Antibodies and Myeloma Flashcards
Vertebral column sections and structure
Cervical 7 Thoracic 12 Lumbar 5 Sacral 5 Coccygeal 4
Intervertebral discs between vertebrae preventing friction and crushing
4 natural curvatures of spine
Function of vertebral column
Protects spinal cord
Supports weight of body
Maintains posture
Facilitates movement
Describe the C1 atlas
First cervical vertebrae
Articulates w head and occiput of the axis
No vertebral body and no spinous process
Transverse ligament secures the dens (C2) to the axis
Describe the C2 Axis
Easily identifiable by the dens
Dens articulates with anterior arch of atlas making the medial atlanto-axial joint and allowing for independent head rotation
Cervical vertebrae
Thoracic vertebrae: Location Body Vertebral foramen Spinous process Transverse processes Functions
Lumbar vertebrae: Location Body Vertebral foramen Spinous process Transverse processes Functions
What are the curvatures of the spine?
- Cervical curvature (lordotic)
- Thoracic curvature (kyphotic)
- Lumbar curvature (lordotic)
- Sacral curvature (kyphotic)
All of these are important for balance, flexibility, stress absorption and distribution.
What are the types of spinal curves?
Kyphosis/Kyphotic curve- concave anteriorly and convex posteriorly
Lordosis/Lordotic curve- convex anteriorly and concave posteriorly
KEELING OVER- kyphotic
LIMBBO- lordotic
What is hyperkyphosis?
– excessive curvature of the spine (>50 degrees)
What is hyperlordosis?
– excessive curvature of the cervical or lumbar regions
Scoliosis
– excessive lateral curvature of the spine (mild: 10-24 degrees, moderate: 25-40 degrees, severe: >50 degrees)
Spinal cord structure
The spinal cord continues from the medulla oblongata and then travels inferiorly within the vertebral canals
The spinal cord is surrounded by the spinal meninges and CSF
At L2, the spinal cord tapers off to become the conus medullaris
The spinal nerves at the end of the spinal cord bundle together to form the cauda equina
Occupies 2/3 of the vertebral canal
Ventral and dorsal…
Anterior (ventral) and posterior (dorsal) roots of the spinal cord
Type, passing through and innervation of: Somatic efferent Somatic afferent Visceral efferent Visceral afferent
What is lymph?
Lymph is formed from interstitialfluid from plasma filtrate
Contains salts, fat, protein, and cells (mainly lymphocytes)
Interstitial fluid drains primarily as lymph rather than venous reabsorption
Function of lymphatic system
Maintenance of fluid balance
Supportstissue immunosurveillance and prevention of infection
Facilitates fat transport
How does fluid get pulled into lymphatic system?
Lymphatic capillaries are blind ended, all flows in one direction
Gaps between cells (mini valves)
Proteins in lymphatic capillary = higher oncotic pressure compared to venous and arteriole
–> this pulls fluid from interstitial space into lymphatic system, going to greater oncotic pressure
Then pushed through mini valves into larger collecting lymphatics
How is fluid pushed through lymphatic system?
No pumps in lymphatics, works via muscle contraction
As you move, muscle contraction squeezes on lymphatics, compresses vessels which pushes lymph up through system
Hence mobility is important for this to occur, and why build up of fluid (lymphoedema) can happen
How does lymph arrive and exit the lymph node?
Lymph arrives through the afferent lymphatic vessels
Lymph drains through the sinus spaces allowing it to run through the entire node (to medullary sinus)
B cells and T cells sample the peptide: MHC complexes
Lymph exitsvia the efferent lymphatic vessels
Diapedesis
Transmigration, or diapedesis, is the process by which T lymphocytes migrate across venular blood vessel walls to enter various tissues and organs
Differences between:
Isotypic
Allotypic
Idiotypic
Isotypic – changes in the constant regions of the heavy and light chains making up the overall class
Allotypic – small genetic variations between individuals/populations (allelic variation)
Idiotypic – the set of epitopes on the variable region of a particular antibody - key for diversity of antibodies
What are the different regions of an antibody
Variable regions at the top- stripey Constant region0 light parts of Y Outer part- light chain Inner part- heavy chain Fc region- the 'stem' of the Y shape, which binds to receptors on cells
Describe the chain structure of antibodies
Each antibody has two heavy chains and two light chains
A light chain has V and J segments
A heavy chain has V, D and J segments
These recombine in different patterns to generate antibody diversity from a small number of genes
Segments of light chain vs heavy chaiin
Light: V and J
Heavy: V D J
What are the 2 variations of light chain?
