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New notes

  • How do microorganisms cause disease? In particular, we're looking at bacteria.
  • We're interested in microorganism pathogenesis. The human body is like a battle every day.
  • Back in the day, we used to deal with infection after it already occurred (e.g. antibiotics, diagnosis in laboratory). We used to attack once it had already set up infection
    • Since the advent of techniques in molecular biology, we have understanding about how they cause disease, allowing us to take steps to prevent them from causing disease (e.g. vaccination against diphtheria with an attenuated toxin to prevent infection in the future)

A balance exists between health and disease

  • Depends on where we live
  • Depends on both exposure and how good our immune system is (e.g. HIV/AIDS wrecks your immune system)
  • Health and host defence are balanced against disease and exposure to microorganisms

What is an infectious disease

  • An infection that produces signs and symptoms

What is the difference between infectious disease and the terms "infection" or "colonisation"?

  • Infection - the successful multiplication of an organism within the host
  • Colonisation - ability of an organism to remain at a particular site and multiply but not cause disease (signs/symptoms)

Pathogens and definitions

  • Pathogens - microorganisms that have the capacity to cause disease
    • Even though they often may not cause disease under normal conditions, they may have the capacity to cause disease given the opportunity
      • Eg pseudomonas aeruginosa (gram negative rod) - when you're in a fire and get severely burned, it can kill you -- in the right conditions it can cause disease
  • Virulence - A measure of pathogenicity, or the likelihood of causing disease
    • Eg Staph epidermis doesn't have a great amount of virulence factors, so it takes a long time to cause disease
  • Virulence factor - A microbial factor or strategy that contributes to virulence (the ability of an infectious agent to cause disease)

Virulence factors

Virulence factors have a range of activities that facilitate colonisation and invasion of the host by facilitating:

  • Attachment to host cells
  • Invasion of host cells
  • Evasion of host defences

Damage the host:

  • Toxin production (and tissue destruction. E.g. diphtheria produces a toxin that produces the major signs/symptoms)
  • Bacterial enzymes


How does the host protect itself against infection?

Host armament

Barrier to infection Specific barriers Examples or actions
Anatomical barriers
  • Mechanical/physical factors
  • Skin/cilia/peristalsis/flushing action/mucus layers/nasal hairs (note mucus layers have many antibacterials in them)

Chemical factors

Tears, saliva, sweat, gastric secretions, defensins

Biological factors

Normal flora

Human barriers

Complement

Recruitment of phagocytic cells, lysis of cells (MAC) and opsonisation (innate immunity)

Lactoferrin, Tranferrin

Limit bacterial growth by binding iron

Lysozyme

Affects bacterial cell wall by attacking the peptidoglycan. Lysozyme is more active against gram +ve bacteria than gram -ve due to differences in cell wall structure:

  • Gram +ve: inner membrane + thick layer of peptidoglycan but NO outer membrane.
  • Gram -ve: inner membrane + thin layer of peptidoglycan + outer lipopolysaccharide (LPS) membrane. Outer LPS membrane prevents lysozyme entering and attacking the peptidoglycan as easily. The LPS acts as an endotoxin and elicits a strong immune response.
Cellular barriers
  • Neutrophils
  • Macrophage
  • Dendritic cells
  • Natural killer cells (mainly viruses once they get inside the host cell; viruses are obligate intracellular organisms)
  • Main line of defense of the innate (non-specific immune system)

Recognition by the innate and acquired immune system

What happens when pathogens break through the host's physical and chemical defence mechanisms?

They encounter the body's immune defences.

Innate (non-specific) immune responses Specific immune response
Response antigen independent Response antigen dependent
Following exposure an immediate maximal response occurs A lag time occurs between exposure and maximal response
Not antigen specific Antigen specific
Exposure results in no immunologic memory Exposure results in immunologic memory (subsequent exposures lead to a response that is harder, better, faster, stronger.
  • First response: IgM first, then IgG
  • Second response: IgG quickly recruited

The innate immune responses

  • Cells and molecules of the innate immune system recognise pathogens and trigger an inflammatory response
  • Pathogen-associated molecular patterns (PAMPS)
  • Unique structural components on microbial cells
  • These are recognised by pattern recognition receptors (PRRs) e.g. Toll-like receptors (TLRs)

Toll-like receptors (TLRs)

  • Different types of TLRs, recognising different patterns on invading microbes
  1. Interaction of PRR's with PAMPS
  2. Activation of macrophage and dendritic cells
  3. Secretion of pro-inflammatory mediators
  4. Initiates adaptive immune response
  • Note that all of the TLRs carry out their inflammatory effect by upregulating NF-κB, a transcription factor that is central in causing the synthesis of pro-inflammatory proteins. This is targeted by glucocorticoids/cortisone/prednisone

Note that we learned about NF-kb in AEA.

