Micro - Things IDK yet... Flashcards
What is the red complex, and what bacteria does it include?
- related strikingly to clinical measures of periodontal disease
- particularly pocket depth & BOP
- includes Poryphormonas gingivalis, Tannarella forsythia, Treponema denticola.
What is the orange complex, and what bacteria does it include?
- also related to pocket depth
- this relationship & the relationship to other clinical parameters is less striking than the red complex
- includes P. intermedia, C. rectus, C. showae, E. nodatum, F. nucelatum, P. micros, P. nigrescens
- need orange complex for red complex to come in and flourish
What is a common trait of Red complex? And what does it do?
the expression of trypsin-like proteases; Trypsin-like proteases interact w/ host cell protease-activated receptor PAR-2
Pg gingipains have been shown to stimulate the secretion of cytokines via activation of…?
PAR-2
What pathogens share the ability to penetrate the gingival epithelium (invasive)? And why?
Aa, Pg, Tf, Td; due to their ability to produce toxic proteases (ex. endotoxins)
What bacteria are useful indicators of progressive periodontitis (in aggressive & chronic disease)?
Aa, Pg, Pi, Td, Tf
- one or more of these species occur in 99% of progressive lesions
- collectively, these species avoid/compromise host defenses and induce tissue dmg
what is one of the first stages of initiation of gingivitis?
bacterial exotoxins (i.e. Hyaluronidase) destroying intercellular connections betw epithelial cells lining the gingival sulcus.
Pg is commonly found in large numbers in gingivitis patients. Why is this?
WRONG! FALSE! NOOOOO! Pg is RARELY found in gingivitis patients and also remember, it is ABSENT from edentulous.
Gingipains are what and do what?
Gingipains are trypsin-like /cysteine proteases that degrade collagen, fibrinogen, fibronectin, complement components C3 & C5a, IgG & IgA.
*also have 2 arginine-specific and 1 lysine-specific
What does Arg-gingipain do?
It agglutinates and lyses the erythrocytes providing heme-a growth factor for Pg
What does gingipain cleave and what is the resulting consequence?
Gingipains cleave monocyte CD14, contributing to hyporesponsiveness to LPS and other bacterial triggers of mac activation.
______ and ______ promote adherence to host cells, contributing to the invasive capacity of Pg.
Pg fimbriae & Arg-gingipain
How does Pg persist in the plaque biofilm on tooth surfaces?
It attaches to Streptococcus gordonii via the major fimbrial subunit protein (FimA)
What is special about Pg’s LPS?
It has hemin-dependent alteration in its structure of Lipid A that may reduce the host’s ability to mount an innate immune response.
How is Aa invasive?
- enter epithelial cells
- escape phagocytic vacuoles
- multiplies in cytoplasm
- spread to adjacent epithelial cells via protrusions
How does Aa affect PMNs and mac’s?
It produces a leukotoxin that can destroy PMNs and mac’s by forming cation-selective pores that lead to the osmotic lysis of these cells.
What are the major Td virulence determinants?
- Dentilisin
- Major outer sheath protein (Msp)
- Sip protein
What is dentilisin?
a major Td virulence determinant that is a serine protease that degrades matrix prot & host cell junctional prot
- also activates host MMPs to degrade matrix
What is the Major outer sheath protein (Msp)?
a major Td virulence determinant that is a porin, causing membrane depolarization in some cells–cytotoxic to others
- alters fibroblast cytoskeleton reorganization, diminishing the cell migration, which prolongs tissue remodeling and wound healing
What is the Sip protein?
a major Td virulence determinant that induces arrest of T-cells in G1 and subsequent apoptosis, suppressing T-lymphocyte proliferation in response to mitogens (substances that induce mitosis)
In order for Td to be present, what else must be present?
Pg! They coaggregate not only w/ F. nucleatum but also with each other.
What is a major virulence determinant for Tannarella forsythia (Tf)?
BspA protein = binds to fibronectin & fibrinogen and stimulates neutrophil recruitment, inflammation, and bone resorption.
