describe the RPR test procedure
1) patient serum mixed on a card with charcoal particles coated with cardiolipin antigen
2) rotate 8 minutes, 100 RPM
3) observe for macroscopic flocculation
Briefly describe VDRL
- quantitative and qualitative slide flocculation
- all sera that is weakly reactive and reactive must be retested using quantitative slide test
Why are patients in the secondary stage of syphilis subject to a false positive?
Prozone phenomenon (antibody excess)
Briefly describe RPR
- cardiolipin - containing antigen suspension is bound to charcoal particle, making results easier to read
- all active tests should be confirmed by retesting using doubling dilutions in a quantitative procedure
- more sensitive than VDRL in primary stage of syphilis
Describe treponemal tests for detecting syphilis
- detect antibody to T. Pallidum
- usually positive before nontreponemal tests
- lack of sensitivity in congenital syphilis and neurosyphilis
- fluorescent treponemal absorption (FTA-ABS)
- T. Pallidum particle agglutination (TP-PA)
- automated immunoassays
—> ELISA
—> CLIA
—> MFI
Briefly describe FTA-ABS
- an indirect immunofluorence test antibody to T. Pallidum
- use Reiter strain
- false positive results may occur in patients with SLE
Describe FTA-ABS test procedure
1) patient serum is incubated with sorbent to remove cross-reacting antibodies
2) absorbed patient serum is incubated with a microscopic slide fixed with T. Pallidum (Nichol’s stain)
3) folllowing a wash step, anti-human Ig conjugated with fluorescein is added
4) after second incubation, and wash, the slide is examined under a fluorescent microscope
Describe TP-PA test procedure
1) patient serum and controls are diluted and incubated with unsensitized gel particles or gel particles sensitized with T. Pallidum
- positive —> agglutination (smooth may covering surface of well)
- negative —> no agglutination (button)
Briefly describe TP-PA test
- used colored gelatin particles coated with treponemal antigens
- more sensitive in detecting primary syphilis
What types of EIAs are used to detect the antibodies to T. Pallidum?
- one or two-step sandwich assays
- one step competitive assays
- immune capture assays
Describe sandwich assays of syphilis
- antibodies in the patient sample to bind to recombinant T. Pallidum antigens coated onto microtiter plate wells
Describe the immune capture of syphilis
- micrometer wells are coated with antibody to IgM or IgG and are reacted with patient serum
What are capture EIAs useful at diagnosing?
Congenital syphilis
Describe competitive EIAs of syphilis
- treponemal antibody in the patient sample competes with an enzyme-labeled treponemal antibody conjugate for T. Pallidum antigens bound to microtiter plate wells
Describe CLIAs of syphilis
- one-step sandwich assay
—> higher sensitivity, faster performance, and more stable reagents
—> patient sample is incubated with paramagnetic microparticles that have been coated with T. Pallidum anitgens linked to chemiluminescent derivative
describe PCR testing of syphilis
- involves isolating and amplifying a specific sequence DNA
- extremely sensitive, more than dark field microscopy
- availability is limited
What is the main disadvantages of nontreponemal testing?
Subject to false positive
What should a reactive nontreponemal test be confirmed by?
A more specific treponemal test
When should treponemal test for syphilis by performed?
- confirmatory test to distinguish false positive from true positive nontreponemal results
- establish diagnosis in late latent or late syphilis
Why are nontreponemal test for syphilis performed?
- screening for syphilis
- monitoring the progress of the disease
- determining the outcome of treatment
Describe traditional testing algorithm for syphilis
1) initial screening with nontreponemal (VDRL or RPR)
2) if reactive, perform treponemal test for confirmation (TP-PA or FTS-ABS). In nonreactive no further testing needed
3) if reactive, patient is positive for syphilis. If non reactive, previous result was false
Describe reverse sequence algorithm of syphilis
- testing order reversed
1) samples are screened by automated treponemal immunoassay
2) if reactive, results are confirmed by nontreponemal testing
What are the advantages of reverse algorithm over traditional?
1) cost
2) automation
3) saves time
4) less errors
5) can detect more early, late and treated syphilis cases
6) high sensitivity
What should be performed for congenital syphilis?
- perform nontreponemal tests on mother, and infant at birth
- titers decline with successful treatment
What should be performed for congenital syphilis?
- perform nontreponemal tests on mother, and infant at birth
- IgM-specific treponemal assays to confirm
What should be performed if neurosyphilis is suspected?
- perform CSF VDRL or ELISA on cerebrospinal fluid
What should be performed if neurpsyphilis is suspected?
- perform CSF VDRL and ELISA on cerebrospinal fluid
What are the tests in order of reverse algorithm sequence?
1) perform EIA or CIA
2) if positive, perform quantitative RPR (or other nontreponemal tests)
3) if RPR positive, indicates past or present syphilis infection. If negative, perform TP-PA
4) If TP-PA is positive, indicates past or present inflection of syphilis
What test is recommended to confirm congenital syphilis?
Western blot
Describe Lyme disease
- caused by the spirochete bacterium Borrelia burdorferi
- multisystem illness involving the skin, nervous system, heart and joints
- transmitted by ixodes ticks
- main reservoirs white-footed mouse
- most common vector-borne disease in the US
Describe the clinical manifestations of Lyme disease
Stage 1: localized rash and hallmark erythema migraines
Stage 2: early dissemination
Stage 3: later dissemination with arthritis, heart and nervous system involvement
What is the most prevalent neurological signs of Lyme disease?
Facial palsy
What are the types of Ixodes ticks?
1) ixodes scapularis —> northeast and Midwest US
2) Ixodes pacificus —> in the west
3) ixodes ricinus —> Europe
4) ixodes persulcatus —> Asia
Describe erythema migrans
- localized rash stage
- hallmark rash of early infection of Lyme disease
- appears 2-14 days after bite
- begins as a small red papule where bite occurred, then radio expands to form a large ring-like erythema and central area exhibits partial clearing
- must be at east 5 cm in diameter to diagnose with Lyme disease
Describe the early dissemination stage
- occurs via bloodstream in days to weeks following rush
- skin, nervous system, heart, and joints involved
- usually migratory pain occur in joints, tendons, muscles, and bones
Describe late dissemination stage of Lyme disease
- can develop in untreated patients months to years after acquiring infection
- major manifestation —> arthritis, peripheral neuropathy and encephalomyelitis
- some develop fatigue, concentration and short term memory problems and muscoskeletal pains
Describe antibody response to Lyme disease
- may not be detectable for 3-6 weeks
- IgM occurs first, followed by IgG
- IgG peaks at 3rd and 4th weeks of infection
Using the standard testing algorithm. What test (in order) are ran to diagnose Lyme disease?
