Exam Bank Questions

Why is it important for DH to have good understanding of microorganisms?

Understand oral diseases → Microbes stick to surfaces & leads to gingivitis and perio → immune system reaction

Prevent infection → dental procedures can expose pts to microbes

Maintain infection control → knowledge of microbes help DH properly use sterilization, disinfection, PPE, hand hygiene

Protect pt → some pt have weakened immune system, infectious diseases

Compare & contrast prokaryotic & eukaryotic cells

Prokaryotic:

• No nucleus

• No organelles

• Cell wall: Peptidoglycan

• Reproduction: Binary fission

• 1 circular chromosome

Eukaryotic:

• Nucleus

• Organelles

• Cell wall: cellulose/chitin

• Reproduction: Mitosis

• Linear chromosome

What are the unique characteristics of bacteria?

Size

• Very small, can 2.4×10^11 bacteria /gm of plaque

• Large surface to volume ratio → rapid growth + transport

• Inhabit vv small ecosystems (dentinal tubules)

• Mechanical removal can be difficult

• Many diverse species @ single site

Metabolically Diverse

• Use full spectrum of nutrients (Carbs, protein, lactate, nylon tires)

  Live w or w/out oxygen (aerobic or anaerobic)

Adaptable

• Great opportunity for genetic change

• Short generation time (time it takes to reproduce) → can divide up to every 40 minutes

• Mutation rate 1/106

• Genetic info exchanged rapidly

  • If you give antibiotics, doctors tell you to take them all at once or within a time period because if you don’t, it gives them time to adapt and genetically change so they can become resistant

  • After becoming resistant, the bacteria can also pass on the gene

Ubiquitous

• Ubiquitous → present, appearing, found everywhere at the same time

  • Present in virtually all environments

    • Hot underwater fissures that are 300 degrees

    • Soil, air

    • Humans → Skin, nose, GI Tract, Genital tract, oral cavit

What does the term “metabolically diverse” mean when describing bacteria?

Explain how bacterial metabolic diversity contributes to their ability to survive in diff environments

Bacteria are able to use full spectrum of nutrients (Carbs, protein, lactate, nylon tires)

Can live w or w/out oxygen (aerobic or anaerobic)

What was the traditional method of classifying bacteria

1. Staining

• Gram stain (Gram + or -)

• Ziehl Neelsen → stain used to ID acid-fast bacteria like Mycobacteria tuberculosis

• Silver Impregnation staining → Treponema pallidum

2. Shape

• Microscopic

  • Spherical (cocci)

  • Rod (baccili)

• Macroscopic

  • Colony

3. Nutritional Characteristics

• What nutrients they consume

• Bacteriodes melanogenicus asaccharolyticus → does not ferment carbohydrates

• Bacteriodes melanogenicus intermedius → ferments carbohydrates

4. Fermentation end product

• Lactobaccilus species ferments glucose to lactic acid

5. Cell Wall Comoposition

• Ex. Streptococci subdivided based on cell-wall antigens (A-H) w/ the Lancefield Serotypes

6. DNA Base Composition

• Molar Fractions G+C/G+C+A+T

• Calculating the % of DNA ratios

7. Bacteriophage Bacterial virus susceptibility

• Classifying bacteria based on whether they are susceptible to certain viruses

8. Plasmid transfer

• Plasmids= small sections of DNA, can be transferred b/w similar grps of bacteria

• This is the reason for why bacteria can become resistant to antibiotics

• Ex. Enterobacteriaceae bcs bacteria in this family carry plasmids

What are the limitations of traditional bacterial classification and culture-based methods

No sensitivity & poor specificity

1. Does not ID huge uncultivated majority of microbes

• Location: Difficulties in sampling due to physical environment

• Organisms mb not capable of adapting in laboratory culture

  • Lack of essential nutrients, physiological needs/growth factors in culture media. overfeeding, toxicity in culture, inhibitory substances produced by other bacteria, metabolic dependance on other species, disruption of quorum-sensing systems (comm system b/w bacteria to track population density) induced by separation of bacteria, cell in low metabolic state (dormant)

• KEY idea is that when you take a sample somewhere, there is thousands of species of bacteria, but you don’t capture all of them, then when you grow them some of them are unable to grow in standard laboratory conditions

2. Even after successful cultivation, may not be successfully identified

• In the traditional method, identification mb heavily based on phenotypic (observable) characteristics, which you compare to reference slides but its so subjective, it might be done incorrectly → Misidentification

Why are some microbes unable to adapt to laboratory conditions?

• Organisms mb not capable of adapting in laboratory culture

  • Lack of essential nutrients, physiological needs/growth factors in culture media. overfeeding, toxicity in culture, inhibitory substances produced by other bacteria, metabolic dependance on other species, disruption of quorum-sensing systems (comm system b/w bacteria to track population density) induced by separation of bacteria, cell in low metabolic state (dormant)

• KEY idea is that when you take a sample somewhere, there is thousands of species of bacteria, but you don’t capture all of them, then when you grow them some of them are unable to grow in standard laboratory conditions

What is 16sRNA and why is it important in bacterial identification and classification?

16sRNA=part of machinary that bacteria uses to make proteins from amino acids

16sRNA when bound to ribosomal proteins make 30S subunit of ribosome

Gene that codes for 16sRNA found in all bacteria, part of sequence = conserved, so once the scientist find the conserved part, they can amplify it and look at the variable gene sections to classify the bacteria

• Stable + present in all organisms

• Sequence must be conserved sufficiently that regions of homology can be clearly established

• Must be sufficiently large that info is adequate to make phylogenetic inferences

What is the modern method used to determine bacterial classification & ID bacterial species?

Using gene and molecular biology with 16sRNA and PCR (Polymerase chain reaction) to amplify the DNA

You can use 16S rRNA to determine phylogeny of bacteria (relationship of organisms based on evolution)

• Can be used for accurate & rapid identification of known bacterial species (PCR)

• 16S rRNA to differentiate, quantify, classify unknown species (esp those that cannot be cultivated)

• Can be used for phylogenetic reclassification (classifying organism based on how evolutionary related they are) because similar 16sRNA= more similar

• Classifying microbes based on evolutionary relationship = more stable, less likely to need additional revision

• 16sRNA= documents evolutionary history >2000 sequences determined for bacteria but 16sRNA makes up only 0/05% of the genome

What is the mechanism of polymerase chain reaction (PCR)?