What type of patients would you find Prevotella intermedia (Pi)?
gingivitis, healthy, and edentulous patients
Pi expresses proteases and resists killing by phagocytes, but it lacks what…?
antiphagocytic capsule & collagenase production
Pi often dominates the subgingival flora in what type of gingivitis?
NUG = necrotizing ulcerative gingivitis
Predisposition to more severe chronic periodontitis occurs with…?
certain genetic polymorphisms (i.e. IL-1 genes)
What opportunistic pathogens/bacteria can infect a patient with a suppressed immune response due to periodontal disease?
cytomegalovirus, Epstein-Barr virus, and herpesvirus co-infections
Peripheral blood monocytes from PD pts release 2-3x more …?
IL-1B (osteoclast activating factor)–this increase due to monocytes stimulated by LPS
What is wrong with treating oral infections w/ systemic antimicrobial agents? And why?
It risks side effects and gives varied results.
- Bc it is difficult to maintain adequate drug levels for a sufficient time at site of action
- Also bc of reduced susceptibility of bact in biofilm compared to planktonic bacteria on which AB MIC (minimum inhibitory conc) values determine
What is the solution for local delivery of antimicrobial agents for PD disease?
deliver an antimicrobial agent directly to the PD pocket by use of drug-containing gels or films
Examples of local delivery of antimicrobial agents?
- metronidazole - gel or cellulose strip
- chlorhexidine - chip; can kill most of red complex
- tetracycline - fiber
- doxycycline - gel
- minocyclone - gel or powder
What is Actisite?
first ex of localized antimicrobial therapy; ethylene vinyl acetate copolymer (EVA) fiber containing 25% tetracycline
What is the amt/yield for normal delivery of tetracyclines in both serum & gingival crevicular fluid?
~10 ug/ml
What are afimbrial adhesins?
outer-memb or surface-exposed proteins that can bind to host cell surface-exposed proteins/carbs or can bind host proteins that then bind to the cell.
- they are made on the surface of the bact
What are coagulases?
enzymes that accelerate the formation of a fibrin clot around the organism which protects against phagocytosis by neutrophils & mac’s
What do Immunoglobulin A proteases allow?
They digest IgA, allowing colonization of mucous membranes.
How does Chlamydia survive inside a host’s cell?
It grows within a phagosome (inclusion) and does not fuse w/ the lysosome; it is obligate intracellular
How does Listeria survive inside a hist’s cell?
It enters the cell in a phagosome, and then escapes the phagosome by digestion of the memb; it is facultative intracellular; motile in cytoplasm bc of actin tails
What are enterotoxins?
exotoxins that result in watery diarrhea and tend to increase cellular lvls of cAMP (release of water & electrolytes > diarrhea)
What produces endotoxins?
ONLY G(-) bacteria; not secreted by exotoxins–released when cell lyses.
At high levels, what can LPS cause?
lethal toxicity, hypotension, septic shock, death
What recognizes LPS?
TLR4 & associated proteins
What is the toxic part of LPS?
Lipid A
Why is LPS not an effective vaccine target?
weakly immunogenic; does not result in an adaptive immune response w/ “memory”
What are the traits of exotoxins? (8 pts)
- secreted from living cell
- produced by G(+) & G(-)
- heat-labile
- highly immunogenic; can be converted to toxoids
- antisera neutralizes toxin
- highly toxic
- doesn’t usu cause fever
- frequently encoded on extrachromosomal elements (plasmids & phage)
What are the traits of endotoxins?
- produced by G(-) only
- heat-stable
- weakly immunogenic (cannot be converted to toxoids)
- antisera does not neutralize toxicity
- moderately toxic
- induce inflamm, fever, and septic shock
- encoded by chromosomal genes
Antisera can neutralize endotoxins, but not exotoxins?
WRONG, They can neutralize extotoxins, NOT endotoxins.
What mechanisms do microbial pathogens use to
adhere to host cells?
pili or fimbriae, and afimbrial adhesins!
What are the effects of base-pair substitutions?
