Tissue typing: HLA Matching
- - ABO
- - HLA-A, HLA-B, HLA-DR (most important for grafting and transplantation)
- - Preformed Abs- previous pregnancies, transfusions, transplantations
- - Donor Ab screening: Sera of potential donors (donor Abs) mixed with lymphocytes of recipient + C’. Lysis is mismatch
- - Cross-matching: Sera of recipient (recipient Abs) mixed with donor’s lymphocytes + C’. Lysis is mismatch
- - Flow Cytometry
- - PCR
Evasion and subversion of the immune system by pathogens
- - Genetic variation within pathogens prevents effective long-term immunity; Mutation and recombination allow influenza virus to escape from immunity
- - Certain pathogens sabotage or subvert immune defense mechanisms
- - Bacterial superantigens stimulate a massive but ineffective T-cell response
Host defense against Streptococcus pneumoniae is type specific
- - When exposed to S.p. for the first time, Ab are made and bacteria are cleared
- - Over time, there is a different type of S.p.
- - Old response no more effective; new response needed
Two types of variation allow repeated infection with type A influenza virus
- Antigenic drift - mutations alter epitopes in hemagglutinin, old Abs no more work
- Antigenic shift - RNA segments are exchanged between viral strains in a secondary host, new hemagglutinin expressed, works if no cross-protective immunity.
Antigenic variation in trypanosomes allows them to escape immune surveillance
- Many inactive VSG genes (antigenic) but only one site for expression
- Gene conversion - inactive genes copied into the expression site
- Rounds of gene conversion allows the trypanosome to vary expressed VSG
Bears have only one V gene. they generate diversity through gene conversion by moving dormant genes to the variable region.
Persistence and reactivation of herpes simplex virus infection - cold sores
- Primary infection - low expression of MHC I
- Latent phase - trigeminal ganglion; low MHC I; evasion from CD8 T cells
- Recurrent infection back at the initial infection site
Some pathogens resist destruction or exploit the host for their good
- Mycobacterium tuberculosis is taken up by macrophages but prevents the fusion of the phagosome with the lysosome.
- Listeria monocytogenes, escape from the phagosome into the cytoplasm of the macrophage, where they multiply.
- Toxoplasma gondii (a protozoan parasite ) generates its own vesicle, unavailable to MHC loading
- Treponema pallidum (spirochete; syphilis) avoids elimination by coating its surface with host proteins until it has invaded tissues like CNS
- Some strains of Borelia burghdorferi (Lyme disease) may coat themselves with complement inhibitory protein H.
Immunosuppression or inappropriate immune responses can contribute to persistent disease
- Staph. enterotoxins and TSST-1 - toxic shock; superantigen, stimulated T cells undergo massive responses and apoptosis, resulting in generalized immunosuppression
- Bacillus anthracis - anthrax lethal toxin (lethal factor and protective antigen). Protective antigen routes lethal toxin to cytosol. MAP kinase inhibition leading to apoptosis of infected macrophages and premature DC maturation.
- The measles virus can cause a relatively long-lasting immunosuppression in malnourished or undernourished children. Infected DC render T and B cells hyporesponsive to Ag.
- Occur when immune system components defective
- Primary immunodeficiencies
- - Caused by mutations in genes involved in immune responses
- - Highly variable clinical manifestations: Recurrent and often overwhelming infections in very young children; allergy, abnormal proliferation of lymphocytes, and autoimmunity may occur
- Secondary immunodeficiencies
- - consequence of other diseases, or secondary to environmental factors such as starvation, or medical intervention.
Inherited immunodeficiency diseases
- caused by recessive gene defects
- Mostly on X-chromosome
- - Females can shut defective X chromosome with defective gene (do better; X-inactivation)
- Defects in either the adaptive or the innate immune system
- - Selective deficiency in IgA is the most frequently
- reported - respiratory infections
Mechanisms used by viruses of the herpes and pox families to subvert the host immune system
- Inhibition of humoral immunity - virally encoded receptor/protein to block Ab/complement functions
- Inhibition of inflammatory response - blocking chemokines and cytokines; EBV causes deficiency of adhesion molecules, e.g. ICAM-1, LFA-3; mimic TLRs to block pathways to NFkb
- Blocking Ag processing and presentation - inhibition of MHC I; inhibition of TAP for peptide transportation
- Immunosuppression - EBV mimics IL-10 to inhibit TH1 and reduce IFN-gamma
Bruton's X-linked agammaglobulinemia (XLA)
- - The first description of an immunodeficiency disease (male child)
- - Defect in tyrosine kinase called Btk (Bruton's tyrosine kinase) -> defect in Ab production
IgG levels in newborn infants fall to low levels at about 6 months of age
- High before birth, passively transferred maternal IgG.