1) screened with sensitive ELISA or IFA
2) it positive or borderline, Webstern blot test for IgM is performed to confirm results of patient with symptoms present less than 30 days. Western blot test for IgG is patient had symptoms that exceeded 30 days
What are characteristics of standard testing algorithm of Lyme disease?
- costly
- western blot can be difficult to interpret
- highly specific
What are tests (in order) performed in the modified two-tiered algorithm?
1) symptomatic patients are tested with sensitive that uses purified borrelia peptide antigens
2) if positive or borderline, test with different ELISA method for confirmation
What are characteristics of the modified two-tiered algorithm?
- easy to perform and interpret
- comparable specificity to standard method
- increased sensitivity to detecting early Lyme disease
Describe immunofluorence assay (IFA) of Lyme disease
- first test used to evaluate the antibody response
1) doubling dilutions of patients seem are incubated with commercially prepared slides coated with antigen from whole or processed borrelia spirochetes
2) following wash step, anti-human globulin with fluorescent tag is added and reacts with any specific antibody bound to spirochetes on slide
3) after second wash, slide is viewed under a fluorescent microscope - test considered positive only if titer of 1:256 or higher is obtained
What can produce false positives in IFA?
- closely related organisms can cross react
- autoimmune connective tissue diseases such as RA and SLE
- fluorescent light pattern is very subjective
Describe EIA testing for Lyme disease
- ELISA is inexpensive and yields timely results
- automation
- high volume testing
- less sensitive in early Lyme disease
Describe western blot for Lyme disease
- also called immunoblotting
- very complex
- patient serum is incubated with nitrocellulose membrane containing electrophoresed B. Burgdorferi antigens
- positive IgM results —> 2-3 characteristic bands
- positive IgG results —> 5-10 characteristic bands
Describe PCR testing of Lyme disease
- involved probes for target DNA that is present only B. Borrellia
- involves extraction of DNA, followed by amplification using specific primers, DNA polymerase and nucleotides
- highly specifc
- skin biopsy is not justifiable
- used to diagnose difficult neurological and arthritic cases
Describe leptospirosis
- caused by leptospirosis species
- zoonotic infection
- humans are exposed by mucous membrane contact with urine-contaminated water
- causes febrile episode that can progress to severe disease involving renal, liver, pulmonary, and CNS
- symptoms
—> febrile episodes of headache
—> myalgia
—> nausea
—> vomiting
—> diarrhea
—> renal involvement
—> hepatic involvement
—> pulmonary involvement
—> CNS involvement
Whar are characteristics of the leptospira species?
- Thin, flexible, and lightly coiled spirochetes
- pointed ends with hook-like shape
Describe the stages of leptospirosis
- incubation period —> 2-30 days
- usually manifest 5-14 days after exposure
- severe cases can lead to Weil’s disease
What are the laboratory tests performed for leptospirpsis?
- specimens —> blood or serum for first week after, urine or serum
- IgM screening by ELISA, immunoDOT, and LFA
- MAT is gold standard for confirmation
What is the purpose of the Reiter treponemes in FTA-ABS?
- prevents cross-reactivity with antibody with antibody to non-pathogenic treponemes
What test is recommended for testing cerebrospinal fluid for detection of neurosyphilis?
VDRL
What are the advantages of direct fluorescent antibody testing to T. Pallidum?
- Reading is less subjective
- monoclonal antibody makes the reaction very specific
- slides can be prepared for later reading
What syphilis test detects specific treponemal antibodies?
FTA-ABS
What can be used to distinguish between IgG and IgM antibodies in syphilis?
Treponemal EIAs
What is toxic shock syndrome associated with?
Overproduction of cytokines
What are ANTIGEN noninvasive tests used to detect H. Pylori?
- urea breath test
- ELISA
- LFA
- DNA
What antibody is associated with M. Pneumonia?
Anti-I (cold agglutinins)
What disease is associated with anti-i?
Hemolytic anemia
What is as rapid urease method for H. Pylori?
CLOtest
What are virsuses?
Submicroscopic pathogens whose size is measured in nanometers
Describe the basic structure of a virus
- a core of DNA or RNA packaged into a protien coat (capsid)
- in some viruses, the capsid is surrounded by an outer envelope of glycolipids and proteins derived from the host-cell membrane
- obligate, intracellular
What are the steps of the basic virus lifecycle?
1) attachment of their virus to a receptor on the host cell surface
2) penetration of virus into host cell through endocytosis
3) degradation of the viral capsid and subsequent release of viral nucleic acid
4) transcription to produce additional viral nucleic acid
5) translation of viral nucleic acid to produce viral proteins
6) assembly of the viral components to produce intact virions
7) budding off the host-cell membrane or host-cell lysis results
8) release of viral progeny
What are the defenses type against a virus?
- innate defenses
- humoral antibody responses
- cell- mediated immunity
Describe innate defense against viruses
- first like of protection
- skin and mucous membrane barriers
- recognition of PAMPs on virus- infected host cells
- interferons alpha and beta
What occurs if the initial barrier of innate defense does not work?
Other innate defense are activated when cells of innate immunity recognizes PAMPS of surface or within virus infected host cells
What are other innate defenses against viruses? (Besides skin barrier)
- viral cells are stimulated to produce IFN-alpha and IFN-beta after recognizing viral replication by inducing transcription of several gene that code for proteins with anti-viral activity
- they also enhance activity of NK cells
How do NK cells fight against viruses?
- bind to virus-infected cells and release proteins such as a perforin and granzymes, causing cells to die and release viruses and are now accesible to antibody molecules
What plays a key role in preventing the spread of viral infection through neutralization?
Antibodies
Describe antibodies role in preventing the spread of viral infection through neutralization
- involves production of antibodies that are specific for a component of the virus that bind to the virus, the prevent it from attaching to and penetrating the host cell. IgA plays a large role in this.
- IgG and IgM can bind to viruses in blood stream
- IgG and IgM activate complement
- IgG also promote phagocytosis of viruses and promote destruction of viruses through ADCC
- IgM also viral particles by agglutinating them
What does intracellular viruses require?
Cell-mediated immunity
What cells have key roles in cell mediated immunity?
- Th1 cells
- cytotoxic T cells
Describe Th1 cells action that occur in cell mediated immunity
- produce IFN-gamma, which induces an anti-viral state within the virus-infected cells
- produce IL-2, which assists in development of effector cytotoxic T cells
Describe cytotoxic T cells actions that occur in cell mediated immunity
- CD8+ cytotoxic T cells become programmed to expand in number and attack the virus infected cells
CD8 is a co-receptor of T-cell receptor on cytotoxic T cells that must bind to viral antigen complexed with MHC class I on infected cells surfaces
What occurs after cytotoxic T cells bind to viral antigen complexed with MHC class I?
