The BioArtificial Liver

The BioArtificial Liver
By:Susana Candia
Jahi Gist
Hashim Mehter
Priya Sateesha
Roxanne Wadia

Biology of the Liver
Left lobe
Right lobe
Kidneys
Gallbladder
Falciform Ligament
Inferior Vena Cava
Abdominal Aorta

What does the Liver do?
Among the most important liver functions are:
* Removing and excreting body wastes and hormones as well as drugs and other foreign substances
* Synthesizing plasma proteins, including those necessary for blood clotting
* Producing immune factors and removing bacteria, helping the body fight infection

Other important but less immediate functions include:
* Producing bile to aid in digestion
* Excretion of bilirubin
* Storing certain vitamins, minerals, and sugars
* Processing nutrients absorbed from digestive tract

Why would someone need a BioArtificial Liver?
Liver Transplantation Now
* Patients are in waiting list ranked according to severity of disease and life expectancy among other variables.
* Can be from a cadaveric donor or from a live donor.
* Involves heavy use of immunosuppressants during and after surgery.
* The risk of rejecion is always present.

What does a BioArtificial Liver need to do?
* Cellular components must be purified and every component in it must be clearly identified.
* The cellular preparation must be clearly shown to not transmit any infectious diseases of any kind.
* The cellular component must stay viable and active
* The synthetic component must be fully biocompatible, integrity of the material and parts must also be demonstrated
* The device must be able to introduce the therapeutic and regulatory molecules that a healthy liver provides, and it must also filter substances from the blood the way that the normal liver does.
* Must be immunocompatible.
* Blood must perfuse properly through system

Enabling Technologies
* Hemodialysis/hemofiltration hollow fibers- controlled interaction of cells and circulating fluids
* Maintenance and creation of a cell line
* Immortalizing cells
* Encapsulation-envelopment of hepatocytes in a polymeric matrix.
* Microcarriers- polymeric particles containing cells

Works in Progress: Points to Consider
Bioreactor designs/Membrane configurations
Cellular vs. Acellular system
Porcine vs. Human hepatocytes
Point in Development
Liver Dialysis Unit
* FDA approved in 1994
* Plate dialyzer with blood on one side, dialysate is a mixture of sorbents, activated charcoal being the essential component.
* For a substance to be removed, must be dialyzable and able to bind to charcoal.
* “Bridge to recovery” for treat acute hepatic encephalopathy and overdoses of drugs
* Post-market trials have shown the LDU to be effective in improving physiological and neurological status.

MARS®
* Limited to investigational use in US.
* Hollow fiber membrane hemodialyzer.
* Blood on one side, human albumin on other.
* Albumin recycled through circuit containing another dialyzer and carbon and anion exchanger adsorption columns.
* Removes both water-soluble and protein bound substances
* Keep valuable proteins
* Trial have found it safe and associated with clinical improvement

ELAD®
* Uses cultured human hepatocytes express normal liver-specific metabolic pathways. hollow fiber dialyzer.
* Dialyzer cartridge connected to continuous hemodialysis machines, like those used for renal therapy.
* Blood separated into a cellular component and a plasma component.
* Plasma through dialyzer, hepatocytes on outside of hollow fibers.
* Currently involved in a phase 2 clinical trial to evaluate the safety and efficiency.
BLSS
* Extracorporeal hemofiltration hollow fiber membrane bioreactor with 100 grams of primary porcine hepatocytes
* Whole blood is filtered
* Contains blood pump, heat exchanger, oxygenator to control oxygenation and pH, and hollow fiber bioreactor
* Currently undergoing phase I/II clinical trials
* Patients show some improvement

HepAssist 2000 System
* Four components: a hollow fiber bioreactor containing porcine hepatocytes, two charcoal filters, a membrane oxygenator, and a pump.
* Must be used in conjunction with a commercially available plasma separation machine
* Blood separated; plasma processed through charcoal filters to remove particulates, bacteria, then enters bioreactor
* Hepatocytes must be heated and oxygenated
* FDA mandated full Phase III trials
LIVERx2000
* Hollow fiber cartridge
* Primary porcine hepatocytes suspended in a cold collagen solution and injected inside fibers
* Blood circulates outside the hollow fibers
* Designed to treat both acute and chronic liver failure
* Phase I/II clinical trials are underway to test the safety of efficacy of this device
* Anyone treated with the LIVERx2000 will be monitored for PERV
MELS
* Parallel plate design
* Human hepatocytes attached to semipermeable membranes on parallel plate
* Plasma separator, then plasma passes into the bioreactor
* In the bioreactor, the plasma flows over the semipermeable membrane where the hepatocytes are adhered.
* Current trials in Europe show promise

Demographics and Cost
* Market for liver support is estimated to be substantial: $700 million in the United States and $1.4 billion worldwide.
* Liver transplants have more than doubled in the past ten years, with the transplant waitlist growing in a similar fashion

Current and Future Challenges
* GOAL: To produce a fully implantable bioartificial liver.