Lambda
Kappa
rearrangement of Ig genes
Randomly select V region on light chain, and one J region
Join V1 to J2 and the middle section inbetween is lost
Same with heavy chain (random V to random D to random J) everything in middle is cut
Gene segment recombination generates Ab diversity
Define antibodies
Antibodies (Ab) = immunoglobulins (Ig) = gammaglobulins
produced in response to foreign structures (antigens, Ag)
the part of an Ag that is recognised by an Ab = epitope, or antigenic determinant
What are the 5 variations of heavy chains?
Mu Gamma Alpha Delta Epsilon
What holds together the antibody 2 heavy chains and 2 light chains?
Disulfide bond
What are the heavy chains associated with each antibody class?
IgG - gamma IgM - mu IgA - alpha IgD - delta IgE - epsilon
Which immunoglobulins are most prevalent in serum plasma?
IgG
then IgA, IgM
Where is the antigen binding site on an antibody?
N terminus of Variable region (heavy and light chain)
Linear vs conformational epitopes
- linear epitopes: adjacent amino acid residues (6aa)
- conformational epitopes: non-sequential amino acid residues spatially juxtaposed in the folded protein
Epitope
Antigenic determinant- portion of antigen that antibody binds to
Two main function of antibody
- Recognition of an infinite number of antigens
- - antigen binding site (Fab) - Effector functions
- - via Fab - bind and neutralise/block entry of Ags
- - other effector functions - mainly mediated by Fc portion
- - interaction with other cells, complement activation –> macrophages, eosinophils have Fc receptors (FcR) => binding of microbes opsonised by Ab
How to achieve recognition of infinite Antigens ?
ANTIGEN BINDING SITE (FAB)
VH and VL domains contain three hypervariable regions
hypervariable regions correspond with protruding loops that make contact with Ag
hypervariable regions of heavy and light chain form the antigen-binding surface
hypervariable regions = complementarity-determining regions (CDR1, CDR2, CDR3)
CDR3 has the highest variability due to genetic mechanisms that ensure Ab diversity
CDRs in primary vs tertiary structure?
Ig primary structure: CDRs are separated
Ig tertiary (3D) structure: CDRs become adjacent to each other
What type of B cell populates lymph nodes/spleen and why?
Naïve B cells- waiting for antigens
Somatic recombination
Combinations of gene segments allow generation of a high number of different immunoglobulins
Where is the locus for the heavy chain gene located?
Chromosome 14
Kappa chain: V, J and C segments
35V (variable)
5J (joining)
1C (constant region)
Lambda chain: V, J and C segments?
30V (variable)
4J (joining)
4C (constant region)
Antibodies generation of diversity
Somatic recombination (V-D-J joining) Addition of N and P nucleotides, transcription and RNA processing in 3 B cell clones
Endonuclease cuts randomly after one D and before one J and then after one V and before DJ (coded by RAG 1 and 2)
The free ends are ligated together to form a functional gene
What mediates VDJ recombination?
Recombinase enzymes coded for by RAG 1 and RAG 2
recognise recombination signal sequence RSS flanking V, D and J gene segments
Junctional diversity
increases the number of Ab generated
The enzyme terminal deoxynucleotidyl transferase (TdT) adds random nucleotides (N nucleotides) to the free ends before the joining
=> causes differences in the sequence of antibodies produced
Most variable portion of Ig molecule
CDR3 – the most variable portion of the Ig molecule is located at the site of D-J (heavy chain) or V-J joining (light chain)
D-J or V-J joining are the sites of N nucleotide addition => maximum variation of Ig sequence corresponds to CDR3
T cell receptor chains
TCRs made up of 2 polypeptide chains (alpha and beta) with variable chains at ends and constant region
Variable regions produce specificity
Ig Gene expression process
Ways of creating antibody diversity
Multiple germ line genes - V,D and J heavy chain and V and J light chain genes
Random recombination of the - V,D and J heavy chain and V and J light chain segments
Imprecise joining of V,D and J segments due to nucleotide deletion or inclusion
Random pairing of different combinations of L and Heavy chain regions in different B cells.