- stimulate granules in the cytotoxic T cells to release perforin and granzymes entering the pores
What occurs once granzymes enter the viral infected cell?
- activate apoptosis in the host cell, interrupting the viral- replication cycle and resulting release of assembled infectious virions
- the free virions can then be bound by antibodies
Describe humoral antibody responses
- antibodies attack free virus particles
- viral neutralization, opsonization, C’ fixation, and ADCC
What are viral escape mechanisms? And examples?
1) mutations results in production in new viral antigen (influenza viruses undergo frequent genetic changes)
2) viruses block action of immune system components (HSV can bind C3b)
3) suppression of the immune response (CMV reduces MHC I)
4) immune function altered (EBV stimulates polyclonal B-cell activation)
5) latent state is established (VZV remains latent in nerve cells)
What laboratory test are ran to detect a viral infection?
- serological tests
- distinguish between current and past infection
- antibody titers used to monitor course of infection
- assess immune status
- molecular
- detect active infection
- quantitative tests —> guide antiviral therapy
How are current and past infections detected?
- IgM (+) and IgG (+/-) —> Current or recent infection. (Congenital)
- IgM (-) and IgG (+) —> past infection
What does the presence of virus-specific IgG indicate?
Immunity to virus
What is the hepatitis virus?
- hepatitis is the inflammation of the liver
- caused by:
—> radiation
—> chemical toxins
—> secondly to other disease
—> cirrhosis
—> drugs
—> hypothermia
—> bacteria
—> fungi
—> parasites
What are the hepatitis are transmitted by fecal-oral route?
Hep A (HAV)
Hep E (HEV)
What types of hepatitis are transmitted through parenteral route?
Hep B (HBV)
Hep D (HDV)
Hep C (HCV)
What are indicators of hepatitis?
- flu-like symptoms early on
- pain in upper right quadrant of abdomen
- hepatomegaly and liver tenderness with profession
- jaundice
- dark urine
- light feces
- Elevated bilirubin and liver enzymes (ALT)
Describe hepatitis A
- nonenveloped, single strand RNA virus
- belongs to hepatovinis genus or picornarviridae family
- acute hepatitis in majority of adults
- infections in children are usually asymptomatic
- formalin- activated vaccine
- HAV immune globulin may be recommended for unimmunized persons exposed to virus
How is Hepatitis A transmitted?
- fecal-oral route
- close person to person contact
- ingestion of contaminated food food or water
What is critical for establishing a diagnosis of hepatitis A?
- serological tests antibody
What sheds from the feces from someone who has hepatitis A?
- HAV antigen
What are hepatitis A most commonly detected by?
EIAs
Chemiluminescent microparticle immunoassays
When is IgM anti-HAV detectable in a patient with hepatitis A?
Onset of of clinical symptoms and declines to undetectable levels by 6 months
Describe tests for total HAV antibodies
- detect IgM but predominantly IgG, which persists for life
Although IgM anti-HAV is primary marker to detect acute hepatitis. What do you have to look out for?
False-negative results due to early phase of infection
What is the most common format of these molecular methods of HAV?
- RT-PCR
- used to test samples of food or water suspected of transmitting virus
Describe Hepatitis E
- nonenveloped, single stranded RNA virus with four genotypes
- HEV-1 and HEV-2 are transmitted primarily through ingestion of feces-contaminated drinking water
- HEV-3 and HEV-4 are transmitted mainly by consumption of infected pork
- most asymptomatic or self-limiting infections
- can detect HEV RNA in blood or stool during acute infection
- belong to hepevirus; in the family hepediridae
What indicates an acute infection of Hep E?
IgM anti-HEV
What indicates past exposures of Hep E?
IgG anti-HEV
What does diagnosis of HEV?
- serology to detect antibodies to the virus (EIAs)
- molecular methods to HEV nucleic acids
What is used to detect later stages, past exposures, and identify seroprevalence of the infection in HEV?
Immunoassays for IgG anti-HEV
What is the gold standard for diagnosis of acute HEV infections?
- qPCR, quantization of HEV nucleic acid
What testing can be done for immunocompromised patients that are suspected of having HEV?
- molecular testing for HEV RNA (qPCR)
- LAMP
Describe Hepatitis B
- DNA virus with 8 genotypes (A-H)
- belongs to hepadnaviridae family
- acute infection —> symptoms increase with age
- chronic infection —> 6 months or more, occurs in 90% of infected infants and 10% of infected adults
- preventable with immunization
- hepatitis B immune globulin (HBIG) may be given to unimmunized persons exposed to HBV
What does chronic hepatitis B increase the risk of?
Liver cirrhosis
Hepatocellular carcinoma
Describe hepatitis B surface antigen (HBsAg)
- first HBV marker to appear (2-10 weeks after exposure)
- protein on outer envelope of virus-like particles in blood
- marker for active HBV infection
- component of hepatitis B vaccine
Describe hepatitis B antigen (HBeg)
- protein in core of HBV
- marker of active viral replication
- indicates high degree of infectivity
What are serological markers of HBV antigens?
HBsAg
HBeg
What are the antibodies markers for HBV?
Anti-HBc
Anti-HBe
Anti-HBs
Describe anti-HBc of HBV
- directed against HEB core antigen
- IgM anti-HBc consists mainly of IgG and can indicate a current or past-infection
What is a core window?
- period when neither HBsAb can be detected in the serum of the patient only the anti-HBc
Describe anti- HBe of HBV
- directed against HBeAg
- indicates recovery from hepatitis B
Describe anti-HBs of HBV
- directed against HBsAg
- indicates immunity to hepatitis B
How is HBV transmitted?
Transmitted through parenteral or perinatal routes:
- sexual contact
- IV drug use
- during birth process
Describe chronic hepatitis B
- Majority of infants, 10% of adults, one-third children
- most likely in immunocompromised patients
- results in liver inflammation and damage
Describe structure of the HBV
- nucleocapsid core surrounded by outer envelope of lipoprotein
- core of virus contains circular partially double-stranded DNA
How are serological markers of HBV used?
- differential diagnosis of HBV
- monitoring course of infection
- assessing immunity to virus
- screening blood products infectivity
Why is HBsAg important marker of HBV?