Requires:

• Template DNA → DNA w/ target sequence

• Primers → short DNA sequence that defines region to be amplified

• DNA polymerase → heat-stable enzyme like Taq polymerase

• Nucleotides (dNTPs) → building block for new DNA

• Buffers + ions like Mg3+

3 Steps

1. Denaturation ~95C

• Double stranded DNA separate into 2 single strands

2. Annealing ~50-65C

• Primer bind to complementary sequence on DNA strands

3. Extension ~72C

• DNA polymerase extends the primers and synthesizes new DNA strands

3 steps repeated 25-40 cycles, bcs DNA approx doubles each cycle, target sequence increases exponentially, generated millions of copies

Rapidly identifies viruses like HIV, Hepatitis…if you get poked by needle this is what is used

What are the two major pathways used by bacteria to generate ATP?

CONFIRM W/ NAJAR

Fermentation

• No electron transport chain

• ATP produced thru substrate-lvl phosphorylation

• Organic molecule= final electron acceptor

• Net gain= 2 ATP

• Used by subg plaque

Respiration

• Electron transport chain

• ATP produced thru oxidative phosphorylation

• External final electron acceptor

  • Aerobic= O2 is final electron acceptor

  • Anaerobic= Nitrate is final e- acceptor

• Net gain= 38 ATP

How much ATP can be generated thru cellular resp compared w/ fermentation?

Cellular resp can generate 38 ATP and fermentation only 2 ATP

Which is more energetically efficient for bacteria: cellular respiration or fermentation?

Cellular resp=more NRG efficient than fermentation bcs it uses

• Glycolysis

• Electron transport chain

• Oxidative phosphorylation

BUT fermentaiton can be advantageous with external electron acceptor=unavailable bcs bacteria can cont producing ATP w/out electron transport chain

Compare & contrast obligate aerobes, obligate anaerobes, facultative anaerobes, aerotolerant anaerobes

Obligate aerobes= Require O2

Obligate Anaerobes= die in O2

Facultative anaerobes= Respire in O2, ferment other wise (SWITCH)

Aerotolerant Anaerobes= Always ferment BUT have limited capacity to detoxify partial reduction products of O2

What are the sources of carbon and NRG for chemoheterotrophic bacteria?

Chemoheterotrophs include animals, fungi, and most other bacteria

Use oxidation-reduction reactions to produce ATP

Use organic molecules to supply itself with oxygen

What are Koch’s postulates

1. Microorganism must be demonstrable in all cases of disease

2. Microorganism must be isolated from diseased animal + grown in pure culture

3. Microorganism from pure culture must cause disease when inoculated into healthy animal

4. Experimentally infected animal must contain microorganism

What are the limitations or problems associated with Koch’s postulates?

Not all diseases are caused by microbes!!!

Auto-immune

Cancer

Asymptomatic

For Perio=

1. Inability to culture all organisms

2. Difficulties defining + culturing sites of active disease

3. Lack of animal model system

What is Socransky’s criteria?

Criteria for whether a microbe can be considered a periodontal pathogen

1. Must be associated w/ disease → increase in # of organisms at diseased site

2. Must be eliminated/decreased in sites that demonstrate clinical resolution of disease w/ treatment (mechanical or antibiotics)

3. Must demonstrate host response (alteration in host cellular or humoral immune response)

4. Must be capable of causing disease in experimental animal model

5. Must demonstrate virulence factors responsible to enable microbe to cause destruction of periodontal tissue

Name 6 bacterial species commonly found in root canals associated w/ teeth w/ necrotic pulp

Eurobacterium

Lactobacilli spp

Enterococcus

Streptococcus

Actinomyces spp

Peptostreptococcus

Name 5 bacterial species commonly associated with periodontal disease CONFIRM if genus or species

Eikenella

Clostridium

Porphyromonas

Selenomona

Actinobacillus

What is the causative organism of oral thrush (oral candidiasis)? Identify the organism and explain what the presence of oral thrush may indicate about the patient's underlying health or immune status.

Candida albicans which is a naturally occuring fungus in the skin, and oral cavity and is typically kept in check by immune system. It is oppurtunisitic so if immune system down it can turn into overgrowth, meaning pt has compromised immune system

What factors affect microbial growth in oral cavity?

1. Temperature

• Human mouth is 35-36C affects bacterial metabolism, enzyme activity, habitat

2. Redox potential (anaerobiosis)

• Gradients of O2 [ ] and Eh(redox potential) exist in plaque in oral cavity

  • Some areas in plaque high in O2, deeper= lower

• Affects type of microbes that can be found

3. pH

• Most microbes require pH ~7 (Regulated by saliva)

4. Nutrients

• Endogenous saliva → Can be both + and -, helps bring in WBCs but also helps microbes grow by regulating the environment for them

• Exogenous saliva → What we eat

5. Adherence

• Chewing + saliva will detach

• Soft tissue desquamation limits bacterial load

• Hard tissue=high biofilm

6. Antimicrobial agents & inhibitors

• Dentifrices

• Mouth rinses

• Antibiotics

7. Host defences

• Innate immunity

• Non-specific immunity

• Specific immunity

What properties of the oral cavity make it suitable habitat for microbial growth?

Oral cavity=suitable habitat for microbial growth bcs it is a continuous culture system, receives constant supply of nutrients from saliva, GCF, desquamated epithelial cells

Also variety of surfaces, like nonshedding tooth surfaces & specialized mucosal surfaces, provide diff environment for microbial attachment and colonization

Saliva provides nutrients + buffering capacity, GCF provides serum-like nutrients + host defense components

What role does saliva play in bacterial growth in oral cavity

Saliva provides nutrients + buffering capacity (buffers salivary pH) for bacteria

BUT saliva also contains antibodies IgG and IgA that can inhibit bacterial growth

What difficulties are encountered when studying oral microbiota?