Problems:
Cell viability
Fibrosis around implanted capsules
Proteins greater than pore size cannot be released

To achieve density of cells needed to replace liver, an estimated 1000m of hollow fibers would be needed

The BioArtificial Liver.ppt

Renal Replacement Therapy

Renal Replacement Therapy

* What is it?
* How does it work?
Where did it come from?
History of Pediatric Hemofiltration
Mechanisms of Action: Convection
* Hydrostatic pressure pushes solvent across a semi-permeable membrane
* Solute is carried along with solvent by a process known as “solvent drag”
* Membrane pore size limits molecular transfer
* Efficient at removal of larger molecules compared with diffusion
* Solvent moves up a concentration gradient
* Solute diffuses down an concentration gradient

Mechanisms of Action: Diffusion
Semi-permeable Membranes
o Urea
o Creatinine
o Uric acid
o Sodium
o Potassium
o Ionized calcium
o Phosphate
o Almost all drugs not bound to plasma proteins
* Allow easy transfer of solutes less than 100 Daltons
o Bicarbonate
o Interleukin-1
o Interleukin-6
o Endotoxin
o Vancomycin
o Heparin
o Pesticides
o Ammonia
* Sieving Coefficient
* Sieving Coefficient is “1” for molecules that easily pass through the membrane and “0” for those that do not
* Continuous hemofiltration membranes consist of relatively straight channels of ever-increasing diameter that offer little resistance to fluid flow
* Intermittent hemodialysis membranes contain long, tortuous inter-connecting channels that result in high resistance to fluid flow

How is it done?
* Peritoneal Dialysis
* Hemodialysis
* Hemofiltration
* The choice of which modality to use depends on
o Patient’s clinical status
o Resources available

Peritoneal Dialysis
* Fluid placed into peritoneal cavity by catheter
* Glucose provides solvent gradient for fluid removal from body
* Can vary concentration of electrolytes to control hyperkalemia
* Can remove urea and metabolic products
* Can be intermittent or continuously cycled
* Simple to set up & perform
* Easy to use in infants
* Hemodynamic stability
* No anti-coagulation
* Bedside peritoneal access
* Treat severe hypothermia or hyperthermia
* Unreliable ultrafiltration
* Slow fluid & solute removal
* Drainage failure & leakage
* Catheter obstruction
* Respiratory compromise
* Hyperglycemia
* Peritonitis
* Not good for hyperammonemia or intoxication with dialyzable poisons

Intermittent Hemodialysis
* Maximum solute clearance of 3 modalities
* Best therapy for severe hyperkalemia
* Limited anti-coagulation time
* Bedside vascular access can be used
* Hemodynamic instability
* Hypoxemia
* Rapid fluid and electrolyte shifts
* Complex equipment
* Specialized personnel
* Difficult in small infants

Continuous Hemofiltration
* Easy to use in PICU
* Rapid electrolyte correction
* Excellent solute clearances
* Rapid acid/base correction
* Controllable fluid balance
* Tolerated by unstable patients
* Early use of TPN
* Bedside vascular access routine
* Systemic anticoagulation (except citrate)
* Frequent filter clotting
* Vascular access in infants

SCUF:Slow Continuous Ultrafiltration
* Pros
* Cons
Continuous Venovenous Hemofiltration
Dialysis Fluid
Continuous Venovenous Hemodialysis
Continuous Venovenous Hemodialysis with Ultrafiltration
Is there a “Best” Method?
Indications for Renal Replacement Therapy
Indicators of Circuit Function
Filtration Fraction
QP: the filter plasma flow rate in ml/min
Blood Flow Rate & Clearance
Pediatric CRRT Vascular Access: Performance = Blood Flow!!!
Urea Clearance
Solute Molecular Weight and Clearance
Cytokines (large) adsorbed minimal clearance
Replacement Fluids
Physiologic Replacement Fluid
Anticoagulation
Mechanisms of Filter Thrombosis
Heparin - Problems
Sites of Action of Citrate
Anticoagulation: Citrate
What are the targets?
Unknowns of Hemofiltration for Sepsis
Pediatric CRRT in the PICU
Renal Replacement Therapy in the PICU Pediatric Literature