- indicator of active infection
- monitoring course of infection and progression
- screening of donor blood
How are serological markers for hepatitis most commonly detected? Describe
Commercial immunoassays (EIAs and CLIA)
- typically automated
- excellent specificity and sensitivity
- false-negatives and false-positives occur
- positive results should be verified by repeat testing following by confirmation with additional assay (HBsAg neutralization test or molecular methods that detects that detect HBV DNA
What is the method of choice of to quantify HBV DNA?
- qPCR
What are molecular methods used to detect HBV DNA?
- PCR
- qPCR
- branched DNA signal amplification
What is considered a successful treatment for HBV?
1-log10 reduction in HBV DNA level`
Describe hepatitis D
- RNA virus that requires presence of HBV with 3 genotypes
- superinfection of chronic HBV carriers
—> chronic liver disease with accelerated progression to cirrhosis and liver failure - co-infection with HBV —> usually results in acute, self limited hepatitis
In HDV, what is the marker for active viral replication?
HDV RNA
What do co-infections of HDV test results look like?
Positive for ani-HDV and IgM anti- HBc
what do chronic cases of HDV results look like?
Positive for anti-HDV and IgG anti-HBC
How is HDV transmitted?
Through parenteral or perinatal routes
What markers appearance would make cause to for HDV?
- HBsAg
- involves detection
- antibodies and HDV RNA
What is testing used to distiniguish HBV and HDV from acute infection or superinfection?
Serology testing
What does the presence of IgG anti-HDV indicate?
Acute, chronic, or past infection
What molecular methods are used to detect HDV RNA?
- RT-PCR
- also provides quantitative results that can be used to monitor the response of patients to antiviral therapy
Describe hepatitis C
- enveloped, single-stranded RNA virus with 7 genotypes
- belongs to flavivirdae family and genus hepacivirus
- most infections are asymptomatic at first but develop into chronic liver
- genotyping is used to determine best therapy
How is HCV transmitted?
- exposure to contanimated blood, sexual contact, and perinatal exposure
What is detection of anti-HCV IgG used for?
Screening and diagnosis
What is qualitative HCV RNA used for?
Confirmation
What are quantitative molecular tests used for?
Monitor viral load during antiviral therapy
What is the most common blood born infection in the US?
HCV
What is the HCV genotype that is most prominent in the united states?
Genotype 1
Why is difficult to create a vaccine for HCV?
- the variability of HCV
- its ability to undergo rapid mutations within its hosts
What does chronic HCV lead to?
- cirrhosis of liver
- increased risk of Hepatocellular carcinoma
- rheumatoid conditions
- glomerulonephritis
- vasculitis
- neuropathy
- dermatological systems
What is anti-HCV most commonly detected by?
- EIA and CLIAs that use recombinant and synthetic antigens
- rapid immunoblot is alternative
When do antibodies become detectable in HCV?
8-10 weeks after exposure and remain positive for a lifetime
What could case a false positive result in serological testing of HCV?
- because of cross-reactivity in persons with other viral infections or autoimmune disorders
What test is recommended for HCV RNA confirmation?
Nucleic acid testing
What do qualitative tests distinguish between in HCV?
- presence or absence of HCV RNA
What are qualitative molecular assays used for HCV?
- to confirm infection in HCV antibody positive patients
- detect infection in antibody negative patients who are suspected of having HCV
What are the types of quantitative molecular assays from HCV? What are they used for?
- RT-PCR
- qPCR
- bDNA application
—> monitor amount of HCV RNA, or viral load, carried by patents before, during and after antiviral therapy in chronic patients
What is the goal of antiviral therapy of HCV?
When patient continuously tests negative for HCV RNA 12 or 24 weeks after therapy is completed
Describe genotyping of HCV
- to determine exact genotype and subtype of viruses responsible for infection
- ran on all HCV positive patient before antiviral therapy
- important to identify genotype because they vary in their response to different antiviral drugs
What can genotyping for HCV be performed by?
- PCR amplification and sequencing of target gene
- PCR followed by identification of the target gene with genotype-specific probes
- qPCR
What is the reference method of genotyping for HCV? Why?
- PCR/direct sequencing (Sanger sequencing)
- because it provides precise information about genomic variability of the virus during course of infection
Describe herpes virus
- large complex DNA viruses that are surrounded by a protein capsid, an amorphous tegument, and an outer envelope
- herpesviridae family —> includes 8 viruses
Describe Epstein-virus (EBV)
- DNA herpes virs most commonly transmitted by intimate contact with salivary secretions
- begins in oropharynx in B cells and epithelial cells and spreads through lymphoreticular system
What are diseases caused by Epstein-Barr virus?
- IM
- lymphoproliferative disorders
- certain malignancies (burkitt lymphoma)
How can Epstein-Barr virus be transmitted?
- salivary sectretions (most frequent)
- blood transfusions
- bone marrow
- solid-organ transplant
- sexual contact
- perinatal exposure
What cells are infected in the oropharynx in a patient with EBV?
Epithelial cells
B cells
How does EBV enter the B cells?
By binding to surface CD21
What are early antigens (EAs) of EBV?
- antigen produced during the initial stages of viral replication in the lyric cycle
- can be classified into two groups: EA-D and EA-R
Describe EA-D
In EBV, early antigens that have a diffuse distribution in nucleus and cytoplasm
Describe EA-R
In EBV, early antigens restricted to cytoplasm only
Describe latent antigens of EBV
- appear during the period of the lyric cycle following viral DNA synthesis
- includes the viral capsid antigens in the protein capsid and antigens in the protein capsid and the membrane capsid in viral envelope
What antigens appear during the latent stage of EBV?
- EBNA-1 through EBNA-6
- LMP-1
- LMP-2A
- LMP-2B
What are the classical symptoms of IM?
- fever
- lymphodenopathypy
- sore throat
- fatigue
- lasts 2-4 weeks
What are the laboratory findings of IM?
- absolute lymphocytes
- 20% or more atypical lymphocytes
- heterophile antbodies —> IgM
What produces can be performed for detection of IM heterophil antibodies?
- Monospot
- paul-Bunnell test
- rapid agglutination tests
- immunochromatographic assays using purified bovine RBC extract as antigen
What can AID in diagnosis of IM?
- testing for EBV- specific antibodies
- helpful is patient that test negative for heterophil antibodies or determine past exposure
- detected by IFAs, blot techniques, ELISA, CLIA or flow cytometric microbead immunoassays
What with the gold standard of EBV serology methods?
- IFAs
- labs prefer to use EIAs or CLIA tests because the are automated and easier to interpret
What is the most useful marker for acute IM?
IgM antibody to the VC
- presence of IgM anti-VCA and anti-EA-D
- absence of anti-EBNA
What types of tests are best to detect EBV in immunocompromised patient?