High species diversity

Surface attachment/Coaggregation → since oral bacteria attach to surfaces, collecting one sample does not guarantee that you sample all

Obligate anaerobes → some oral microbiota do not grow well in lab bcs they die in O2 but will get exposed to O2 if put into lab

Fastidious nutrition → some oral microbiota have specific nutritional requirements

Generation time → some microbes take a vv long time to divide

Identification problem

Difficultues w/ sampling environments

Describe the process of bacterial distribution and colonization in the oral cavity.

6 Stages

1. Transmission → saliva from mother, food, milk, water

2. Acquisition → microbes enter mouth and colonize surfaces (epithelial & hard surfaces)

3. Pioneer species → first organisms to colonize usually (Streptococci, staphylococci, neisseria, veilonella)

• Grow until they meet environmental resistance

  • Physical forces (epithelial shedding, shear forces)

  • Chemical factors (saliva, Eh <redox>, pH, nutritional requirements

4. Succession → Metabolic activity of pioneer community modifies environment, makes it suitable for new bacterial populations to colonize

• Allogenic factors:

  • Physical environement

    • Eruption of teeth

    • Acrylic obturators for cleft palate (used in pts to mimic palate, provides hard surface for bacteria)

• Autogenic factors

  • Toxic end products

  • Cryptotypes (receotors)

    • Receptors/binding sites where pioneer species bacteria attaches and modifies it so other bacteria can stick on it too

  • Metabolism/Eh

    • Ex. pioneer bacteria consume o2, Eh decreases, environment becomes more anaerobic and anaerobic bacteria can grow there

5. Increasing species diveristy

6. Climax community

• Not static (highly dynamic)

• Stable due to complexity

What are pioneer species?

First organisms to colonize oral cavity from transmission, typically with saliva from mother, food, milk, water, and grow until they meet environmental resistance factors

Usually streptococci, staphylococci, neisseria, veilonella

What factors cause pioneer species to stop growing or become replaced by other

microorganisms?

Pioneer species=first microbes to grow in oral cavity, initially grow bcs environment is suitable but then as they grow, they encounter environmental resistance factors

Two main categories

1.Physical forces

• Epithelial shedding → oral mucosa shed and take other microbes with it

• Shear forces → chewing, swallowing, can remove microbes

2.Chemical forces

• Saliva → wash away microbes + have antimicrobial components

• Eh (redox potential) → environment changes as oxygen in consumed, less suitable for microbes that need oxygen

• pH → certain microbes grow in specific pH

• Nutritional requirement → Nutrients can become limited or diff nutrients become available

Name 3 examples of pioneer species found in oral cavity

Streptococci, staphylococci, neisseria, veilonella

What factors are responsible for maintaining the stability of a climax community?

Explain the factors that contribute to the development and maintenance of a stable

microbial community.

1. Competition for receptors/ligands for adhesion

• Bacteria need to attach to surfaces/other bacteria

• Established bacteria=limits ability of other bacteria to colonize

2. Competition for essential endogenous nutrients/cofactors

• Bacteria need nutrients + other substance to grow, established bacteria can use up resources, limit whats available

3. Production of inhibitory substances

• Some bacteria produce substance that inhibit/kill other bacteria

4. Creation of micro-environment that discourage growth of endogenous specieis

• Established species can change local environment and make it difficult for other bacteria to grow like pH, O2 [ ]

What factors can cause/interrupt/disrupt climax community?

EXCEPTIONS

• Pregnancy → hormonal changes alter environment

• Tooth eruption in children → creates new surfaces

• Disease (cancer/immunosuppression) → change in host defense

• Denture wearing → artificial surface that changes oral environment

What is the difference between allogenic and autogenic factors?

During the succession stage where the environment changes and the pioneer species evolve there are allogenic and autogenic factors that contribute to the change.

Allogenic factors= environmental changes caused by external physical factors like eruption of teeth, and use of acrylic obturators

Autogenic factors= envionmental factors caused by microbes itself

• production of toxic end products

• Cryptotypes (receotors)

  • Receptors/binding sites where pioneer species bacteria attaches and modifies it so other bacteria can stick on it too

• Metabolism/Eh

  • Ex. pioneer bacteria consume o2, Eh decreases, environment becomes more anaerobic and anaerobic bacteria can grow there

What are the major factors involved in the development of dental caries?

Host → have to be susceptible, saliva, tooth anatomy, OH, other host factors

Time → the other factors need to interact in sufficient duration

Microflora → need to have the bacteria that causes caries

Diet → fermentable carbohydrates (which lowers pH of mouth)

Name five cariogenic bacteria

S. mutans

Lactobacilli

S. sobrinus

S. sanguis

S. Mitis

Actinomyces naeslundii

A. casei

Describe the development of dental plaque in the oral cavity.

Explain the process from initial bacterial attachment through the development of a mature plaque biofilm.

a) What factors facilitate attachment of bacteria to acquired pellicle?

b) What are major stages/layers involved in plaque development

a) Factors= Enamel pellicle which is a thin protein rich film formed from saliva & GCF, contains receptors & binding sites for bacteria to attach

• Provides host ligands for attachment=, Acidic proline rich (S. mutans), Amylase, Lysozyme, Immunoglobulins, Albumin

AND

electrostatic interactions (Divalent cations, hydrogen bonding, ionic interaction, hydrophobic bonding)

---

b) First stage= development of supragingival plaque

• After tooth is clean, acquired dental pellicle is formed, saliva deposits host proteins & glycoproteins on enamel, creating acquired enamel pellicle. Pionner species bacteria bind to host ligands on dental pellicle (adherence)

  Once bacteria attach → begin dividing and aggregate

  THEN allow diff bacterial species to attach and COaggregate

Second stage= development of subgingival plaque

• Forms when supragingival plaque migrates into sulcus then inlammation creates subgingival environment

• Mostly motile bacteria that carry non-motile bacteria in

What is dental plaque?

general term for complex microbial community found on tooth surface

Embedded in matrix of polymers of bacterial & salivary origin

Plaque being calcified is called calculus/tartar

Bacterial members cooperate + signal to each other

• Fluid channels carry nutrients + remove waste

What is the difference between aggregation and coaggregation

Aggregation= same type of bacteria clumping together

Co-aggregation= clumping w/ others, diff types of bacteria clumping w each other

• One bacteria species attaches to another species within dental plaque

T/F: A 4-mm periodontal pocket contains the same amount of bacteria as a 7-mm

periodontal pocket

FALSE

7mm periodontal pocket generally more bacteria than 4mm because it provides larger, more protected environment for plaque accumulation

• Less oxygen= good for subg bacteria

Deeper pocket depth affects amnt + composition of bacteria

T/F: Supragingival plaque can attach more readily than subgingival plaque.