Bacterial recognition and host response

  • Adaptive immunity -- antibody-mediated and cell-mediated
  • Antigen-presenting cells = macrophages and dendritic cells. This presents to T cells
    • B cells produce and secrete antibodies, that bind to the antigens
    • T cells produce cytokines, inflammation, killing, etc
  • COPY DIAGRAM WHEN IT COMES OUT

The urinary tract

Ascending pathway for cystitis and pyelonephritis.
  • UTI = cystitis (inflammation in the bladder) and pyelonephritis (inflammation in the kidney)
  • Generally the ascending pathway (via urethra)
  • E Coli is the major cause of UTI (link to previous lecture)
    • Causes 80% of UTI

Host defences in the urinary tract

  • Mechanical processes
    • Flushing effect of urination
    • Epithelial cell shedding
    • Sphincter action (prevent organisms ascending to bladder/kidney)
  • Biological
    • Immune system: Less important than mechanical processes

Virulence factors for E Coli for UTI

  • First requirement
    • Source of Urinary pathogenic E. coli (UPEC) in colon
      • This is a particular strain of E. coli with virulence factors specific to UTI
  • Second requirement
    • Entry and colonisation of vagina and/or urethra
  • Most occur in women because:
    • Urethra is shorter
    • Anus is closer to urethra than in the case of men

Adhesion in UTI

  • Most important virulence factor in UTI
  • Adhesion due to adhesisns protects E. Coli from urinary lavage, thus increasing ability to multiply and invade
  • E. coli adhesion
    • Pyelonephritis-associated pili (P pili)
    • Type 1 pili
    • Bind to mono-mannose residues. Abundant in 1. Bladder and 2. Urinary tract
      • E coli attaches to mannose receptors present on uroplakins that cover the surface of uroepithelium
    • Not only attach, but also invade the cells of the uroepithelium -- binding triggers engulfment of E coli and the formation of intracellular communities
    • They form intracellular bacterial communities
    • Early stage - flattened, start to multiply
    • Middle stage - shorten, multiply more
    • Late stage - differentiate
    • Then they escape to environment, to find more cells to attach to (susceptible to lavage)
    • This forms a cycle of infection that can be hard to break
    • Organism can remain dormant for long periods of time

Effect of invasion

  • Invasion
    • Triggers uroepithelial cells to slough off rapidly
    • Excretion of bacteria in urine
    • But this leads to inflammation, causing symptoms (e.g. 1. Dysuria; pain on urination, due to inflammation of the sphincters 2. Frequency 3. Urgency)

Pyelonephritis

  • These organisms have an extra mechanism that allows them to cause this disease
  • They have P pili (fimbrae) - these pili allow the organisms to attach well to cells in the kidney
    • Important in kidney infection
    • Attach to alpha-D-Gal(1-4)-beta-D-Gal (globobiose) receptor in the kidney
      • Present on many cell types in bladder and kidney
  • Note that the kidney is very well vascularised, so pyelonephritis can lead to sepsis (rapidly cause death). This is a big problem.
  • Attachment and multiplication of E. coli leads to damage to kidney cells and inflammation --> Symptoms

Respiratory tract infections

The respiratory tract
The mucociliary elevator

Respiratory barriers

  • Upper respiratory tract
    • Nasopharynx - nasal hairs to trap microorganisms
    • Cough response - expels microorganisms
    • Throat - normal microflora
  • Lower respiratory tract
    • Lined with a mucus layer
      • Goblet cells - produce mucus
      • Ciliated cells - propel mucus upwards

Normal flora

Normal flora of the gut
  • Microorganisms found on or in the body of healthy individuals
    • There are more bacteria on and in your body than there are cells of your body
    • These compete with pathogens for space and nutrients
  • Streptococcus pneumonia colonises the throat, and can spread down to the lower respiratory tract

Protective effect of mucus

  • Traps bacteria
  • Secretory IgA binds to:
    • Bacterial antigens
    • Mucin (is the protein to which the IgA's Fc portion is attached. This protein exists in the mucus)
  • Antibacterial action
    • Lysozyme
    • Lactoferrin
    • Lactoperoxidase - form toxic molecules that can be antibacterial. Can initiate a reaction that forms hydroisocyanide that is toxic to cells
    • Rapid turnover of mucosal cells - shed attached organisms
  • Hence pneumonia is not that common

Virulence factors of Strep pneumonia

Action of IgA protease.