Molecular tests
How is cytomegalovirus transmitted?
- DNA herpes virus is transmitted through oral secretions, genital secretions, congenitally, or by transfusion/transplantation
Describe cytomegalovirus
- healthy individuals may be asymptomatic or develop a mononucleosis-like syndrome
- in immunocompromised persons, CMV can disseminate to lungs, liver, GI tracts, CNS, and eyes and cause life-threatening infections
- healthy individuals cause congenital defects and decreased survival in infants
What are types of testing for CMV?
- direct virus detection
- serology
Describe direct virus detection of CMV
- viral culture
- sample placed with a cell type that the virus being tested for can infect
- if the cells show changes, known as cytopathic effects, then the culture is positive
- ID of CMV antigens
- molecular tests for CMV DNA
Describe serology methods for CMV
- used to screen blood and organ donors; pregnant women
- presence of IgG anti-CMV indicates infection
- low avidity antibodies indicate recent infection
- most beneficial in determining past exposures
- usually semi-or fully automated EIAs
What is the most common cause of congenital infections?
CMV
What indicates CMV?
Characteristics cytopathtic effects (CPEs) that produce enlarged, rounded, refractile cells
Describe the CMV antigemenia assay
- uses immunocytochemical or immunofluorescent staining to detect the CMV lower-matrix protein pp65 in infected leukocytes from peripheral blood or cerebral spinal fluid
- 2-4 hours to perform
Describe VZV
- DNA herpes virus
- cause of: varicella (chicken pox) and here’s zoster (shingles)
- preventable by vaccine
How is VZV transmitted?
- by inhalation of infected respiratory secretions or aerosols from skin lesions
Describe varicella
- highly contagious illness
- blister-like rash
- intense itching
- fever
What are the activities of VZV in the primary infection?
- thought to travel from the skin lesions and blood to sensory neurons, where it deposits its DNA and establishes a lifelong latent state in the dorsal root, autonomic and cranial ganglia
Describe herpes zoster
- painful vesicular rash caused by the virus moves down the sensory nerve to the dermatome supplied by that nerve
What is the definitive diagnosis of VZV?
- identifying VZV in skin lesions, vesicular fluids or tissue
- used for atypical cases
What is the method of choice to detect VZV DNA?
QPCR
How is serology testing useful with VZV?
- determines immunity to VZV in health-care worker, pregnant women, and organ transplant candidates
What cells can be detected in VZV patients?
Tzanck cells
What is quantitative PCR useful with VZV?
Monitoring response of immunocompromised patients to antiviral drugs
Describe serology testing for VZV
- most of them detect total VZV antibody, which consists of primarily of IgG
- most reliable and sensitive method —> FAMA (reference method)
- most commonly used—> ELISA
Describe rubella virus
- enveloped, single-stranded RNA virus
- cause of German measles
- can be prevented with immuzation
- genus rubivirus and belongs to family Togaviridae
- after incubation, virus replicates in upper respiratory tract and cervical lymph nodes, then travels through blood stream
How is rubella transmitted?
- respiratory droplet
- across the placenta
What can rubella virus cause?
- deafness
- eye defects
- cardiac abnormalities
- mental retardation
- motor disabilities
- miscarriage
Stillbirth in infants
What are the symptoms of rubella virus?
- erythematous (macropapular rash). Appears first on face, then spreads to the trunk and extremities
- low grade fever
- malaise
- swollen glands
- upper respiratory infection lasting 1-5 days
- 50% infected with Rubella are asymptomatic
- arthritis
What populations have severe consequences with rubella virus? Why?
- pregnant women, especially in first trimester
- may cause miscarriage, stillbirth, or congenital rubella syndrome
What can occur if infants are born with congenital rubella syndrome (CRS)?
- many abnormalities
—> deafness
—> eye defects
—> cardiac abnormalities
—> mental retardation
—> liver and spleen damage
—> motor disabilities
What type of methods are ran for rubella virus?
- serology
- viral culture
- molecular methods
Describe serology testing of rubella virus
- presence of IgG is used to screen for immunity
- congenital infection is indicated by rubella-specific IgM or fourfold rise in IgG
- low avidity antibodies indicate recent infection
Describe viral culture of the rubella virus?
- diagnosis can be obtained this way
- viral growth is slow
- may not produce characteristic CPE
If CPE is absent in the viral culture of rubella. What should be done next?
- viral nucleic acid can be identified by RT-PCR or viral proteins can be detected with IFA or EIA
What is the most widely used test for rubella virus?
- molecular methods —> RT-PCR
- used to detect rubella RNA
What are the most common methods used to confirm rubella?
- serology tests (rapid and low cost)
- detect rubella antibodies
- ELISA- most widely used
- hemagglutination inhibiton (HI)
- latex agglutination
- immunoassays
Describe ELISA testing of rubella
- specific solid phase capture ELISAs can be used to detect IgM rubella antibodies
What assays can simultaneously detects measles, mumps, rubella, and varicella?
- automated chemiluminescence assays
- multiplex bead immunoassay
How is a primary rubella infection indicated?
- presence of rubella-specific IgM antibodies
OR - four-fold or greater rise in rubella specific IgG antibody titers collected at least 10-14 days apart
What can cause a false negative in rubella?
Obtained sample too earlier
Describe test ran to confirm positive IgM results of rubella
- EIA —> measures avidity of rubella IgG antibodies, which helps distinguish between recent and past infections
- low antibody avidity —> recent infection
- high antibody avidity —> past infections
Describe laboratory testing of congenital rubella infection
- begins with serological evaluation of the mothers antibodies and measurement of rubella-specific IgM antibodies in fetal blood, cord blood, or neonatal serum
-any positive IgM results should be confirmed by viral culture, RT-PCR amplification, or demonstration of persistently high titers of rubella IgG antibodies after 3-6 months of age
Describe rubeola
- RNA virus single-stranded RNA virus
- genus morbillivirus in the paramyxoviridae family
- after initial infection of the epithelial cells in upper respiratory tract, rubeola virus is disseminated through the blood to multiple sites in body, such as the skin, lymph nodes and liver
- preventable by immunization
- diagnosis is usually based on clinical presentation and confirmed by serology
How is rubeola transmitted?
Through respiratory droplets
What does rubeola cause?
- measles
- subacute sclerosing pancephalitis (SSPE)
What are the symptoms of rubeola?
- fever
- cough
- runny nose
- conjunctivitis
- koplik spots appear on mucous membrane of inner cheeks or lips
What is the rubeola virus characterized by?