TRUE

Supragingival plaque can adhere to the enamel pellicle which provides ligand and electrostatic bonds directly o the exposed enamel.

Subgingivally, there is less space and oxygen. Access to nutrients and space is more restricted.

T/F: Saliva can have both beneficial and detrimental effects on bacteria in the oral cavity

TRUE:

• Saliva provides nutritents

Saliva provides nutrients + buffering capacity (buffers salivary pH) for bacteria

BUT saliva also contains antibodies IgG and IgA that can inhibit bacterial growth

T/F: The generation time of Mycobacterium tuberculosis is approximately 40 minutes

FALSE

Mycobacterium tuberculosis= vv slow gen time ~15-20 hrs

Long generation time makes some microbes difficult to study

What is the average temperature of the oral cavity that supports microbial growth?

Approximately 36–37°C.

Temp is important bcs it affects enzyme activity + metabolic processes

If temp too low= grow too slow

If temp too high= dmg or denature cellular proteins

What are the four electrostatic/physicochemical interactions involved in bacterial adherence and colonization of dental plaque

Divalent cation interactions

Hydrogen bonding

Hydrophobic bonding

Ionic interactions

Explain how changes in the oral environment can influence the microbial community and contribute to dental disease.

In your answer, integrate concepts such as pioneer species, microbial succession, climax communities, plaque development, saliva, diet, and changes in the host environment

Oral cavity= dynamic environment where changes in host environment, saliva, diet, & oral surfaces can alter microbial community.

Pioneer species are are one of the first microbes to colonize the oral cavity.. As they grow, their metabolic activitiy modifies the environment & allows other microbes to colonize. This process is called microbial succession where microbial community changes overtime and develops into a stable climax community.

As microbes attach to tooth surfaces + interact w each other → plaque biofilm develops. Changes in oral environment alter which microbes=favoured.

For example, frequent carbohydrate intake provides nutrients for acid producing bacteria which can lead to caries.

Changes in host environment like salivary flow/composition can affect nutrient availability, buffering capacity, and antimicrobial defenses. Changes in gingival margins and periodontal pockets can create environment that is favourable for species, especially anaerobic species in subgingival pocket areas. Microbial shifts can lead to periodontal disease.

Impaired immune system can also lead to opportunistic infections and allow microbes that usually do not grow there to begin growing

Poor oral hygiene can lead to accumulation of bacteria too because less disruption

What are viruses

1. Acellular (lacking cells/cell parts)

2. Obligate intracellular parasites → viruses can only live & reproduce in host cell, not alive

3. No ATP generating system

4. No ribosomes or means of protein synthesis

Describe the basic structural components of a virus

Nucleic acid

• DNA or RNA (never both)

Capsid (coat protein)

Others: (only some)

• Envelope

• Enzymes, RT

  • Reverse transcriptase (enzyme that uses RNA to synthesise complementary DNA strand)

Fill in the blank: Viral specificity is determined by receptor-ligand interactions. ______ are found on the viral capsid or envelope, and _____ are found on the host-cell membrane

Ligands

Receptors

Where are viral binding sites and host-cell receptor sites typically located?

Viral binding sites usually located on surface of virus either capsid or envelope of virus

• Uses specific receptors called “spikes”/”peplomeres”

Binding sites on virus must match the receptor site on host

Viruses can only infect narrow range of cells (specific range)

What is a viral capsid?

Made up of capsomeres

Protect viral genome from host endonucleases (enzymes that cut DNA/RNA)

Provide binding sites

What are the 3 major criteria used to classify viruses?

1.Nucleic acid

• Viruses can be single stranded or double stranded, and made of DNA or RNA

• ssDNA, dsDNA, ssRNA, dsRNA

2.Morphology

• Viruses can be 4 shapes: Helical, Polyhedral, Enveloped, Complex

3.Strategy for replication

What are the different methods used to grow animal viruses?

1.Injecting the virus into live animals

• Mice, rabbits, guinea pigs

2.Chicken embryos

• Most common method, used to make the flu still

3.Cell cultures

• Using technology to grow and maintain living cells

• Three types → primary cell lines, secondary cell lines, continuous cell lines

Primary cell line

• Freshly taken from tissue but die out after a few generation

Diploid cell line

• Cell has normal 2 sets of chromosomes

• Usually human embryos (high activity, replicate super fast)

• Maintained up to 100 generations

What is a continuous cell line

• Transformed cancerous cells

• Maintained indefinitely

• Ex. HeLa cells → piece of tissue from Henrietta ax is 1951 who had cervical cancer (still maintained today)

List and explain the major stages of viral replication

Lytic cycle:

1.Attachment

• Binding site on virus must matches receptor sites on host cell (ligand interactions)

2.Penetration

• Capsid is removed and viral DNA injected into bacterial cell

3.Biosynthesis

• Genome replication, transcription, translation

4.Assembly (maturation)

• viral particles assembled

5.Release

• Lysis → cell plasma membrane bursts

Lysogenic cycle:

1.Attachment

2.Penetration

3.Integration

• Viral genome is integrated into host cell genome

• So when host cell replicates itself, it is passively replicating the virus BUT the virus is latent and just hides there

• Virus is in a stage of prophage (stage bfr being a complete virus)

• *NO BUDDING/NO ASSEMBLY OF NEW VIRUS

4.Biosynthesis

• Genome replication

• Transcription

• Translation

5.Assebly

6.Release

• Either through…

  • Lysis

  • Budding

    • Virus can stay latent in the body cells and just budd and create a lot of viruses but then it can become activated by environmental factors and start lysising host cells

What is the difference between the lytic and lysogenic life cycles of viruses?