Allelic exclusion: heavy chain
B cells are diploid ie two alleles of all Ig genes
Heavy chain:
- two alleles
- in theory could make two different heavy chains
This never happens = Allelic exclusion
Mechanism: as soon as one allele rearranges successfully and the heavy chain protein is produced the heavy chain rearrangement is switched off
Allelic exclusion: light chain
Light chain:
- 2 alleles for κ chain and 2 alleles for λ chain
- in theory could make 4 different light chains
This never happens = Allelic exclusion
Mechanism: as soon as one allele rearranges successfully and the light chain protein is produced => switches off light chain rearrangement
Each B cell/B cell clone makes either κ or λ chains never both
Polyclonal B cells are a mixture of cells making κ or λ chains
=> light chain restriction (importance in identifying B cell tumours ie monoclonal)
Light chain restriction
Each B cell/B cell clone makes either κ or λ chains never both
Polyclonal B cells are a mixture of cells making κ or λ chains
=> light chain restriction (importance in identifying B cell tumours ie monoclonal)
Clonal selection
Antigen-specific clones of lymphocytes develop before and in the absence of antigens in central (generative) lymphoid organs
lymphocyte clones specific for >107-109 antigens
present before exposure to antigen
B cells leave the ‘central’ lymphoid organ and function in the ‘peripheral’ lymphoid organs
when an antigen enters, it activates (‘selects’) the specific lymphocyte clones that recognise the Ag
generation of Abs specific for that Ag only
expansion of antigen-specific clone
What happens to plasma cells after they get rid of antigen?
they DIE
Why are normal immune responses polyclonal?
More than one clone of B-cells is activated
More than one antibody is synthesised
Because
- Multiple antigens on organism
- Multiple epitopes on each antigen
- More than one Ab may recognise the same epitope
What generates the switch of B cells to secreted form of antibodies?
Differential splicing of exons
- secreted IgG doesn’t have hydrophobic transmembrane portion like membrane IgG does so can be released
Production of secreted IgG
Primary transcript has membrane coding sequence- this is spliced out for secreted IgM and so whole molecule is instead secreted
Antibody actions
Bind to extracellular microbes and toxins:
- neutralise (block adherence/entry)
- eliminate
- opsonisation = ↑ phagocytosis
- complement activation = opsonisation, lysis
Antibody isotope switching
During an immune response B cells become capable to produce Abs of different classes but without changing specificity (respond to the same Ag)
- ability to perform different effector functions
- can deal better with pathogens
- isotype switch needs signals from helper T cells
- does NOT alter specificity or alter light chain
What does IgM switch to?
IgG, IgA, IgE
What does IgG switch to?
IgA, IgE
How do T cells help with isotope switching?
- CD40L on T cell interacts with CD40 on B cells
- cytokines produced by T cell
- IFN-gamma => switch to IgG1, IgG3
- IL-4 => switch to IgE
TGF-beta (and other cytokines) => switch to IgA
Isotope switching process
Between constant regions are switch regions that allow splicing out of different constant region genes
Done via endonucleases like activation induced cytidine deaminase (AID)
Sequence with variable part and different heavy chain classes of genes along chromosome → so if B ell switches from IgM to IgG3 then Cmu and Cdelta cut out = Cgamma 3 (produced from class switch)
then to produce IgA from Cgamma 3, switch out Cgamma 1 and 3 for Calpha 1
ALL MEDIATED BY TGF BETA
What happens in patients with AID immunodeficiency?
AID: activation induced cytidine deaminase
DONT HAVE
- Class switch recombination (CSR)
- Somatic hypermutation (SHM)
Polymeric pentameric IgM
J chain (joining chain present in all polymeric Igs) Pentameric IgM: high avidity for Ag – 10 Ag binding sites
1st Ab produced in a primary immune response
very efficient at complement activation
–Leading to promotion of opsonisation and lysis
IgG properties
Highest concentration in circulation
monomeric
complement activation
promotes opsonisation and lysis
neutralises/blocks entry microbes and toxins
Only Ab to cross placenta -> neonatal protection
IgA dimer properties
J chain (joining chain present in all polymeric Igs)
In circulation: mostly monomer
Major Ab in mucosal secretions
GI / respiratory tract, breast milk
Dimer (polymer) – has a J chain
Prevents adherence and entry of pathogens
How are IgA antibodies secreted?