- Drops shortly before birth and own IgG starts to rise at birth at a very slow rate -> lowest level reached at 6 mo pp
X-linked Hyper IgM syndrome
- - Commonest of hyper IgM syndromes
- - Defect CD40L -> no adequate costimulation, defective class switching
Common variable immunodeficiency (CVID)
- - Usually a deficiency in IgM, IgG, and IgA together
- - Decreased serum Ig and abnormal Ab responses
- - Develops recurrent infections
Severe congenital neutropenias
- - Persistently low neutrophil counts (<200 per ul; normally 3-5.5k/ul)
- - bone marrow transplant needed
Defects in complement components are associated with susceptibility to certain infections and the accumulation of immune complexes
- MAC (C5-9) Deficiency -> Neisseria infection
- C3 deficiency -> infection w/ pyogenic bacteria and Neisseria, sometimes immune-complex disease
- Classical pathway (C1,2,4) deficiency -> immune-complex disease
- MBL pathway (MBL, MASP1,2, C2,4) deficiency -> childhood infections
- Alternative pathway (Factors D, P) deficiency -> infection w/ pyogenic bacteria and Neisseria, NO immune-complex disease
Defects in phagocytic cells: persistence of bacterial infections
- TLR signalling defects (e.g., MyD88 or IRAK4) -> severe cold pyogenic bacterial infections
- G6PD deficiency -> defective respiratory burst, chronic infection
- Myeloperoxidase deficiency -> defective intracellular killing, chronic infection
Severe combined immunodeficiency (SCID)
- Commonest is XSCID
- Mutation in the gene for IL-2R common gamma chain (γc) shared by:
- - IL-2, 4, 7, 9, 15, and 21
- - T cells and NK cells fail to develop normally
- - B cell numbers normal, but non-functional
Grafting of bone marrow can be used to correct immunodeficiencies caused by defects in lymphocyte maturation, but two problems can arise
- GvHD, systemic immune disease
- Host-vs-Graft response -> graft failure
Acquired immune deficiency syndrome (AIDS)
- - The most extreme immune suppression caused by a pathogen - the human immunodeficiency virus (HIV)
- - opportunistic pathogens infection, aggressive form of Kaposi's sarcoma or B-cell lymphoma, profound decrease in CD4 T cells number
- - Two types of HIV: HIV-1 (chimpanzee, common in western countries, more aggressive) and HIV-2 (monkey)
- - Groups of HIV-1: M (main), O (outlier), and N (non-M, non-O)
- - M group is classified (according to genetic diversification) into subtypes, or clades, A to K
- - Virus is mainly carried by cells w/ CD4, which acts as the receptor for the virus, along with a co-receptor, usually the chemokine receptors CCR5 or CXCR4; Dendritic cells and macrophages are also infected; or as a free virus in blood, semen, vaginal fluid, or mother's milk.
Typical course of untreated infection with HIV
- Flu-like (majority) in the first 2-6wk after infection, CD4 T rises slightly then drops quickly w/ some rebound.
- Secoconversion happens, CD4+ PBL, aymptomatic for years, CD4 drops below 500/ul
- Symptomatic, CD4 keeps dropping to 200/ul
- AIDS starts and CD4 keeps dropping to zero, death.
Virion of HIV
- within nucleocapsid: dsRNA, reverse transcriptase, integrase (integrate viral DNA into host genome, stays forever)
- envelope proteins: gp120 and gp40 bind to CD4, use CCR5 or CXCR4 as coreceptor; MHC proteins capture something and put it on to hide from the immune system .
Dendritic cells initiate infection by transporting HIV from mucosal surfaces to lymphoid tissue
DC-SIGN used to bind the virus
- - Zidovudine (AZT)- nucleoside analogs that inhibits reverse transcriptase
- - Highly active antiretroviral therapy (HAART) - combination therapy with a cocktail of viral protease inhibitors and nucleoside analogs; has lead to great increases in survival
- - Vaccines are attractive but difficult
- Active immunization: Deliberate induction of adaptive immunity to a pathogen by injecting a dead or attenuated (nonpathogenic) form of the pathogen (vaccine).
- “Passive immunization”: Immunity is transferred by administration of an immune serum or Ig
- Are compounds that enhance the immune response when administered with antigen, thereby producing higher antibody titers and prolonged production.
- The distinction between primary and secondary immune responses become blurred when adjuvants are used.
Adjuvants that enhance immune responses
- Only two adjuvants - alum and MF59 - are licensed for use in vaccines given to humans
- - Incomplete Freund’s: oil in water emulsion, antigen diffuses slowly, constantly stimulating immune response
- - Complete Freund’s: also oil in water
- - Freund’s adjuvant with MDP: mimics the same result
- - Alum (aluminum hydroxide gel)
- - Alum plus killed B.p.