An extra integration stage in cell replication where Viral DNA/RNA mergers into host cell genome and can remain dormant, copies itself passively along host DNA, and can start replicating and releasing viruses when environmental factors are favourable. Lytic life cycle typically follow through to lysis right away while in the lysogenic stage, once the virus becomes activated, it can also bud or lysis to release the viruses.

What are retroviruses and what is unique about them?

Retroviruses are RNA viruses that make DNA copies of their RNA genome in host cells. Use enzyme called reverse transcriptase to do that and it allows them to hide within our cell’s dd DNA. Is what allows the virus to enter to lysogenic stage

RNA → DNA → mRNA → Protein

Example of retrovirus: Human immunodeficiency virus (HIV)

Explain the process of HIV infection

1.Attachment

• HIV gp120 binding site match CD4 receptor sites

• CD4 receptor sites found on macrophages, some cells of CNS, T4 Helper cells

2.Penetration

• Viral membrane + host cell membrane merge (fusion)

3.Uncoating

• Capsid removed, viral genome exposed

4.Integration

• Once viral genome integrated = 2 possibilities

• Using the reverse transcriptase to convert RNA into DNA to integrate within our cells

1. Nothing → virus is “latent”, can be latent for days, weeks, months, years

Median latency time= 10 years

---

ONCED TURN ON=

5. Biosynthesis

• Genome replication

• Transcription

• Translation

6. Assembly

• Virus particles put together

7. Release

• Budding or lysis

• Progressive loss/dysfunction of the WBC

What factors can trigger activation of the integrated HIV genome?

Other infections

Stress/shock to immune system

Drug abuse

Alcohol abuse

Malnutrition

Exercise → lack of/too much

Sunburn

• Herpes simplex 1

What are the different routes of HIV transmission?

1.High risk sexual contact

• Unprotected vaginal, oral, anal sex

2.Needle

• Intravenous drug abuse (sharing dirty needles)

• accidental needle sticks

3.Blood to blood contact

• Open sores/wounds

• Transfusions

• Organ transplant

• Artificial insemination

4.Mother to child

• Placenta

• As baby passed thru birth canal

  • Nowadays if mother has HIV, usually c-section done to protect baby

• Breast milk

Once the HIV genome has been integrated into the host-cell genome, what are the two possible outcomes?

1.Nothing

• Virus remains latent, can be latent for days, weeks, months, years

• Median latency time= 10 yrs

2.Virus is turned on

• If environmental factors favourable, the virus can be turned on and enter biosynthesis, assembly and release

What components of saliva can inhibit HIV?

HIV RNA and Proviral DNA have been detected by PCR in saliva BUT there is poor transmission of it through saliva bcs of salivary HIV-1 inhibition

HIV is neutralized

• Anti-HIV Antibodies

• Broad spectrum innate activities

  • Lysozyme

  • Lactoferrin

  • Defensins

  • Lactoperoxidases

• Viral entrapment + aggregation by mucins (heavy sugar-coated proteins that form the structural component of mucus membranes), inhibits HIV entry into target cell

Really the only way for an HIV infected individual to transmit the virus through saliva is if they had a ulcer on/in the mouth that is bleeding and than that has to come into contact with the healthy person’s ulcer

• The virus HAS TO enter the bloodstream

If a person is suspected of being infected w HIV, what is the appropriate testing process?

ELISA is usually If initial ELISA is (+), same sample tested again

If ELISA is (+) AGAIN, then Western Blot test or PCR done

• Western blot= Lab method to detect + ID specific proteins, takes the blood proteins and separates them, trying to detect HIV antibody proteins

• PCR= amplifies and looks for HIV gene

**In the early stages like if a clinician is poked by a needle, PCR is typically more accurate and is better and catching it sooner than it looks for the HIV gene

T/F: During the first several weeks following HIV infection, tests may produce a false-

negative result even though the person is infected and capable of transmitting the virus.

TRUE because there is a window period between getting infected and when individuals begin producing antibodies. Tests may be negative because the level of HIV antigens, proteins, antibodies are still undetectable. The seroconversion rate is typically 6-8 weeks for antibodies to appear. During that time, the person is considered infected and capable of transmitting HIV

Why has development of an effective HIV vaccine been particularly difficult?

No, HIV mutates too rapidly

Everytime reverse transcriptase is used, it causes at least 1 mutation SO

• 1 million variants during asymptomatic disease

• 100 million variants during advanced disease AIDs

So there is no way a single vaccine will be able to target all the genes of HIV

Describe clinical stages of HIV infection

1.Acute infection

• Initial infection of HIV from exposure to bodily fluids

• Viremia (virus present in blood stream)

  • Fever

  • Headaches

  • Weakness

  • Muscle & joint aches

• Last for ~weeks

• Normal CD4= 1200mm^³

2.Asymtomatic disease

• Virus= “latent” in CD4 cells

• Median latency period-10 years

• No signs/symptoms (asymptomatic)

• HIV positive → antibodies can be detected in blood

• Seroconversion= 6-8 weeks (time to make antibodies)

3.Symptomatic disease <can occur after 10 years>

• CD4 count <600m³

• Viral genome turned on, symptoms begin to appear

  • Chronic fatigue

  • Low grade fever

  • Night sweats

  • Diarrhea

  • Weight loss

• Susceptible to infection <conditions that we usually don’t see in people, only appearing bcs the immune system is supressed>

  • Bacterial pneumonia

  • Meningitis

  • Oral + vaginal yeast infections

  • Tuberculosis

4. Advanced disease (AIDs)

• CD4 count <200m³

• Severe opportunistic infection

  • Pneumocysitis carinii pneumonia (PCP) Fungi

  • Kaposi’s Sarcoma (Cancer of skin + blood vessels)

  • Toxoplasmosis (brain) protozoan

  • Cryptosporidiosis (GI tract) protozoan

  • Other bacterial, fungal, viral infections

What are different types of viral infection?