- none (no AA change)
- missense (AA change)
- nonsense (stop codon created)
What is the difference between forward and reverse genetics?
Forward genetics is an approach used to identify genes (or set of genes) responsible for a particular phenotype of an organism.
Reverse genetics analyzes the phenotype of an organism following the disruption of a known gene.
***simplified: reverse genetics- force a mutation in DNA and look for change in phenotype. forward- see a change and then map the DNA
What are the types of mutations?
- base-pair substitutions
- addition/deletion
- DNA rearrangements
What is an example of a chemical mutagen?
base analogs - chemicals that mimic bases & get incorporated in DNA; usu results in improper base pairings (ex. intercalating agents, modifying agents)
What is an example of physical mutagens?
ionizing radiation, ultraviolet light (they result in distortion of DNA molecule itself
What does ionizing radiation do to cause mutations?
double and single-stranded DNA breaks
What does ultraviolet light do to cause mutations?
produce pyrimidine dimers on the same strand
What are the causes of mutations?
- chemical mutagens
- physical mutagens
- replication errors
- DNA insertions
What is a cistron? Operon? Regulon?
- cistron: DNA encoding a prot
- operon: DNA encoding > 1 prot (usu linked together)
- regulon: group of genes that co-regulate (don’t have to be directly next to each other)
The binding of certain promoters is directed by what?
sigma-factors (that form part of the RNA polymerase)
- sigma factors directs polymerase to promotor–process = promoter recognition
What does a repressor protein do?
Binds near a promoter and blocks transcription (negative control)
What are examples of positive control?
- activator protein binding near promoter and enabling transcription
- molecule binding to repressor protein causing its release
What does the operon consist of?
promoter (P), operator )O) sites, & structural genes which code for protein.
The operon is regulated by what?
the product of the regulatory gene (I)
Where does the repressor protein generally bind?
to the operator sequence, which is downstream of the promoter and upstream of the structural gene(s).
What is a direct repressor? Co-repressor?
- Direct repressor - controls expression of operon
- Co-repressor - requires binding of a 2nd molecule before binding the operator sequence
What is the role of tryptophan in the Trp biosynthetic operon?
- When Trp (co-repressor) is present, it binds to Repressor protein, which then binds to the operator–results in no expression of operon!
- However, when Trp is absent, no binding of the repressor protein, and there will be expression of genes to synthesize Trp
- Trp = repressor! (oppos of Lactose)
What do inducers do?
They inactivate repressor proteins by binding to them, thus preventing them from binding to the operator sequence.
Enzymes encoded by ____ are required for lactose catabolism.
lacZYA
What happens when lactose (allolactose) is absent? What happens when it is present?
- absent: LacI repressor binds to operator, no expression
- present: LacI repressor binds to allolactose, is inactivated & released from operator
- allolactose = inducer!! (oppos of Trp)
What is signal transduction a response to?
an environmental condition
What is the role of a sensor? Role of transducer?
Both are part of the 2 component system of Signal Transduction
- Sensor: on outer surf of bact will phosphorylate the intracellular transducer when a signal is detected
- Transducer: activated by the phosphorylation will bind to upstream region of genes & activates transcription (basically turns expression on)
What is the PhoP/PhoQ System?
- system that pathogens have evolved to determine the inside/outside of cells
- PhoQ = sensory of extracytoplasmic Mg2+ (which is in high conc outside of a cell, and low conc in cytoplasm of mammalian cells)
- PhoP = is phosphorylated by PhoQ and controls expression of a large number of genes under low Mg2+ conc by binding a direct hexanucleotide repeat
- PhoP controls a system that activates a second 2 component sys that controls biosynthesis of LPS
What is the autoinducer?
Molecule that controls quorum sensing system; produced by bacteria
- acylhomoserine lactones in G(-)
- peptides in G(+)
What is “Quorum Quenching”?
use of autoinducer analogs to control expression of bacterial virulence factors
What are the 3 primary mechanisms of horizontal transfer of DNA? Briefly elaborate on the 3 mechanisms.