Virulence factors of Strep. pneumoniae:

  • Secretory IgA protease
  • Polysaccharide capsule
  • Pneumolysin
  • Hyaluronidase

IgA protease

  • Protects against host secretory IgA1 antibodies present in the respiratory mucus
  • Destroys this host defence mechanism
  • Breaks IgA1 in hinge region
  • IgA predominant immunoglobulin in mucosal defence

Polysaccharide capsule

  • Pneumococcal polysaccharide capsule. >90 different capsular serotypes
  • Capsule is extremely thick (looks ridiculously thick under the microscope when compared to the size of the organism!... see wikipedia:File:Pneumococcus_CDC_PHIL_ID1003.jpg)
    • 200-400nm
  • Conceals inner structure of S. pneumoniae (hidden from the immune system!)
Rough and smooth capsules for Strep. pneumoniae.
  • Properties of the capsule
    • Anti-phagocytic - negatively charged
    • Blocks complement attachment (opsonisation etc)
    • S. pneumoniae NOT phagocytosed
    • Hence it continues to multiply in lung
      • Leads to inflammation
  • Exist rough and smooth capsules -- they differ in their ability to cause disease
  • We learn all this stuff from studies on animals :(

Pneumolysin

  • Cytoplasmic protein - released on bacterial cell lysis
  • Binds to cholesterol on host cell membrane --> pore formation
    • -->Killing of host cells (RBC, WBC, respiratory epithelial cells)
  • Stops cilia beating
    • Build up of mucus in the lungs
    • Coughing reflex compromised
    • Prevents expulsion of pneumococcus
  • Activates classical complement pathway
    • Binds Fc portion of antibody to LTA (liptechoic acid; present in cell wall of gram-positive microbes)
    • Complement activation
    • Increases inflammation

Hyaluronidase

  • An enzyme
    • Breakdown of hyaluronic acid (ground substance of CT)
  • Allows organism to move through lung
    • Further bacterial dissemination within the lung

Events occurring in the lung

  • Influx of PMNs to the site of infection
    • Alveoli engorged with oedema and WBC
  • LTA on surface of pneumococcus
    • Activates alternative complement pathway
      • Production of C5a
  • C5a further mediates inflammatory response
    • Further influx of PMNs
  • PMNs release reactive oxygen species
    • Damage to host tissues
    • "Surfs on wave of inflammation"
  • Not until the host can produce an adaptive response and have antibodies to the organism
    • "Crisis period" before this point -- may die
    • Prior to antibiotics, many people died to pneumonia because their immune system didn't act quickly enough

Relationship between S.pneumoniae virulence factors and the pathology observed in the lung

  • S. pneumoniae enters the lung
    • Capsule prevents phagocytosis
  • S. pneumoniae continues to multiply in lung
    • Inflammatory response
  • LTA --> further inflammation via C5a
  • Pneumolysin --> Further inflammation
  • Hyaluronidase --> Spread of organism in lung

Together, these produce the signs and symptoms of pneumonia

Lower respiratory tract infection

  • Staphylococcal pneumonia
  • Nowhere near as common as that above
  • Rarely a primary event
  • Previous viral infection is a predisposing factor
    • e.g. influenza.
    • Viruses are obligate intracellular organisms. Therefore multiply in and cause lysis of the goblet cells etc, so the mucociliary elevator fails, predisposing to lower respiratory tract infection

Major virulence determinants of S. aureus

Virulence factors of S. aureus.