Two main categories

Acute

• Short-term, rapid onset and relatively rapid resolution

• Follow lytic sequence of events

• Cell exhibit cyotpathic effect (kills cells)

• Immune system activated to neutralize infection + eliminates virus

• EXAMPLE: INFLUENZA

Persistent

• Latent

• No infectious virus present

  Viral genome present

  • Can stay latent in nerve ganglion

  Virus can be reactivated/gene can be expressed if environmental factors are favourable

  • Ex. Stress, pregnancy

  • EXAMPLE: Retroviruses, HSV (Herpes), VZV

  
  

• Chronic

  • Infectious virus present

    • Virus is in circulation, usually not causing too much harm

    Usually no tissue pathology

    Common for RNA viruses

    EXAMPLE. HIV

• Slow

  • Infectious virus present only after LONG incubation period

  • EXAMPLE: Measles-Subacute Scholerosing, Pan-encephalitis

Does an acute viral infection generally follow a lytic sequence of lysogenic cycle?

Acute viral infection usually follows a lytic sequence because they are short-term, rapid onset and rapid resolution. Similarly, the lytic cycle allows viruses to rapidly replicate, release viruses, and lyse cells. In an acute infection, cells typically exhibit cytopathic effect and there is no incubation phase, which is present in the viruses that follow the lysogenic cycle.

What are prions?

Proteinaceous infectious particle

• Misfolded protein

Contains no envelope, capsid coating, or nucleic acids

Usually there are healthy proteins that the brain secretes and releases, but then if a misfolded protein called a prion appears (sporadic, genetic, acquired), it causes other proteins in the brain to become misfolded which creates plaques in the brain and damages neurons

Name 2 diseases caused by prions?

• Scrapie (degenerative disorder in sheep)

• Creutzfeldt-Jacob disease (CJD)

• Kuru (Occurs in tribes in New Guinea)

• Bovine Spongiform Encephalopathy (BSE_

  • Mad cow disease

How does the immune system function to eradicate an infection from the body?

The immune system has two pathways it uses to eradicate an infection from the body. The innate immune system consists of many cells that provides a rapid, non-specific response that targets the microbe. These cells are typically divided into four categories

• Phagocytosis: process where a microbe is engulfed by cells like dendritic cells, macrophage, monocytes, polymorphonuclear granulocytes

• Complement: Cascade of protein that increase inflammation, opsonize microbes, and forms the MAC (membrane attack complex)

• Natural killer cells: kill modified self cells like virus infected or cancerous cells

• Soluble proteins

  • Acute phase proteins → found in blood circulation and increase during infection (Prekallikrein → vasodilation), CRP

  • Lysozyme → enzymes in tears

  • Cytokines →

    • IFN (release by lymphocytes to signal infection)

    • IL → modulate immune response

    • Haemopoietic growth factors → regulate haempoiesis, stimulates bone marrow to produce immune cells

The innate immune system typically works minutes to hours following infection. If the pathogen is not irradiated, than the adaptive immune system is activated which is stronger, specific, and has memory.

The first step in the adaptive IR is antigen presentation, this is when antigen presenting cells (APCs) like dendritic, monocytes, and macrophages take up the antigen, process it, and display it onto their MHC Class II molecules. A CD4+/T-helper cell than binds to it through the Th receptor and reads the antigen. Depending on the cytokines and pathogen, it releases cytokines to go down one of two routes.

If the antigen is intracellular, the T-helper cell sends cytokines causing naive T cells to differentiate into Th1 and releases cytokines IFN-gamma and IL12. This activates CD8+/Cytotoxic T-lymphocytes. The infected cells also process the antigen and load it onto MHC Class I molecules, which is read by CD8+/CTL with the help of T-helper cells. The CTL than uses peforins and granzymes to lysis the cell, or uses a death-litigation receptor which programs cell death.

If the antigen is extracellular or soluble, the T-helper cell sends cytokines to activate naive T cells into Th2. The Th2 cells than secrete cytokines like IL4,6,13 which acitvates B cells. Once the antigen binds to the population of B cells with that specific antigen receptor, the B cells also process the antigen by taking it in and loading it onto MHC Class II molecule. This is read by Th2 cells which activate B-cells to proliferate and differentiate into plasma cells which produce antibodies, and memory B cells. Antibodies help eliminate pathogens by neutralizing it, opsonizing it for phagocytosis, and activating the complement system.

How does immune system respond differently to intracellular and extracellular infections?

If the antigen is intracellular, the T-helper cell sends cytokines causing naive T cells to differentiate into Th1 and releases cytokines IFN-gamma and IL12. This activates CD8+/Cytotoxic T-lymphocytes. The infected cells also process the antigen and load it onto MHC Class I molecules, which is read by CD8+/CTL with the help of T-helper cells. The CTL than uses peforins and granzymes to lysis the cell, or uses a death-litigation receptor which programs cell death. This is called the cell-mediated immune response.

If the antigen is extracellular or soluble, the T-helper cell sends cytokines to activate naive T cells into Th2. The Th2 cells than secrete cytokines like IL4,6,13 which acitvates B cells. Once the antigen binds to the population of B cells with that specific antigen receptor, the B cells also process the antigen by taking it in and loading it onto MHC Class II molecule. This is read by Th2 cells which activate B-cells to proliferate and differentiate into plasma cells which produce antibodies, and memory B cells. Antibodies help eliminate pathogens by neutralizing it, opsonizing it for phagocytosis, and activating the complement system. This is called the humoral immune response.

What are the two major antigen-presentation pathways.

MHC Class I, which occurs at the site of infection, it involves infected cells loading the antigen onto MHC Class I molecules and presenting it to Cytotoxic T-lymphocytes

MHC Class II, which occurs in peripheral tissues and lymph nodes where APCs like dendritic, monocytes, macrophages capture antigens and load it onto MHC Class II molecules and present it to T-helper cells

Where does antigen presentation occur?

The first antigen-presentation pathway takes place when the adaptive immune system gets activated. APCs like dendritic, macrophage, monocytes take up the antigen, load it onto their MHC Class II molecules, which is read by T-helper cells which helps sends cytokines to activate either the cell-mediated immune response or humoral immune response.

The second antigen-presentation pathway takes place in either the cell-mediated or humoral immune response. In the cell-mediated immune response, infected cells that have intracellular pathogens in them, process the antigens and load it onto MHC Class I molecules, which is read by Cytotoxic T-lymphocytes that eliminate them. In the humoral immune response, in order for B cells to make antibodies, they have to also process the antigen. B cells will take up the antigen and load it onto MHC Class II molecules which are read by Th2 helper cells to activate them to proliferate into plasma cells to secrete antibodies.