- - ISCOMs (immune stimulartory complexes) are made from a protein Quil A (comes from bamboo in South America) that contains viral proteins to deliver stimulation to T cells
Approaches to vaccine develpment
- Live attenuated or killed bacteria- Innate & Adaptive (e.g. BCG, Cholera)
- Live attenuated viruses – E.g. Polio, rabies
- Subunit (Ag) vaccines – inactivated toxin (“toxoid”). E.g. Tetanus toxoid, diphtheria toxoid. Polysaccharide vaccines have protein conjugate for
- induction of B and T cell responses
- Conjugate vaccines- E.g. Haemophilus influenzae, pneumococcus
- Synthetic vaccines- Recombinant DNA. E.g. Hepatitis (recombinant proteins)
- Viral vectors- Clinical trials of HIV antigens in canarypox vector
- DNA vaccines- Plasmid of cDNA, CpG DNA (TLR9 agonist)- Clinical trials ongoing for several infections
Vaccination with cowpox virus elicits neutralizing Abs that react with antigenic determinants shared with ________.
Attenuated viruses are selected by growing human viruses in non-human cells
- 1. Isolate pathogenic virus from patient
- 2. grown in human cell culture
- 3. cultured virus infect monkey cells
- 4. to grow well, acquire many mutations; no longer grows well in human cells (attenuated)
- 5. used as vaccine
Production of live attenuated viral strains by recombinant DNA techniques
- 1. isolate virus
- 2. isolate genes for receptor-binding proteins, virrulence, core proteins
- 3. mutate/delete virulence gene
- 4. resulting virus is viable, immunogenic, but avirulent, used eaas vaccine
Influenza virus vaccination complication
- Reye's syndrome - an acute pathological condition affecting the central nervous system.
- Principally associated with children who have taken aspirin
- no known direct cause-and-effect relationship between aspirin use and Reye's syndrome
Diseases w/o effective vaccine
- malaria, tuberculosis, ...
- Measles - IgM based, heatsensitive, difficult to use in tropical countries w/o refrigeration
2010 Milestone in Cancer Vaccine
- Provenge: Advanced hormone resistant prostrate cancer vaccine. April 30, 2010: FDA Approval to Dendreon
- DC from patient activated with PAP (Prostatic Acid Phosphatase), GM-CSF (granulocyte monocyte colony stimulating factor) fusion protein
- Vaccination of patient
- - 3 year survival increased by 38%
The three most important elements in relation to caries
- host and teeth
- substrate (diet)
- also important, time, saliva
Association of S. mutans with Caries - clinical data (mid 70’s)
- • Strains isolated proved cariogenic in animals
- • Relationship of S. mutans to Carious Lesions
- – a. Lesion First
- - S. mutans found in plaque over incipient lesions involving pits and fissures of smooth surfaces.
- - 71% had > than 10% S. mutans.
- - 70% no caries little or no S. mutans
Bacteria Produce Acid
- During acid attack, plaque fluid pH is lowered.
- pH drop changes the ecological environment close to tooth
- Aciduric - endures acid, favors overgrowth of S. mutans
- Acidogenic - S. mutans produces acid
Saliva Neutralizes Acid
- Stephan Curve: pH drops after rinsing with sucrose more in caries active vs. caries inactive areas.
- Critical pH is 5.5, causes plaque to demineralize the enamel
___________ results in cavitation.
Parotid duct is located next to the _______.
- upper first molar
- there will be IgA and other salivary components, but no serum factors including IgG
Sublingual duct pours saliva on the ____ surface.
Gingival crevice fluid
- Fluid exists into saliva from crevice containing:
- – Serum elements
- – PMNs
- Crevice contains both
- – Innate factors: Lysozyme, lactoferrin, lactoperoxidase
- – Acquired factors: IgG, IgA
- Bacterial adherence and production of acid from carbohydrates.
- Demineralize enamel
- Saliva can buffer acid and provide many sources of proteins that can effect the outcome
- Microbial habitat or niche is important
- Bacteria can accumulate on a variety of enamel surfaces:
- – Buccal
- – Lingual
- – Proximal
- – Occlusal, most important, w/ pits and fissures
- Surfaces are different
- Smooth surfaces require bacteria to attach via adhesins and glucans
- Proximal surfaces are exposed to both saliva and crevice fluid
- Occlusal caries - bacteria are impacted into deep crevices where Saliva can't reach
Highest concentration of IgG would be found _________, and the highest concentration of IgA is found __________. There is a huge difference in pits and fissures in adult and child teeth - Difficult to penetrate saliva into this.
- in proximal areas of teeth
- on smooth areas of teeth
Difficulty of bristle penetrating the fissure
Saliva can interfere with caries by many mechanisms:
- – Buffering with bicarbonate
- – Dilution by flow
- – Healing by calcium deposition
- – Lubricating soft tissues
- – Antibacterial mechanisms: both innate and acquired immune capabilities
LAP and Proximal Decay
- Children with LAP have minimal proximal decay.