List includes:

  • Protein A
  • Surface polysaccharide
  • Extracellular enzymes and toxins
    • Coagulase
    • Lipase
    • DNAase
    • Staphylokinase (fibrinolysin)
    • Superoxide dismutase
    • Catalase
    • Leukocidin
    • Alpha toxin

Protein A and surface polysaccharide

Structure of IgG
Exploitation of the structure of IgG by S. pneumoniae, which uses Protein A to bind the Fc region of the antibody, thus preventing phagocytosis.
  • Protein A
    • Organism has to bind to the Fab (variable region) to cause engulfment. But because it binds to protein A on the Fc region, it won't get phagocytosed.
  • Surface polysaccharide
    • Majority of clinical isolates of S. aureus express surface polysaccharide. This impedes phagocytosis in the absence of complement

Extracellular enzymes and toxins

  • Coagulase - most important
    • Important virulence factor
    • Encloses S. aureus in fibrin (by breaking down fibrinogen)
    • Walls off infection
    • Protects S. aureus from PMNs
    • Fibrin deposition acts as a barrier
    • Leads to abscess formation in lung
  • Staphylokinase (fibronolysin)
    • Produced by all Staph aureus
    • Dissolves fibrin clot, so it can move to another area
  • DNAase
    • Reduces viscosity of abscess material and facilitates spread
  • Hyaluronase
    • Local dissolution of host extracellular matrix
  • Superoxide dismutase (SOD)
    • Detoxifies oxygen radicals released by PMNs
    • Equation: 2O2- + H2O --> H2O2 + O2 (catalysed by SOD)
  • Catalase
    • Protects cells from H2O2
      • Can directly and indirectly cause
        • DNA damage
        • Protein damage
        • Lipid damage
    • 2H2O2 --> 2H2O + O2 (catalysed by catalase)
  • Leukocidin
    • Lyses neutrophils and macrophages
  • Alpha toxin
    • Forms pores in human cell membranes, causing lysis
    • This causes cytokine production (because lysed cells release factors that stimulate cytokine production and inflammation)

Relationship between virulence factors and the pathology in the lung

  • Coagulase leads to the deposition of a layer of fibrin around the organism
    • --> walled off lesions (abscesses)
  • Spread facilitated by:
    • DNAase and staphylokinase (fibrinolysin)
    • Leukocidin, alpha toxin, hyaluronidase

Old notes

Introduction

  • The body is in a constant battle between health and disease
    • Based on the host defences (immune system, antibodies, CD8 cells, phagocytosis, barriers (skin/mucosa)) vs microorganisms

Infectious disease

  • The ability for microorganisms to cause disease depends on 2 factors:
    • Ability to spread from host to host
    • Ability to survive and multiply in the host
  • Definitions:
    • Infectious disease: an infection that produces signs (eg. runny nose) and symptoms (eg. headache)
    • Pathogen: a microorganism that has the capacity to cause disease
  • Regular pathogens vs opportunistic pathogens
    • Regular pathogens cause disease regularly (Corynebacterium diphtheriae – diphtheria, streptococcus pneumoniae)
    • Opportunistic pathogens cause disease rarely
      • Pseudomonas aeruginosa – gram –ve rod
        • Found in the environment,
        • Cystic fibrosis predisposes, often kills, creates a biofilm that is hard to get rid of
      • Candida albicans
        • Immunosuppressed are predisposed
        • Cant get systemic infection or with antibiotics, vaginal thrush
        • Pathogenicity – capacity of a microorganism to produce disease
    • Depends on microorganisms ability to:
      • Gain entry to host
      • Attach to host cells
      • Survive in host and multiply
      • Evade host defences
      • Damage tissue (invade tissue) and produce disease symptoms
    • Dependent on virulence: a measure of pathogenicity or the likelihood of causing disease
      • Virulence factor: a bacterial factor or strategy that contributes to virulence