- Conjugation: direct cell-to-cell transfer of plasmid DNA (highly efficient)
- Transduction: viral transfer of DNA (smal chromosomal pieces, low freq event)
- Transformation: transfer of free DNA (inefficient process, but some bact are “naturally competent” & have high transformation rates”
What is f-factor movement?
plasmid transfer via sex pilus results in conversion of F- cell to F+ cell
What is HFR formation?
HFR = high frequency recombination
- following plasmid transfer, the plasmid is integrated into recipient cell’s chromosome
What is resistance transfer factors (RTF)?
conjugative plasmids that contain 1 or more transposons that carry AB resistance markers; spread rapidly in bacterial pop.
What occurs in phage transduction?
Bacteriophages (virus-like bact) during lytic cycle degrade host chromosomal DNA
- small fragments incorporated in newly formed phage particle
- transferred to a new host & can be incorporated by recombination–slow process
What is the lytic pathway?
“living phase”
- DNA circularizes after entry & rapid replication and synthesis of new virions occurs.
- Bact cell lyses, new virions released
What is the prophage pathway?
DNA recombines in chromosome
- induction: excision of prophage & lytic cycle will start then
- waiting for the right moment to enter lytic cycle again! usu when host cell is immunocompromised
How is naked DNA taken by by the bacterial cell in transformation? (2 ways)
- natural bacterial systems (bacterial competence)
2. physical means (electroporation)
What is the difference between transformation with plasmids and with DNA fragments?
plasmids can replicate independently, but chromosomal DNA fragments must be incorporated into bacterial chromosome by DNA recombination before replicating.
3 types of DNA recombination?
- homologous (generalized) recombination
- site-specific recombination
- transposition
Tell me briefly about homologous (generalized) recombination.
- requires extensive sequence homology
- involves host recombination pathways
- major mechanism for DNA repair
Tell me briefly about site-specific recombination.
- requires small regions of homology
- specific integrases (DNA-joining enzymes)
Tell me briefly about transposition.
- involves simple or complex transposable elements
- transposase = enzyme for movt for specific element
- movt = nonspecific or target DNA sequences
what is the process called when there is replacement of 1 strand of DNA w/ a very similar strand to correct errors and to move genes?
homologous recombination
What does homologous recombination require in most bact?
- introduction of a ssDNA nick
- strand invasion directed by recombination enzymes
- resolution of the exchange point
What is lambda integrase?
DNA joining enzyme that is involved in site-specific recombination
- will add lambda DNA into bacterial cell chromosome, making a prophage (bacterial stress will cause induction & lytic cycle of Lambda)
What is a transposase?
Transposase ‐ an enzyme that recognizes the inverted repeats of transposons and catalyzes their insertion (including the intervening DNA) into a new DNA site - with no sequence homology requirement
What is an insertion sequence (IS)?
minimal transposons that contain only sequences necessary for their own transposition; can even lack transposase gene
What is a complex transposons?
have one to several genes in addition to genes for transposition (often are antibiotic resistance genes)
Transposition mechanism?
“Cut and Paste” transposition: 1. Transposase binds inverted repeat to form Synaptic Complex 2. Cleavage of the transposon 3. Re-integration at a new site (certain transposons may have target recognition sequences) *can be random or specific
Pathogens appear to have evolved from nonpathogens largely by the _____ rather than by the adaptive evolution of preexisting genes.
acquisition of pathogenicity islands
Enteropathogenic E.coli (EPEC) and cause attachment-effacement
(A/E) lesions on intestinal epithelial cells. What does attachment refer to? Effacement?
- Attachment: bacteria intimately attached to a cuplike pedestal structure, the result of extensive rearrangement of the host cell cytoskeleton.
- Effacement: loss of microvilli on enterocytes adjacent to the bacteria
LEE pathogenicity island (PI) of E. coli does what to an infected cell? What secretion system does it use?
forces the infected cell to cooperate in its own infection; incorporates a Type 3 secretion system (syringe-like mechanism)
*Background info: “Enteropathogenic Escherichia coli (EPEC) produce attaching and effacing lesions. The genes responsible for this lesion are clustered on the chromosome forming a pathogenesis island called LEE.”