Found in lamina propria Produce IgA attaching via J chain Then attach via poly Ig receptors Pass into mucosal epithelial cells Proteolytic cleavage -> secreted into lumen where they function
IgE properties
very low levels in healthy individuals
monomeric
Fc receptors on eosinophils, mast cells- degranulation
Defence against particles
Responsible for allergies (type 1 hypersensitivity)
IgD properties
Monomer
Mainly B cell receptor together with IgM
Co expression of IgM and IgD
IgD is co-expressed with IgM (works as Ag receptor)
IgM is the 1st immunoglobulin to be produced
IgD is produced at the same time with IgM
Mechanism: differential splicing
Exons for Cμ and Cδ are transcribed as part of a single precursor RNA
Differential splicing can remove Cμ exons => now Cδ exons are used => IgD (same VDJ as IgM joined to Cδ)
Myeloma pathogenesis
Clonal expansion of immunoglobulin secreting, heavy-chain class switched, terminally differentiated B cells
Bone marrow based disease associated with lytic bone disease, anaemia and bone marrow failure
PC secrete monoclonal protein. IgG (60%), IgA (20-25%), light chains only (15-20%)
Tests required to diagnose myeloma
Lab tests:
FBC and blood chemistry
SPEP (Serum protein electrophoresis) and UPEP (Urine protein electrophoresis)
- looking for abnormal light chains
Bone marrow biopsy
–> flow cytometry
Imaging: CT, MRI, PET
MM diagnostic criteria
At least 10% bone marrow cells being clonal abnormal plasma cells or biopsy of bone proving a clonal plasma cell disorder
Must also demonstrate patient has one or more SLiM CRAB criteria
MGUS diagnosis
Less than 10% clonal plasma cells or less than 30g light chain/paraprotein and NONE SLiM CRAB criteria
SLiM criteria stands for:
S- BM >60% clonal plasma cells
Li- SFLC (light chain) ratio >100
M- 2 or more lesions on MRI
Myeloma prognostication
Abnormalities like 17p deletion or translocation of 4;14 puts them in a high risk group
What pattern characterises multiple myeloma?
Remission and relapse
Patients become symptomatic so have first line therapy
→ longest period of remission before disease relapses and need further treatment
→ with each relapse time the remission becomes shorter until = refractory disease and they succumb to their illness
What is pain due to in MM?
- Bone pain
- Nerve pain - nerve root, spinal cord compression
Pain as a complication of the treatment
- Peripheral neuropathy
NICE recommendations for MM imaging
Offer imaging to all people with a plasma cell disorder suspected to be myeloma.
Consider whole‑body MRI as first‑line imaging.
Consider whole‑body low‑dose CT as first‑line imaging if whole‑body MRI is unsuitable or the person declines it.
Only consider skeletal survey as first‑line imaging if whole‑body MRI and whole‑body low‑dose CT are unsuitable or the person declines them.
Do not use isotope bone scans to identify myeloma‑related bone disease in people with a plasma cell disorder suspected to be myeloma.
Why do bone scans have low sensitivity in myeloma?
Bone scans have low sensitivity in myeloma due to low osteoblastic activity
Common sites for bone involvement in MM
Skull
Spine
Pelvis
Long bones
Skeletal lesions in MM
pepper pot
Lytic bone disease
bone weakened, little stress can cause pathological fracture
T2 weighted MRI showing myeloma- related fractures at L3 and L4
Common cause of back pain in MM patients?
Vertebral compression fracture
75% of the patients have bone pain1
55-70% have VCFs or history of vertebral body abnormalities.2
Back pain in patients correlates with Vertebral Compression Fracture (VCF) in >50% of patients at time of diagnosis1
Why does the bone pain occur?
Origin of pain from sensory and sympathetic neurones in medulla of bone
Marrow contains rich plexus of nerves
Cancer cells release cytokines sensitizing neurones. This can lead to up regulation of pain receptors, central sensitization in spinal cord and long-term potentiation
Cause of MM bone disease
RANK is protein receptor found on osteoclast precursor cells
RANKL is a protein expressed by bm stromal cells - binds to RANK and promotes osteoclast differentiation
OPG decoy receptor - inhibits binding of RANK and RANKL.