Urinary tract infection

  • Host defence mechanisms
    • Mechanical:
      • Flushing effect of urination
      • Epithelial cell shedding
      • Sphincter action (prevent entry to bladder)
    • Biological:
      • Immune system is less important than mechanical processes
  • Majority of infections infect via the ascending pathway
    • Enter via the urethra and multiply in bladder
      • May remain in bladder or ascend the ureters to the kidneys themselves
        • If damages blood vessels and enters blood stream, can cause sepsis
  • Definitions:
    • Cystitis – infection of the urinary bladder
    • Pyelonephritis – infection of the kidney
  • Several requirements for urinary tract infection:
    • A source of urinary pathogenic E. coli (UPEC) – a strain that can cause infection
      • Gram –ve rod common in normal GIT flora
      • Not all strains are the same, some can infect UT others can’t
    • Entry and colonisation of vagina/urethra
  • Adhesion
    • Important factor in facilitating colonisation of urethra
      • 10-36% of faecal E. coli adhere to vaginal, uroepithelial cells, 22-36% of asymptomatic bacteruria E. coli, 50-60% cystitis E. coli, 70-100% of pylonephritis bacteremia e. coli
      • Thus, adhesion is an important factor in determining invasiveness
    • Adhesion protects the bacteria from urinary lavage thereby allowing multiplication and invasion
      • Adhesins: Type 1 pili (fibrae); pyelonephritis associated pili (P pili)
  • Type 1 pili
    • Produced by most strains of E. coli
    • Binds to mono-mannose residues/receptors on host cells
      • Found in the bladder, vaginal tract
      • Faecal isolates of e. coli interact poorly with mono-mannose receptors (GIT doesn’t have receptors)
  • Uroepithelial cells (bladder epithelium, umbrella cells)
    • Secrete uroplakins that sits on the top layer of the urepithelial cells
      • These are transmembrane proteins that cover the uroepithelial cell surface
      • Mannose receptors attach to the uroplakin
  • Invasion process:
    • E. coli enters bladder, multiplies and attaches to mannose residues attached to uroplakins via type 1 pili
    • Binding leads to engulfment of e. coli allowing avoidance of the immune system
    • Bacterial invasion triggers cells to slough off rapidly and be excreted in urine
    • Inflammation
    • Symptoms – increased urine frequency (sphincter control loss) and dysuria (acidic urine irritation)
Binding of P-pili to globobiose.
  • P pili
    • Attach to the Globobiose receptor that is present in many cell types in the bladder/kidney
      • Attachment and damage to kidney cells leads to an inflammatory response
  • This may lead to damage to blood vessels, if bacteria in this way enters blood stream, can cause sepsis

Respiratory tract infection

  • Defence mechanisms:
    • Nose
      • Nasal hairs – trap organisms/particles
      • Sneeze reflex – flush organisms/particles
    • Mouth
      • Flow of saliva
      • Antibacterial properties of saliva (lysosyme, antibacterial to gram +ve)
      • Resident microflora – take up nutrients/space
    • Mucous
      • Mucin – traps bacteria
    • Cilia
      • Ciliated cells in the respiratory tract, beat upwards to flush out foreign particles
    • Antibacterial action
      • Lysozyme
      • Secretory IgA – binds to bacterial antigens
      • Lactoferrin – iron acquisition (bacteria need iron, prevent this, bacteria have siderophores however)
      • Lactoperoxidase – antibacterial
    • Rapid turnover of mucosal cells
    • Immune system – Peyer’s patches

Bacterial sore throat

  • Normal major cause is viruses
  • Streptococcus pyogenes can cause a bacterial infection
    • Gram +ve cocci
    • Virulence factors:
      • Non-fibrillar adhesins (doesn’t use pili or fimbrae, uses proteins instead)
        • Attaches to Fibronectin (in the host cell) via Fibroenctin binding proteins F1 and F2
      • Hyaluronic acid (HA) capsule
  • Colonies freshly isolated appear mucoid
  • Aids attachment to pharynx
    • Attaches to CD44 receptor on epithelial cells
  • Antiphagocytic properties
    • Acidic (negatively charged),
  • HA appears similar to host cell CT and is not recognised as foreign
    • Thus, antigens are hidden below capsule
      • Prevefns opsonised phagocytosis by neutrophils or macrophages
      • M-protein
  • Binds to mammalian proteins
    • Fibrinogen – results in fibrin production and walling off of the infection
      • Causes pus formations to build up: fibrin, PMNs and cell debris
    • Fc region of IgG – prevents opsonisation by antibodies
  • Opsonisation: aided phagocytosis, neutrophils recognise the Fc region of IgG
    • M protein binds Fc, thus no phagocytosis
      • Extracellular enzymes
        • Streptolysins – damage membranes of susceptible cells (epithelial cells)
        • Streptokinases – activate plasmin to dissolve fibrin clots and facilitate spread
        • Hyaluronidase – digests host CT and facilitates spread