- One injected protein, the LEE-encoded ____, is modified by the host cell then inserted into the host cell plasma membrane, becoming a high-affinity receptor for LEE-encoded ____, an EPEC outer-membrane protein
Tir; Initimin
- The ______ triggers host cell accumulation of actin polymers beneath the adherent bacteria, resulting in ‘pedestal’ formation
Tir:Intimin interaction (intimate adherence)
How is site specific recombination different from homologous (generalized) recombination?
SSR:
- only short stretches of homologous sequence required
- breakage & union occur only at specific sites
- alters the arrangement of chromosomal sequences
What do these 3 enzymes do?
- NADPH oxidase
- Superoxide dismutase
- Myeloperoxidase
- NADPH oxidase makes superoxide radicals
- superoxide dismutase turns the superoxide radicals into H2O2
- myeloperoxidase converts H2O2 & Cl- into hypochlorite (bleach)–used to kill bacteria
Which TLR binds to lipopeptides?
TLR1:TLR2; can form a heterodimer
Which TLR binds (viral) dsRNA?
TLR3
What is NF-kB’s functions?
once in nucleus, it regulates transcription of hundreds of genes w/ important functions in inflamm & infection
- stimulates the release of TNFa, IL1, & IL6.
When signalling with TLRs, what is the order of proteins and factors that is involved? Or what is the pathway of the signalling?
receptors (TLRs) > Adaptors (MyD88) > Kinases > Transcription factors (NF-kB) > Effectors (cytokines, chemokines, etc)
What is the role of TNF-a?
activates vascular endothelium & increases vascular permeability, which leads to inc’d entry of complement & cells to tissues & inc’d fluid drainage from lymph nodes
What is the role of IL-1B?
activates vascular endothelium and lymphocytes.
- local tissue destruction increases access of effector cells
What is the role of CXCL8?
chemotactic factor that recruits neutrophils & basophils to site of infection using cytokines
What are the ways/mechanisms our body uses to protect itself?
- basically asking for effects of IL-1/IL6/TNF4?
- in liver: acute phase proteins (C-reactive prot, mannose-binding lectin)
- activates complement opsinization. - in bone marrow, Neutrophil mobilization > phagocytosis
- hypothlamus: inc body temp; dec viral/bacterial replication
- fat, muscle: protein & energy mobilization to generate increased body temp
What are the 3 complement pathways (in order)?
- Alternative = pathogen surface creates local environ conductive activation; 1st to act
- Lectin pathway = mannose-binding lectin binds to pathogen surface
- 2nd to act
classical pathway = even more creepy; C-reactive protein or AB binds to specfic AG on pathogen surface
Sequential insertion of complement proteins into pathogen membrane results in ____.
“Membrane Attack Complex” (MAC).
What is the diff between C3a & C2b?
C3b is covalently found on the surface of components of pathogens
What are the 4 outward signs of inflamm?
redness (rubor), heat (calor), swelling (tumor), pain (dolor)
What recognition mechanism are used by innate pathogen receptors?
- TLRs
- LPS receptors (CD14)
- mannose receptors
- glucan receptors
- scavenger receptors
- complement receptors (CR3)
Where do neutrophils come from and how do they reach site of infection?
bone marrow; chemotaxis/diapedesis
What events precede and follow the act of phagocytosis?
Precede: recognition mechanisms
- sensing of bact
- mov’t twd them
Phagocytosis: binding, uptake, degradation
Follow: activation of adaptive IR
- remains of bact are used to further signals to other cells
What are the characteristics of innate immunity?
- rapid response
- invariant receptors
- limited # of specificities
- limited expansion during response
What are the characteristics of acquired immunity?
- slow response
- variable receptors
- numerous highly selective specificities
- improves during response & has memory
Antibodies activate what pathway of complement activation?
C-c-classical!