Describe innate and adaptive immunity

The major difference between innate and adaptive immunity is that innate immunity is rapid and non-specific. It does not require antigen-presentation and can target a wide variety of pathogens and can respond as quickly as a few days to hours. However, it is weaker and if it cannot get rid of the pathogen, it will switch to the adaptive immune response which is stronger, specific (antigen-presentation to target specific antigen), and has memory so subsequent exposure can warrant a faster and stronger response.

What components make up the innate immune system?

Typically categorized into four components

Phagocytosis where microbes are engulfed by phagocytes like monocytes, macrophages, PMNs and dendritic cells

Complement system is a cascade of soluble proteins that activate to increase inflammation and bring more lymphocytes over, opsonize pathogens for phagocytosis, and activates a membrane attack complex that lysis pathogens

Natural killer cells do not have antigen receptors, attack modified self cells, and have cytotoxic granules.

Soluble proteins

• Acute phase proteins which are found in blood and increase during inflammation like prekallikrein which vasodilates, and CRP

• Lysozyme → tears

• Cytokines

  • IFN produced by infected cells to signal immune system

  • IL → modulate immune system

  • Haemopoietic growth factor → regulate haemopoesis in bone marrow, can stimulate formation of WBCs

Name 6 types of cell that make up the innate immune system

1.Mast cell

• Found in submucosa/dermis, protect epithelial surface from pathogens

• Releases substances like

• Histamine, SRS-A , Leukotriene → inflammation

• Proteases → Break down proteins

• Cytotoxic peptides

• Heparin → anti-coagulant

2.Macrophage

• Monocytes move to tissues can differentiate into macrophages called tissue-bound macrophages, or macrophages already there are resident macrophages

• Phagocytose and antigen-presentation

• Brian → microglial

• Lung → alveolar macrophages

• Skin and lymph node → dendritic cells

• Liver → kuppfer cells

3.Monocyte

• Found in blood circulation

• Two activation pathways

• 1.Classifically activated → monocytes will migrate to tissue if under presence of TFN-gamma or LPS to becoem macrophages → pro-infammatory, antigen-presenting and phagocytoses, recruiting other WBCs. Can also use respiratory burst where it generates reactive oxygen species

• 2.Alternatively activated → anti-inflammatory, tells immune system to calm down, support tissue regeneration and repair

4.Basophil

• Inc in blood during parasitic or allergic rxn

• Release enzymes, raise inflammation, chemotaxis of other WBCs

5.Eosinophil

• Inc in blood during parasitic or allergic rxn

• Release enzymes, raise inflammation, chemotaxis of other WBCs

6.Dendritic

• Tissue bound in T-cell region of lymphatic tissue, blood, and other tissues

• Phagocytosis & antigen-presenting

7.Polymorphonuclear granulocyte

• Eliminate pathogens w/ 3 methods

• 1.Phagocytosis

• 2.Lysis of bacteria outside of cell: releasing reactive oxygen species

• 3.Nuclear extracellular traps (NETs) → PMNs sacrifice themselves, releasing nuclei content w/ bactericidal enzymes that form lethal network outside cell

8.Natural killer cell

• No antigen receptors

• Target modified self cells like virus cells and tumour cells

• Have cytotoxic granules

T/F → With innate immunity, repeated exposure to same pathogen leads to faster and stronger immune response

False, the innate immune system does not target specific antigens, its cells are designed to take on all of them. The cells do not have an antigen-presentation system and no memory system. Therefore, even once the pathogen has been eliminated, the innate immune system has no memory cells that will remember the pathogen and create a stronger and faster response. This is something the adaptive immune system has.

What are the major advantages of adaptive immunity compared with innate immunity

Specific → through antigen-presentation process, the immune cells are directed towards eliminating the specific antigen

Stronger → Activates stronger WBCs like B and T lymphocytes which produce antibodies or activates cytotoxic T lymphocytes

Memory → Memory B and T cells are also produced which helps the immune system “remember” the antigen and what response it used to eliminate it. Subsequent exposures will lead to stronger and quicker response.

What mechanical and chemical barriers contribute to innate immunity

Skin provides physical barrier against pathogens

Mucus membranes like nasal passages and in airway/bronchioles can catch pathogens

Lysozymes in tears, saliva

Acidic pH in vagina, urine, stomach, seminal fluid

What mechanisms do polymorphonuclear leukocytes (PMNs) use to destroy/immobilize bacteria?

1.Phagocytosis

2.Lysis of bacteria outside cell → PMNs releasing reactive oxygen species

3.Nuclear extracellular traps (NETs) → PMNs sacrificing themselves to release nuclei content with bactercidal enzymes creating lethal network

What are the major characteristics and functions of natural killer (NK cells)

Characteristics:

• Come from lymphoid lineage and circulate in blood

• Part of the innate immune system

• No antigen receptors

• Contain cytotoxic granulues

Function:

• Eliminate modified self cells like virus-infected cells or cancerous cells

• Modulate immune system

• Produce cytokines

What are the roles of MHC Class I and MHC Class II activation?

MHC Class I interacts with CD8+ or cytotoxic T-lymphocytes. Infected cells load the antigen onto the MHC Class 1 molecule which is read by the T receptors on cytotoxic T-lymphocytes and results in the elimination of the cell

MHC Class II interacts with CD4+ or T-helper cells. APCs like dendritic, macrophage, monocytes process the antigen and load it onto their MHC Class II molecules and is read by the T receptors on Thelper cells which aids in determining whether it is the cell-mediated, or humoral immune system activation

What receptors are involved in phagocytosis?