Streptococcus pneumoniae

Lobar pneumonia
  • Causes pneumonia (25-60% of pneumonia)
  • Alpha haemolytic, gluggy, gram +ve dipolococci. Causes lobar pneumonia
  • Virulence factors:
    • Polysaccharide capsule
      • >90 capsular serotypes, antibodies against one may be useless against another
      • Extremely thick
        • 200-400nm (vs: peptidoglycan 20nm, plasma membrane 9nm)
        • Conceals inner structures of S. pneumoniae
      • Anti-phagocytic – negatively charged
        • Anionic surface is not well recognised by host defences
        • Prevents complement activation
      • Allows bacteria to survive in the lungs, cause damage to epithelial cells and thus inflammation and an immune response
      • Fragments of the capsule shed in the replication process
        • These are bound by antibodies and activate the complement pathway away from the bacteria, causing inflammation and damage
    • sIgA1 protease
      • Breaks down the host secretory IgA1 antibody at the hinge regions
        • IgA is the predominant immunoglobulin in mucosal defence
    • Pneumolysin
      • Stops beating of cilia
        • Leads to mucus build up in the lungs
        • Compromises the cough reflex – can’t expel pneumococcus
      • Activates classical complement pathway
        • Binds Fc portion of antibody to lipotechoic acid, activates the complement system
        • Increases inflammation
    • Hyaluronidase
      • Enzyme, breaks down hyaluronic acid – the ground substance of CT
        • Thus promotes further bacterial dissemination
        • Lung tissue damage
    • Influx of PMNs to site of infection
      • Need antibodies for phagocytosis, therefore ineffective
      • Alveoli become engorged with oedema and immune cells
    • Teichoic acid on the surface of bacteria activates the alternate complement pathway
      • Leads to production of C5a that further mediates the inflammatory response
  • Further influx of PMNs
    • PMNs release reactive oxygen species like H2O2 hydrogen peroxide
      • Causes damage to host cells
  • Invasion process:
    • S. pneumoniae enters the lung and causes an inflammatory response
      • Capsule prevents phagocytosis and increases the inflammatory response
        • Pneumolysin further increases the inflammatory response
    • Hyaluronidase further facilitates the spread of the organism

Staphylococcus aureus

  • Staphylococcal pneumonia – a lower respiratory tract infection
    • Rarely a primary event, often follows a previous viral infection
      • Eg: Spanish flu 1918, deaths were mostly due to secondary Staph. aureus bacterial pneumonia
  • Virulence factors
    • Protein A
      • Extracellular protein (90% of strains)
      • Covalently bound to the cell wall
      • High affinity for Fc region of IgG
        • Prevents opsonisation of organism, and thereby facilitation of phagocytosis
    • Surface polysaccharide
      • Majority of clinical isolates have this factor
      • Impedes phagocytosis without complement system aid
    • Enzymes and toxins
      • Coagulase
        • Important virulence factor
        • Encloses S. aureus in fibrin and walls of the infection
    • Protects S. aureus from PMNs, fibrin deposition acting as a barrier
    • Abscesses form diffusely in the lung
      • Lipase
      • Staphylokinase (fibinolysin)
        • Produced by all Staph aureus
        • Dissolves fibrin clot allowing organism to spread
      • DNAase - reduces viscosity of abscess material and facilitates spread
        • Hyaluronidase - local dissolution of host extracellular matrix facilitating spread
      • Superoxide dismutase
        • Used to detoxify oxygen radicals used by PMNs to destroy bacteria
        • 2O2. + H2O --SOD--> H2O2 + O2
      • Catalase
        • Used to protect cell from H2O2 that can cause DNA, protein, lipid damage
        • 2H2O2 --catalase--> 2H2O + O2
      • Leukocidin
        • Lyses neutrophils and macrophages
      • Alpha toxin
        • Forms pores in human cell membranes that disrupts the cells (esp. in the lungs)
        • Induces proinflammatory cytokine production:
        • IL-1, TNF-α, --> IL-8 --> neutrophils
  • Invasion process:
    • Coagulase leads to deposition of fibrin around the organism causing a walled-off abscess
      • Spread is facilitated by DNAase and staphylokinase (fibrinolysin)
      • Tissue damage is caused by leukocidin, alpha toxin and hyaluronidase