What gives rise to lymphocytes? And what are the effector cells it can change in to?
lymphoid progenitor
- B-cells (into plasma cells)
- T-cells (into cytotoxic or helper T-cells)
- NK cell
* all cells from this progenitor are part of adaptive immunity except NK cell
What is a mechanism to prevent “self-reactivity” in B & T cells?
clonal selection
Difference betw how B-cell & T-cell receptors recognize Ag?
B-cells can bind to native antigen (at epitope).
T-cells only bind to processed antigen (peptide:MHC complexes).
What are MHC molecules in humans called?
Human Leukocyte Antigens (HLA)
What cells are MHC Class I in? MHC Class II?
MHC Class I = infected cell; bact cleaved in ER; CD8
MHC Class II = phagocytes; bact cleave in lysosome; CD4
How do cytotoxic T-cells fight infection?
recognizes complex of viral peptide w/ MHC class I and kills infected cell
How do Helper T cells help fight infection?
TH1 recognizes complex of peptide Ag w/ MHC class II & activates mac’s or B cells
What cells arise from the myeloid progenitor?
Blood: basophil, eosinophil, neutrophil, monocyte
Tissues: dendritic cell, mast cell, macrophage
Which WBC is best to kill multicellular parasites? And what Ig’s do they bind to?
eosinophil; have receptors to bind to IgE antibody
*OR basophils bc they function like eosinophils
What is the least abundant immune cell?
granulocytes
Mast cells have a high affinity for what Ig?
IgE
What substance do the granules of mast cells contain?
histamines & other inflamm mediators
What are examples of bridges between innate & acquired immune systems?
- macrophages
2. dendritic cells
What receptors do mac’s have?
- Fc receptors that help it bind & destroy Ab-opsonized microbes
- has PRR receptors (pattern recognition receptors)
- it also have both MHC I & II
Why are NK cells not considered lymphocytes?
they bear NO Ag-specific receptors.
What is a central/primary lymphoid organ? Examples?
- tissue where lymphocytes develop & mature
- ex. bone marrow, thymus
What is a peripheral/secondary lymphoid organ? Examples?
- tissues at which IR are initiated (APCs & lymphocytes interact!
- ex. spleen, lymph nodes (i.e. tonsils), GALT, MALT/BALT, lymphatic vessels
In the lymph node, which lymphocytes are closer to the center/core, and which ones are closer to the periphery?
B-cells on the outside. T-cells near the inside.
How does a lymphocyte enter through a lymph node? Which lymphocyte would they encounter first?
through high endothelial venule (HEV); encounter T-cell first, then B-cell.
What makes ABs stable?
disulfide bonds
what happens to IgM’s structure once it has encountered an Ag?
it goes from a monomeric structure to a pentameric design
IgA is pumped out into the mucosal layers of the body. What is the process it goes through to be released out onto the apical face of epithelial cells?
transcytosis
What is the difference betw when Ab (IgM or IgG) activates
IgM - pentameric; C1 binds to a signle IgM molecule
IgG - monomeric; C1q binds to 2 or more IgG molecules
*both activate classical pathway–recall that Ab’s are involved with classical pathway
Importance of IgA, IgE, IgG, IgM.
IgA - present in mucous membrane—transport across epith
IgE - important in triggering Mast cell degranulation—sensitization of mast cell (degranulation)
IgG - all of the IgGs can be transported across the placenta (mainly IgG1)—also does opsonization & diffusion into extravascular sites
• Allows baby to be born with some antibodies
IgM - activation of complement system
there are two antibody isotypes that incorporate the J chain. Which ones?
IgA (dimeric) & IgM (pentameric)
Histamine is released from what?
platelets during clotting and from mast cells
kinins are released from?
clotting and granulocytes
What is CD14?
a LPS receptor that is involved in a complex with MD2 & TLR4 as well. They all bind to LPS bound to LBP.
what cytokines does NFkB code for?
TNF-a, IL1, IL6
______ act locally to increase capillary blood flow and permeability. ____ also activates endothelium and increases adhesion of white blood cells.