- erythamatous (maculopapular eruption) that begins at hairline, then spreads to the face and neck and gradually moves down to trunk, arms, hands, legs, and feet
What complications can measles result in?
- Diarrhea
- otitis media
- croup
- bronchitis
- pneumonia
- encephalitis
What is SSPE associated with?
Extremely high titers of rubeola antibodies
What is preferred method to detecting IgM antibodies in rubeola?
- IgM capture ELISA method
- 3-4 days after symptoms
What is the preferred method of detect IgG antibodies of rubeola?
- ELISA
- detectable 7-10 days after symptoms and persists for life
- IgG antibodies indicate immunity
When are molecular methods used to detect rubeola RNA?
- in which serological tests are inconclusive or inconsistent
- can be used to genotype the virus
What is the preferred molecular method for detecting rubeola?
RT-PCR
Describe mumps virus
- Single stranded RNA virus
- paramyxoviridae family, genus ribuavirus
- most common clinical manifestations is parotitis
- replicates initially in nasopharyngeal and regional lymph nodes
- preventable by immunization
- diagnosis is usually based on clinical presentation
- confirmation is done by culture or RT-PCR
How is mumps transmitted?
Respiratory droplets, saliva formites
How does the mumps virus function?
- virus spreads from blood to various tissues:
—> meninges of brain
—> salivary glands
—> pancreas
—> testes
—> ovaries - produces inflammation at site
What is parotitis?
- inflammation of the parotid glands
- occurs in 30-40% of patients with mumps
What is the gold stranded for detecting the mumps virus?
- culture
What is the primary diagnostic test for mumps? Why?
- RT-PCR
- because it is more sensitive than serology
Describe the serological testing of mumps
- provides simple means of confirming mumps diagnosis but has some important limitations
- EIAs and IFA mostly only measure IgG mumps virus antibodies
- most commonly used —> ELISA
- use of solid-phase IgM capture assays reduces incidence of false positive because of rheumatoid factor C
What is a current or recent infection of mumps indicated by?
- presence of mumps-specific IgM antibody in a single serum sample or by at least a four-fold rise in specific IgG antibody between two specimens collected during the acute and convalescent phases of illness
Describe human T-cell lymphoytropic virus type I and type II (HTLV-1 & HTLV-2)
- closely related retroviruses that preferentially infect T- lymphocytes (usually CD4+)
- serological tests for antibodies to HTLV-I/II are used to diagnose infections and screen blood donors
- have RNA as their nucleic acid and enzyme reverse transcriptase
- has 3 structural genes: gag, pol, and env and region called pX
- ELISA and CLIA are used to screen
-western blot or LIA are used for confirmation of positive results
What does HTLV-1 cause?
- adult T-cell leukemia/lymphoma(ATLL) and HAM/TSP
How are HTLV types I and II transmitted?
- mainly blood borne
- sexual conduct
- perinatal (especially breastfeeding)
What is “gag”?
Codes for viral core proteins
What is “pol”?
Codes for viral enzymes
What is “env”?
Encodes proteins in viral envelope
What is pX?
Encodes several regulatory proteins, including tax and Rex
What is reverse transcriptase function of HTLV?
- to transcribe the viral RNA into DNA
What are the 4 subtypes of ATLL? What are they characterized by?
- acute
- T-cell non-Hodgkins lymphoma
- chronic
- smoldering
- a monoclonal proliferation of mature T cells that express the surface markers CD3, CD4 and CD25
How is HAM/TSP characterized?
- slowly progressive weakness and stillness of legs
- back pain
- urinary incontinence
What has been associated with HTLV-1?
- variety of autoimmune and inflammatory disorders
- uveitis (intraocular inflammation of the eyes)
- infective dermatitis
- myositis (inflammation of the muscles)
- arthropathy (inflammation of the joints)
When do HTLV antibodies develop?`
- 30-90 days after exposure to the virus and persists for life
When is a sample considered positive for HTLV-1 performed with western blot?
- if visible bands are produced for one of the env proteins (either gp46 or gp62/68) AND one of the gag proteins (either p19, p24, or p53)
Describe PCR methods for HTLV testing
- detects HTLV type I and II DNA
- can be used to monitor the proviral load in patients during therapy
Describe toxoplasmosis
- found in feces of house cats and rodents (other mammals)
- transmitted:
—> accidental ingestion of oocyst (fecal contamination of meat, raw milk,etc)
—> transplacental transmission
How does one individual prevent congenital toxoplamosis?
- avoid touching mucous membranes of mouth and eyes while handling raw meat
- wash hands and kitchen surfaces after coming in out with raw meat
- not allow flies and cockroaches access to food
- avoid contact (or wear gloves) when handling cat litter boxes or gardening
What are signs and symptoms of toxoplasmosis?
- usually asymptomatic organism that can multiply in any organ of body
- similar to mono ( if symptoms are mono)
- spontaneous recovery
Describe congenital infections of toxoplasmosis
- of most concern
- result in CNS malformation and mortality of the neonate
- 75% have no symptoms at birth, disease is dormant, discovered only when neurological problems such as blindness occurs
What is the method of choice for a laboratory diagnosis for toxoplasmosis?
EIA
Describe TORCH testing
- consists of tests for antibodies to four organisms that cause congenital infections transmitted from mother to fetus
T- toxoplasmosis
O- others
R- rubella
C- CMV
H- herpes simplex virus
What are the “others” of TORCH?
- syphilis
- HBV
- coxsackie virus
- EBV
- VZV
- human parvovirus
What viruses affect the repiratory system?
- adenovirus
- influenza
- mumps
- measles
- RSV
What virus affects the skin?
Arbovirus
What virus effects the HI tract?
Rotovirus
What is the role of CTLs in immune responses against viruses?
Destroy virus-infected host cells
A newborn suspected of having a congenital viral infection should be tested for virus-species antibodies of which class?
IgM
The serum of an individual who received all doses of the hepatitis B vaccines should contain what?
Anti-HBs
Heterophile antibodies are routinely detected by their reaction with what?
Bovine erythrocytes antigens
What is the method of choice for detecting VZV infection in immunocompromised hosts?
RT-PCR
Describe human immunodeficiency virus (HIV)
- causes AIDS (acquired immunodeficiency syndrome)
- two types: HIV-1 and HIV-2
Describe HIV-1 (will be referred as that for the rest of the set)
- cause most HIV infections worldwide
- 4 groups —> M, O, N, P
- 9 subtypes in group M —> A, B, C, D, F, G, H, J, K
- groups M is responsible for most cases
- predominant subtype —> C
- most prevalent is US —> B
- belongs to genus lentivirinae of virus family retroviridae
- decrease in cell production is a hallmark feature
Describe HIV-2
- originated in west Africa
- causes fever cases
- less pathogenic
- lower transmission rate
What are ways HIV can be transmitted?