- Children with LAP have saliva that kills S. mutans, not due to difference in pH
- An active antimicrobial factor found in LAP saliva is Lactoferrin (Lf)
- Lf in LAP patients has a SNP (Single nucleotide polymorphism) in amino acid position 29 (or 47) that codes for lysine (K); Control group had arginine (R) at 29.
- Lf with lysine killed SM (also S. mitis, S. sanguis), whereas arginine one didn’t
Move from crevice to saliva thru vasculature and junctional epithelium
- Primary (azurophilic):
- Secondary (specific): Toxic oxygen, Lactoferrin, Lysozyme
Lf Causes membrane to leak proteins and causes them to die?
First line of Defense
- Influence of Co-Inhabiting Bacteria
- Acid produced by S. mutans = Lactic acid
- Cohabitated by Veillonella, also early plaque former
S. mutans FIL Produces
lactic acid, w/ lactic acid dehydrogenase (LDH)
S. mutans C67 Produces
Veillonella alcalescens Ferments _______ to Produce __________
S. mutans FIL cohabitates w/ Veillonella alcalescens, S. mutans C67 does not. W/ cohabitation, ____ caries happened.
- acid was not available to cause demineralization of teeth in rats with Veillonella
impractical to eradicate oral flora from the mouth before inoculating the replacement
Vaccination -> Ag?
- Serotype carbohydrate
- Antigen I/II
- 1. Transmission
- 2. Secretory IgA system discovered
- 3. A microbe amenable to vaccination
- – Serotype
- – Attachment factors; GBP; GTF
- 4. Result of Immunization
- – Agglutination
- – Killing enhanced
- Ways to Vaccinate:
- – Swallowing
- – Intra gastric – GALT
- – Intra nasal – NALT
- – Intra ductal – salivary gland
- – IM and IV
- Salivary (IgA) from oral; or Serum (IgG) Response (from IV injection)
- Active or Passive Immunization:
- – Active: Stimulate Lymphocytic response
- – Passive: Apply already synthesized Ab to prevent colonization: In egg yolk; In milk; Apply to teeth
- Goal: Reduce number of microbes
- Inject S. mutans into monkeys --->serum IgG response ---> reduced caries
- Inject S. mutans into rat salivary glands --> Secretory IgA response ---> reduced caries
- Feed S. mutans to rats in capsule develop a mucosal immune response
- –> salivary IgA without detectable IgG ---> less caries.
- –> Produced secretory IgA in tears
- These experiments led to the concept that there was a common mucosal immune system, Monitoring response in serum, saliva and tears
- S.mutans challenge: Gut Associated Lymphoid
- Tissue (GALT)
- S.mutans challenge: Nasopharyngeal Associated Lymphoid Tissue (NALT)
- - Antigen delivery by oral (swallowing) or nasal routes results in the migration of antigen-specific IgA-producing B-cells to effector organs (salivary
- – Then get differentiation and maturation of these B cells and secretion of IgA in the lamina propria, where it crosses the ducts into the saliva
Took Salmonella typhii, knocked out toxins, cloned in S. mutans I/II antigens and gave to rats; produced mucosally immunized rats to S. mutans I/II via IgA
- Topical application of monoclonal antibody to I/II antigen has been effective.
- Studies of application of mouse monoclonal IgG specific antibodies for antigen I/II showed that recolonization of S. mutans was postponed for 2 years after a 9-day treatment with Chlorhexidine prior to admin of antibody.
- Julian Ma T. Lehner Nat Med 1998
- Production of salivary IgA in a tobacco plant
- Constructs encoding four proteins
- – Heavy chain mouse gamma1 and alpha domains, K light chain and J chain also in mouse, secretory component from rabbit.
- – Purified from tobacco plant by ammonium sulfate precipitation and protein G affinity column --> 10-80 mg/kg of plant.
- – Application to humans who had S. mutans on their teeth
- – 9 days of application of CX
- – Apply antibody or control to their teeth, two applications per week for three weeks.
- Recolonization of S. mutans took 58 days in controls
- No colonization by 118 days in either monoclonal IgG or in plant derived IgA topically applied
Conclusions Caries Research and Practice:
- 1. Early Evidence: (1900-1950);
- Animal experiments implicate an coccal microbe
- 2. Seminal Experiments (1960’s)
- a. Followed Koch’s postulates
- b. Strep was isolated and transferred from caries resistant to caries susceptible hamsters.
- 3. Clear assoc. between carious lesion and S. mutans (1970’s)
- 4. Genetic attributes of S. mutans identified (1980’s -present)
- 5. Importance of sucrose as a substrate shown (1980’s - present)
- 6. Diagnostic and Preventive Strategies Developed (1990’s)