In myeloma RANKL expression upregulated and OPG switched off – promotes osteoclast activity
RANK Ligand upregulated by myeloma cells + downregulation of its natural antagonist OPG = increased interaction of RANKL and RAN on osteoclasts with upregulation of osteoclasts
= bone reabsorption and imbalance of osteoclasts and osteoblasts
MM treatment goals
Induce remission, if possible
Prevent immune suppression
Prevent hematologic abnormalities (anemia, low platelets)
Prevent end-organ damage
Prevent fractures
Maintain function, quality of life, control symptoms
Painkillers to use in MM patients
Act on different receptors Ca2+ - Gabapentin
opioid - Opiates/ Fentanyl GABA - Benzodiazepines
Effect may be synergistic
Potential toxicities:
Nausea/ constipation, opiate induced hyperanalgesia, metabolite toxicity, potential immunosuppression
Novel drugs that have helped myeloma patients
Proteasome inhibitors
HDAC inhibitors
VEGF inhibitors
When do we need orthopaedic input?
Stabilisation of pathological fracture Decompression of cord Vertebroplasty Balloon kyphoplasty Radiotherapy
Role of bisphosphonates
Role in treatment of malignant hypercalcaemia and bone pain
Inhibitors of osteoclastic bone resorption
Reduction in vertebral and non vertebral fractures in MRC myeloma VI trial
How do bisphosphonates work?
High affinity to bone – rapidly absorbed onto bone surface and ingested by osteoclasts
2 classes of bisphosphonate – work in different ways
Non-nitrogen containing (eg clodronate) metabolised by osteoclast and disrupt cell metabolism leading to apoptosis
Nitrogen containing (aminobisphosphonates) eg pamidronate and zoledronic acid and some orals eg alendronate and ibandronate) – induce apoptosis and inhibit osteoclast function by disrupting signalling of key regulatory proteins
What is a complication of using bisphosphonates long term?
Osteonecrosis of jaw
Exposed bone in maxillofacial area often in association with dental surgery (ONJ can also happen spontaneously), with no evidence of healing
What are the different causes of peripheral neuropathy when associated with MM?
Disease related: Amyloid, cryoglobulin, nerve root compression, POEMS, autoimmune
Co-morbidities: Diabetes, EtOH, weight loss and malnutrition
Drug related: Thalidomide, IMiDs, proteosome inhibitors, platinum,
Assessment and management of neuropathic pain
Peripheral neuropathy is associated with newer agents thalidomide and bortezomib
Assessment through use of neurotoxicity assessment tool, neurological examination and EMG studies, antibody studies, biopsies
Management includes dose reduction, pharmacological agents, massage, patient education
Incidence of neuropathic pain in patients with multiple myeloma
- 11-20% of newly diagnosed patients
- 83% in relapsed, refractory patients
- High incidence due to neurotoxic drugs used including vincristine, platinum, thalidomide, bortezomib
Management of neuropathic pain in a MM patient?
Close monitoring of patients (neurology assessment/ tools)
Actively seeking out symptoms through asking appropriate questions
Prompt action (dose reduction, switching to alternatives)
Symptom relief (amitriptyline, duloxetine, gabapentin or pregabalin )
12/23 rule
Only a Ig gene segment with a 12 base spacer RSS can be joined to an Ig gene segment with a 23 base spacer
In heavy chain never just join a V segment to a J segment- so because both V and J have 23 they cant join together due to 12/23 rule,
(needs to be V 23 and D 12, or D 12 and J 23- needs to be in right order so 12 first)
Joining segments in heavy chains
There are V, D and J segments in a heavy chain
A D segment will be randomly selected and joined with a J
The DJ segment will then be randomly joined with a V segment
The 12/23 base spacer rule makes sure that V joins to a D and a D joins to a J
How does rearrangement occur in light chians?
V and J segments will randomly join to generate a VL domain
There are specific recombinases (RAG enzymes) which recognize the RSSs and allow joining
There will be a coding joint generated between the V and J domain that has been randomly selected
There will also be a signal joint which is the lost intervening piece of DNA
There may also be receptor editing of the light chain genes
Antibody structural regions are…
1 Fc fragment region + 2 Fab fragment regions