- sexual contact involving exchange of body fluids —> responsible for majority of cases
- contact with blood or other body fluids
- perinatal —> before , during and after (breast milk)
What body fluids are infective with HIV?
- blood
- semen and vaginal secretions
- synovial, pleural, peritoneal and pericardial
- CSF
- breast milk
What are characteristics of HIV
- retrovirus
- contains two copies of ssRNA
- reverse transcipatase transcribes the viral RNA into DNA
- surrounded by a protein coat (capsid)
- outer envelope of glycoproteins embedded in a lipid bilayer
Why is HIV considered a retrovirus?
Because it contains RNA as its nucleic acid and a unique enzyme called reverse transcriptase
Describe the HIV structure
- spherical particle
- includes 3 structural genes: gag, pol, and env
- 100-200 nm in diameter
- contain inner core with two copies of single stranded RNA surrounded by protein coat (capsid) and an outer envelope of glycoproteins embedded in lipid bilayer
- glycoproteins are knob-like structures that are involved in binding the virus to host cells during infection
Briefly describe replication of HIV
- attachment of hIV to host cell
- main target: CD4 Th
- coreceptor required
What are the steps of HIV replication?
1) binding- HIV binds to receptors on surface of CD4 cell
2) fusion- HIV envelope and CD4 cell membrane fuse and allow virus to enter cell
3) reverse transcription- inside cell, HIV uses and releases reverse transcriptase, which HIV RNA into HIV DNA. This allows HIV to enter nucleus of CD4 cell
4) integration- inside nucleus, HIV releases integrase. Uses intergrase to insert its viral DNA into the DNA of the CD4 cell
5) Replication- HIV uses machinery of CD4 cell to make long chains of HIV proteins. These proteins are building blocks for more HIV
6) Assembly- new HIV proteins and HIV RNA move to surface of cell and assemble into immature HIV
7) Budding- immature HIV pushes out of host CD4 cell surface. New HIV release proteases, which break down immature HIV, forming mature HIV
What is T-tropic or X4 strains of HIV?
They preferentially infect T-cells
What is M-tropic or R5 strains of HIV?
Can infect T-cells and macrophages
What chemokine receptor is required for HIV to enter T-lymphocytes?
CXCR4
What chemokine receptor is required for HIV to enter macrophages?
CCR5
When does HIV not virally replicate?
During the latent stage
What are the type of immune responses for HIV?
- innate
- humoral antibody production
- cell-mediated immunity
describe the innate defenses against HIV
- NK cells mediate cytolysis of HIV infected cells
- dendritic cells stimulate release of cytokines that have antiviral effects
Describe humoral antibody production against HIV
- antibodies are detected by 6 weeks after infection
- antibodies produced later may prevent HIV from infecting host cells and participate in ADCC
Describe cell-mediated immunity against HIV
- T cells produce cytokines with antiviral activity
- CTLs destroy HIV-infected host cells
What are the HIV escape mechanisms?
1) genetic mutations rapidly occur, generating new viral mutants with altered antigens
2) down regulates expression of MHC-1 molecules on infected host cells
3) can survive as a patent provirus for prolonged periods
4) As result, HIV persists and destroys the immune system
5) CD4 T cells are the prime targets of destruction, resulting in reduced effectiveness of antibody and cell-mediated immune responses
How is HIV suspected to kill or render function of CD4 Th cells?
1) loss of plasma-membrane integrity because of viral budding
2) destruction by HIV-specific CTL
3) viral induction of apoptosis
What is the central role in the immune system for CD4 T cells?
Regulating the activities of B and T cells
What is the acute stage of HIV characterized by?
- rapid burst of viral replication before the development of HIV-specific immune responses
- high levels of viremia is found in this stage
- decrease in CD4 T-cell number
What are the symptoms of acute stage of HIV-1?
- flu like symptoms
- IM-like symptoms
- many are asymptomatic during this stage
Describe HIV latent stage
- decrease in viremia
- increase in CD4 T-cell number
- symptoms are subtle or absent
Describe the last stage of HIV
- full blown AIDS
- characterized by profound immunosuppression with very low numbers of CD4 T cells
- resurgence of viremia
- patients demonstrate neurological symptoms
- appearance of life-threatening opportunistic infections and malignancies
What are the symptoms for infants with AIDS?
- failure to thrive
- persistent oral candidiasis
- hepatosplenomegaly
- lymphadenopathy
- recurrent diarrhea
- recurrent bacterial infections
Describe antiretroviral therapy (ART)
- drugs that block various steps of the HIV replication cycle
- nucleoside analog reverse transcriptase inhibitors
- proteases inhibitor
- integrase inhibitors
- fusion inhibitors
- CCR5 antagonists
- post attachment inhibitors
- are most effective when used in combination
- has significantly improved morbidity and mortality of HIV-infected persons and has reduced the rate of perinatal transmission
What are ways to prevent HIV?
- screening blood/organ donors for HIV
- education of the general public on HIV transmission, safety measures
- precautions for health-care workers
- vaccine-being researched
Describe screening and diagnosis of HIV
- previous algorithms and tests methods
- current algorithm and test methods
Describe previous testing algorithm of HIV
- screen for HIV-1/HIV-2 antibodies by ELISA or rapid EIA
- confirm positive test results by repeating ELISA, followed by western blot
Describe western blot interpretations of HIV
- no band: negative
- positives are harder to interpret
- CDC says must have antibody against two or three of the following bands:
—> p24
—> gp41
—> gp120/160 (these bands are very close together and is hard to distinguish between the two.
Describe the current CDC testing algorithm for HIV
- initial screening: HIV-1/2 antigen/anitbody combination immunoassay
- if positive, run HIV-1/2 differentiation immunoassay
- HIV-1(+) & HIV-2 (-): HIV-1 antibodies present
- HIV-1(-) & HIV-2(+): HIV-2 antibodies present
- HIV(+) & HIV-2(+): HIV-1 and HIV-2 antibodies present
- if both HIV negative, run HIV-1 NAT
—> HIV-1 NAT(+): acute HIV-1 infection
What is the principle of the fourth generation HIV-1 antibody/HIV-2 antibody/p24 antigen combination immunoassay?