Tumor necrosis factor (TNF) and IL-1 ; IL-1
*TNF induces the synthesis of IL-1 which in turn induces IL-6 and CXCL8. TNF and IL-1 act systemically to induce the fever response.
_____ is an important chemotactic factor, acting to induce the migration of leukocytes into the site of infection.
CXCL8 (older name IL-8)
IL-6 is an important cytokine that acts systemically. In the liver, IL-6 along with TNF-α and IL-1 induce the synthesis of _____.
acute phase proteins
- These include C-reactive protein (CRP), serum amyloid A, fibrinogen, and mannose-binding lectin (MBL).
Cleavage of C3 (3rd component of complement) generates the cleavage products C3a and C3b. ____ is covalently bound to the surface of a bacterium where it assists in macrophage binding and phagocytosis of the bacteria. The other, ___, acts to recruit more phagocytes to the site. This mechanism of complement activation received the name of _____.
C3b, C3a, “Alternative Pathway”
Antigen receptors are assembled by ______ that randomly recombine their DNA sequences in order to generate the complete coding regions for the antigen-binding portions (the variable domains) of the (B and T) lymphocyte receptor.
association between gene segments
BINDING TO ANTIGEN ALONE IS NOT ENOUGH TO ACTIVATE B AND T CELLS
Activation of B cells and T cells requires two separate stimuli (“signals”): __2__.
one requires binding to the surface antigen-receptor, the other is stimulation of a second cell surface receptor.
- B-cell: Once the B cell receptor binds to its specific antigen (1st signal of activation), the B cell ingests the antigen, breaks it into pieces (peptides), and presents the peptides to an antigen-specific T helper cell. The T helper cell supplies a 2nd signal of activation to the B cell.
- T-cell: binding of cognate antigen (1st signal of activation) must be accompanied by co-stimulation (2nd signal of activation) provided by T cell binding to a counter- receptor on the antigen presenting cell.
Spleen’s white pulp is divided into areas that contain primarily _____, and adjacent areas containing primarily _____.
T cells; B cells
Examples of MALT?
larynx, tonsils, bronchus, nose, some along intestinal wall, peyer’s patch
Remember that all immunoglobulins produced by a B cell have what…?
the same antigen-specificity
Both C and V domains are a roughly barrel-shaped and are formed by_____ that are stabilized by a disulfide bond.
two sheets of antiparallel β-strands
When the sequences of many variable domains are compared, the greatest sequence variability is observed in 3 “hypervariable” regions of the heavy and light chain variable domains. These hypervariable regions make up the combining site. What are these regions called?
complementarity-determining regions
less variable regions betw CDRs are known as framework regions ______.
framework regions
two isotypes of light chains of antibodies?
kappa (κ) and lambda (λ)
*Approx. 60% of human IgG contain kappa light chains, the rest contain lambda chains
what is a hapten?
an Ag too small to induce an IR
Why is T-independent Ag-elicted responses limited?
T-independent antigen-elicited responses are usually limited to production of IgM antibody and do not elicit B cell memory.
Which route of Ag administration elicits a potent IR? Which one induces non-responsiveness/tolerance? How about ingestion of an Ag–what happens? Respiratory tract?
subcutaneous administration; intravenous administration; systemic tolerance; results in allergic rxns
Antigens delivered to the respiratory or gastrointestinal tracts can also induce antigen-specific ______ responses.
secretory IgA Ab (sIgA)
What is an adjuvant?
any substance that enhances immunogenicity of an Ag
- they usu inc length of exposure of immune sys to Ag
Cytoplasmic sensors of the presence of bacteria belong to the family of ___ that form an ______.
NOD proteins; inflammasome
benefits and limitations of precipitation and agglutination?
- Benefits: No specific treatment of antibodies required, direct method.
- Limitations: Not particularly sensitive, relatively large amounts of antigen required.
benefits and limitations of Western blot?
- Benefits: purified antigen or antibody are not needed; highly sensitive.
- Limitations: non-quantitative
benefits and limitations of ELISA?
- Benefits: very sensitive, quantitative.
- Limitations: must have specialized reagents and a 96-plate spectrophotometer.