1.FC receptors: FC receptors on phagocytic cells bind to the FC portion on antibodies

2.C receptor: C receptors on phagocytic cells bind to C3a protein molecules which coat pathogens when the complement system is activated

3.TLR: “Toll-like receptors” bind to components found on pathogen cell surfaces

• TLR1/2 → lipoproteins (bacteria)

• TL3 → Double-stranded dsRNA

• TLR5 → flagellin

• TLR9 → unmethylated CpGDNA

Describe the process of phagocytosis

1. Chemotaxis + adherance of microbe to phagocyte

2. Ingestion of microbe by phagocyte

3. Formation of phagosome

4. Fusion of phagosome w lysosome to form phagolysosome

5. Digestion of ingested microbe by enzymes

6. Formation of residual body w/ indigestible material

7. Discharged waste material

What are neutrophil extracellular traps (NETs)

PMNs sacrifice themselves, releasing nuclei and mixing it with bactericidal enzymes to create lethal network outside of the cell to eliminate microbes

Name 4 types of phagocytic cells

Dendritic cells

Monocyte

Macrophage

PMNs

What are diff types of lymphoid tissues?

Primary lymphoid tissue where lymphocytes mature

1.Thymus → where T lymphocytes mature through positive and negative selection

2.Bone marrow/GALT → Where B lymphocytes mature

Secondary lymphoid tissue → where matured ymphocytes circulate and look out for antigens, move through circulation b/w various organs

• Lymph node

• Spleen

• Skin

Name 5 types of tissue macrophages and ID tissue/organ it is found

1.Brain=microglial

2.Liver=Kupffer cells

3.Blood=circulating monocytes

4.Bone=osteoclasts

5.Skin=Dendritic (Langerhan cells)

EXTRA= Lung=alveolar macrophages

What are the major functions of dendritic cells

Tissue bound, found where in T-cell lymphoid tissues, and other organs

Play a role in circulating to detect for antigens and antigen-presentation

Also phagocytosis

Langerhan cells

Differentiate b/w classically activated and alternatively activated monocytes/macrophages

Classically activated=

• When in presence of LPS or TFN-alpha, circulating monocytes will enter tissue where infection is present and differentiate into macrophages. They increase inflammation and tissue damage through non-specific phagocytosis, and releasing cytokines to recruit other WBCs.

Alternatively activated=

• Circulating monocytes can also enter area where infection has resolved and release cytokines to promote tissue regeneration and repair, and inflammatory to slow the immune system

What are the different functional roles or modes of blood monocytes?

Circulating monocytes can

1.Phagocytose microbes and debris

2.APC → search for microbe antigens

3.Releasing cytokines to promote inflammation or recruit other immune cells, can also be anti-finflammatory

What are the major roles of anti-inflammatory monocytes

Monocytes can be recruited to areas where infection has resolved, releasing cytokines that slows the immune response (anti-inflammatory), promotes tissue regeneration and repair

What are the major functions of the complement system in innate immunity

1.Increase inflammation → C3a and C5a molecules increase vascular permeability and recruit lymphocytes and other WBCs to area

2.Opsonization → C3b molecules coat pathogens, marking them for phagocytes which can bind to them through C receptors

3.MAC (Molecular attack complex) consisting of C5b6789 which cytolysis microbes by forming pores in the membrane allowing plasma membrane to leak out and water/salt to flow in and compromise the cell integrity

Explain the classical pathway of complement activation

Can be activated via 3 pathways (classical, lectin, alternative)

Classical pathway

• Begins with C1 protein complex which has 3 subunits-C1q, C1r, C1s

• During infection, C1q can bind to some bacterial surfaces, antibodies on pathogen or CRP *MUST SAY STARTS WITH ANTIBODY ON NAJAR EXAM

• C1q binding causes conformational change that activates C1r which activates C1s

• C1s than cleaves circulating C4 and C2 into C4a/C4b and C2a and C2b.

• C4b covalently binds to pathogen or antibody, than C2b binds to C4b, forming C4bC2b complex called C3 COVERTASE

• C3 Covertase than cleaves C3 into C3a and C3b

• C3b can do one of two things

• First is that many C3b molecules can coat the pathogen surface in process called opsonization, marking it for destruction by phagocytes. Phagocytes have C receptors that recognize the C3b molecule

• Second is that C3 binds to C4bC2b complex creating the C5 covertase (C4bC2bC3b) which than cleaves C5 into C5a and C5b

• C5a is also released into circulation to recruit phagocytes + increase inflammation

• C5b nucleates MEMBRANE ATTACK COMPLEX, binds to C6 forming C5bC6 which then binds to C7 and causes conformation change allowing the C5bC6C7 complex to insert in the pathogen cell membrane

• C8 then binds to the complex and causes another conformational change that allows it to insert into the plasma cell membrane

• Then C9 polymerizes within cell membrane forming pore that allows water and salt to flow out the cell causing lysis

What are the functions of the complement by-products C3a and C5a?

C3a and C5a play a role in increasing inflammation. It gets released into circulation, increasing vascular permeability and recruiting other WBCs/lymphocytes to the site

What is the membrane attack complex (MAC)

Complex of complement proteins C5bC6C7C8C9 that causes cytolysis in the pathogen. It inserts into the pathogen’s cell and plasma membrane, causing the cell cytoplasm contents to leak out and water and salt to flow in compromising cell integrity.

Define cytolysis

Breaking open/rupturing cell membrane causing cell to die

What is the first requirement for activation of the classical complement pathway

Presence of antibody bound to antigen

2 isotypes IgG and IgM that activate classical pathway

• 1 IgM= enough to activate complement

• 2 IgG in close proximity required

What is the sequence of complement protein activation in classical pathway?

C1 → C4 → C2 → C3 → C5 → C6 → C7 → C8 → C9

142356789

What are the functions of antibodies in a Th2-mediated immune response?

1.Neutralize pathogens

2.Enhance phagocytosis by making the pathogens for phagocytes

3.Activating complement system

What are the 2 types of T-lymphocytes?

There are two major types of T-lymphocytes. T-helper cells which are CD4+ assist the immune response by binding to APCs and releasing cytokines to help direct the immune response towards cell-mediated, or antibody mediated response.

In the cell-mediated pathway, they can activate CD8+ cells and enhance co-stimulation b/w APCs and the cyotoxic T-lymphocyte.

In the antibody pathway, they activate B-cells by binding to their MHC Class II molecules with the antigen on it to induce proliferation and differentiation into plasma b cells and memory b cells.

The second type is CD8+ or cytotoxic T lymphocytes. These T cells target cells infected by an intracellular pathogen. They eliminate them by producing perforins and granzymes which kills cells through apoptosis. Another method is through a death-litigation receptor, programming cell death.