1) incubate patient serum with solid phases onto which HIV-1 antigens, HIV-2 antigens and antibody to HIV-1 p24 have been bound
2) following incubation, HIV-1 or HIV-2 antibodies in the patient sample will bind to their respective antigens
3) HIV-1 p24 antigen in the patient sample will bind to anti-p24 solid phase
4) wash, then add conjugate consisting of labeled anti-24 labeled HIV-1/HIV-2 antigens
5) following, incubation, wash and addition of trigger solutions or substrate/stop solution, relative light units or optical absorbance are measured
6) confirm positive result with rapid EIA
What are some ways the HIV is monitored?
- peripheral blood CD4 T-cell counts
- quantitative viral load assays
- drug resistance and tropism
Why do false-positives occur int he 4th generation HIV-1/2 antibodies and p24 antigen test?
- heat inactivation
- repeated freezing and thawing
- presence of heterophil antibodies
- passive immunoglobulin administration
- administration of an experimental HIV vaccine to patient
What is seroconversion?
- the change of a serological test result from antibody negative to antibody positive
What are the two markers used to monitor HIV for disease progression and guide the treatments?
- the peripheral blood CD4 T-cell count (best indicator of immune functions)
- HIV-1 RNA level (viral load)
Describe CD4 T-cell enumeration
- CD4 T-cell numbers are the best indicator of immune function i HIV-infected individuals
- incubate peripheral blood with fluorescent-labeled anti-CD4; analyze results by flow cytometry
- in untreated patients, CD4 T cell number declines progressively, and CD4 T: CD8 T cell ratio is less than 1:1
- CD4 T cell count of less than 200/Ul indicates stage 3 HIV
- a significant decline in CD4 T cell count over time may indicate a need to change ART or administer prophylactic therapy for certain infections
What is the gold standard for enumerating CD4 T cells?
Immunophenotyping with data analysis by flow cytometry
Describe the CDC classification list of HIV
0-5
- 0: early infection of patient who tested positive for HIV in initial screening but has a negative or indetermine confirmatory test
- 1-3: based on peripheral blood CD4 T-cell count or percentage. If this information or percentage. If this information is missing, classified as unknown
Describe quantitative viral load assays
- measure amount of HIV RNA circulating in patient plasma
- methods: qPCR and bDNA
- HIV RNA detectable about 11 days after infection
- successful therapy with ART results in the decline in the viral load to an undetectable antigen
- patients with persistently increased viral load should undergo drug resistance testing and may need a possible change in ART
Why are viral load tests used?
- help predict disease progression
- monitor patient response to ART
- guide treatment plan
Describe the viral load assays of HIV RNA
- based on amplification methods that increase the number of HIV RNA copies
- most common: PCR and bDNA assay
What are the 2 PCR methods that have been developed to detect HIV nucleic acid?
- RT-PCR (not usually used)
- qPCR
What is the basic principle of RT-PCR for HIV RNA?
- to amplify a DNA sequence that is complementary to a portion of the HIV RNA genome
What are the disadvantages of RT-PCR for HIV RNA?
- limited dynamic range
- highly susceptible to cross-contamination with extraneous nucleic acid
What is the basic principle of bDNA assay?
- based on amplifying the detection signal generated in the reaction
- accomplished by using a solid-phase sandwich hybridization assay that incorporates multiple sets of oligonucleotide probes and hybridization steps that produce a series of branched molecules
What are disadvantages of bDNA?
- requires larger sample volume
- lacks internal controls
- lower specificity
Describe genotype resistance assays of HIV
- performed in clinical laboratory settings
- HIV reverse transcriptase and protease genes from RNA in patient plasma are amplified by RT-PCR
- products are sequenced and analyzed with software for mutations
- results are reported as: Resistance, possible resistance, no evidence or resistance
Describe phenotype resistance assays of HIV
- determine ability of HIV from clinical samples to grow in the presence of antiretroviral drugs
- involve sophisticated technologies only performed by specialized reference laboratories
Describe tropism testing of HIV
- genotypic or phenotypic assays are performed to determine if the patient has virus that will bind to the CCR5 co-receptor and be responsive to CCR5 antagonists
Decribe testing of infants younger than 18 months in HIV
- all pregnant women should be tested for HIV
- maternal antibodies in infant serum can complicate serological tests results
- molecular methods are used for diagnosis
- Qualitative HIV-1 DNA PCR using infants peripheral blood mononuclear cells is the preferred method
- serological testing at 12-18 months of age may be used to confirm the diagnosis
HIV virions bind to host T cells through which receptors?
CD4
CCR5
What is a typical of the latent stage of HIV infection?
- proviral DNA is attached to cellular DNAq
Why would false negative results in a test from HIV?
Collection of the test sample before seroconversion
What does the conjugate used in the 4th-generation immunoassays for HIV consists of?
- HIV-1 and HIV-2 specific antigens plus antibody to p24
What is the first detectable antibody in serum after infection with hepatitis B?
HBc
What is the difference between HAV and HBV incubation periods?
- HAV —> short
HBV —> long
How do you interpret these results?
Anti-HAV (IgG) —> neg
Anti-HAV (IgM) —> neg
HBsAg —> neg
Anti-HBc —> pos
Anti-HBs —> neg
HBe —> neg
HBV in core window phase
What is the most common cause of congenital infections?
CMV
What populations does VZV cause severe complications in?
- immunocompromised patient
- pregnant women
What is the clinical importance of CMV?
Life threatening for immunocompromised patients and infants
A patient has IgG and IgM antibodies against viral capsid antigens of EBV. What does this patient have?
Current EBV infection
What are heterophil antibodies routinely detected by their reaction with this agent?
Bovine RBC antigens
What does the presence of IgM anti-rubella antbodies in serum from an infant born with a rash?
Congenital infection with rubella virus
What are the most commonly used tests to detect mumps virus?
RT-PCR
ELISA
What is the method of choice for detecting VZV in immunocompromised hosts?
RT-PCR
Describe Tzank smear
Scrap lesion with scalpel but put on slide and stain, the look for giant multi-nucleated cells
What does p24 encode for?
Gag
How should these results be interpreted for HIV?
ELISA: pos
Repeat ELISA: neg
Western blot: no bands
Negative for HIV
What type of cells can HIV infect?
- primarily lymphocytes
- monocytes
What does the HIV core consists of?
Two identical strands of RNA
Why is HIV called a retrovirus?
Can transcribe RNA into DNA
A 22 year old male sees his physician for an annual checkup. He reported having flu-like symptoms including fever, sore throat, and lymphadenopathy several months ago. He has lost 10 pounds but otherwise fee fine. HIV testing is recommended and ordered
- ELISA —> pos
- western blot —> POs
- CD4+ T-cell count is 500/ml
What disease and stage is this?
HIV positive in latency stage 1