Steps in ELISA procedure?
Steps in the Procedure:
• Wells are coated with purified antigen.
• Dilutions of Ab sample (primary Ab) are added to wells and incubated for binding.
• Unbound Ab are washed out of the wells.
• Bound antibodies are detected with anti-immunoglobulin antiserum conjugated to enzyme (secondary antibody).
• Substrate is added to each well and incubated until color change is observed.
• Color development is read quantified in an ELISA reader (spectrophotometrically).
• Benefits: very sensitive, quantitative.
• Limitations: must have specialized reagents and a 96-plate spectrophotometer.
_____ is a method to use fluorescence and laser light excitation to perform single cell analysis. In an adaptation, this method is capable of separating (“sorting”) positive cells.
Flow cytometry
What are the benefits and limitations of flow cytometry?
- Benefits: very sensitive; quantitative
- Limitations: costly; requires sophisticated software
Ab’s are covalently bound to Ag. T/F?
FALSE! The Ab/Ag binding is a reversible thermodynamic equilibrium.
What is the diff between affinity & avidity?
Affinity: measure of strength of a single Ab binding to a single site on an Ag
Avidity: sum of the strength of Ab binding to Ag in multivalent fashion
What are monoclonal antibodies? And how do they usually occur?
MAbs are Ab’s tjat are produced by a cloned (immortalized) B-cell line, a pop. of Ab-secreting cells that all descended from a single B-cell.
- occur naturally as a myeloma or generated by fusing a B cell to a myeloma cell
The production of which Ig does not require T cell help?
IgM
What are the 2 stages of B cell maturation?
- Ag-independent
2. Ag-dependent
What is the order of the segment joining in the construction of an Ig heavy chain variable-region exon?
joining of a D-region gene segment w/ a J-region gene segment, then joining of a V-region gene segment w/ the combined DJ sequence
what occurs in clonal deletion? in anergy?
Clonal deletion: immature b cells that interact w/ “self”-Ag’s undergo apoptosis
Anergy: immature B cells that interact w/ soluble Ag in bone marrow are signaled to down-regulate cell surface expression of IgM and become anergic (or unable to respond to specific Ag)
_____ is a costimulatory protein found on antigen presenting cells and is required for their activation.
CD40
Tell me about light chain gene construction.
- random joining of a single V gene segment w/ a single J gene segment
- C gene segment does not move and joins VJ after transcription and splicing of the non-coding (intronic) RNA
- transcription
Tell me about heavy chain gene construction.
- random joining of a D gene segment w/ a J gene segment
- random rearrangement of single V to DJ to form DNA sequence coding for complete variable domain of heavy chain (VDJ)
- RNA transcription followed by processing of introns and splicing of exons, brings VDJ closer to C
* as in the light chain, C gene segment is “downstream” of the J segment but is separated from the VDJ junction by non-coding introns
What are the proteins that catalyze DNA recombination?
recombinase activating genes 1 & 2 (RAG 1 & 2)
Joining of gene segments is imprecise. What is the consequence of this?
Some of the VJ and VDJ joints are out-of-frame (they do not follow the triplet codon reading frame and cannot be translated into a full-size protein).
When does rearrangement stop?
when the functional V region coding sequences have been formed
All progeny of a single B cell will express the same V region genes. T/F?
true
What are the 4 mechanisms that generate diversity in B-cells?
- different combinations of V, D, J
- imprecise joining of gene segments during somatic recombination process
- pairing of many possible light chains and many possible heavy chains
- somatic hypermutation
How does the same gene encode a membrane-bound Ig receptor and a secreted Ab?
alternative RNA splicing
Switching between isotypes of Ab’s occurs through a specialized DNA recombination mechanism that is guided by _______. As switching occurs, the recombination event loops out and removes the DNA between the original and the newly accessed constant regions.
switch recombination sequence
The Fc portion of Ab molecules can interact w/ which of these components? A) complement proteins B) other Fc domains C) Fc receptors on mac's & cells D) A & C E) all of the above
E) all of the above