Renal Replacement Therapy.ppt

Renal Replacement Therapy

Renal Replacement Therapy
Trauma Conference
By:Amanda Wheeler, MD

Principles
4 Main Modalities in ICU
* HD
* PD
* CVVH
* CVVHD

Definition of Terms
* SCUF- Slow Continuous Ultrafiltration
* CAVH- Continuous Arteriovenous Hemofiltration
* CAVH-D- Continuous Arteriovenous Hemofiltration with Dialysis
* CVVH- Continuous Venovenous Hemofiltration
* CVVH-D- Continuous Venovenous Hemofiltration with Dialysis

Indications for Continuous Renal Replacement Therapy
* Volume Overload
* Electrolyte Imbalance
* Uremia
* Acid-Base Disturbances
* Drugs

Hemodialysis vs Hemofiltration Membrane
The hemofiltration membrane consists of relatively straight channels of ever-increasing diameter that offer little resistance to fluid flow.
Hemodialysis membranes contain long, tortuous inter-connecting channels that result in high resistance to fluid flow.
Hemodialysis allows the removal of water and solutes by diffusion across a concentration gradient.

Hemodialysis
* maximum solute clearance
* best tx for severe hyper-K+
* ready availability
* limited anti-coagulation time
* bedside vascular access
* hemodynamic instability
* hypoxemia
* rapid fluid + solute shifts
* complex equipment
* specialized personnel

advantages
disadvantages

Peritoneal Dialysis
* simple to set up + perform
* easy to use
* hemodynamic stability
* no anti-coagulation
* bedside peritoneal access
* unreliable ultrafiltration
* slow fluid + solute removal
* drainage failure, leakage
* catheter obstruction
* respiratory compromise
* hyperglycemia
* peritonitis

advantages
disadvantages

CVVHD vs CVVH
CVVH
* 1. near-complete control of the rate of fluid removal (i.e. the ultrafiltration rate)
* 2. precision and stability
* 3. electrolytes or any formed element of the circulation, including platelets or red or white blood cells, can be removed or added independent of changes in the volume of total body water

CVVH
* easy to use in ICU
* rapid electrolyte correction
* excellent solute clearances
* rapid acid/base correction
* controllable fluid balance
* tolerated by unstable patients
* early use of TPN
* bedside vascular access routine
* systemic anticoagulation *
* citrate anticoagulation new
* frequent filter clotting
* hypotension

advantages
disadvantages

Renal Replacement Therapy.ppt

Bacteria Pathogenicity Ability to Cause Infection

Bacteria Pathogenicity Ability to Cause Infection

Infectious Diseases
* Encounter-bug meets host (reservoir)
* Bug adheres to host
* Entry-bug enters host
* Multiplication- bug multiplies in host
* Damage to host
* Outcome- bug or host wins or
* Coexist- chronic infection

Reservoir
* Exposure to microbe
Virulence Factors
Adherence
* Prevent infection
* Influenza changes adhesions over time
* Neisseria gonorrhoeae -variety of adhesions

Portals of Entry
* Mucous membranes
* Conjunctiva
* Skin
* Bugs have preferred portal
* C. tetani spores in soil --- anaerobic wound

Inoculum

* Number of microbes-dose
* Greater dose, more chance infection will occur
* ID50 or LD50 expresses virulence

Invasins
* Adherence of microbe to surface
* Activates factors that let microbe in-penetration
* Microbes produce invasins (proteins)
* Endocytosis
* Requires multiplication
* Compete with normal flora for space & nutrients
* Overcome local host defenses
* Avoid IgA

Multiplication
* Need Fe to multiply
Avoid Phagocytosis
* Components of cell wall –virulence
Surviving Within Phagocyte

Tuberculosis
* Ancient disease
* 1/3 of world population infected
* 8 million develop active TB each year
* 2 million die each year
* AIDs increases activation of latent TB
* Dependent upon virulence of strain & host resistance
* Produces cell mediated immunity which prevents active disease in many people
* Multi drug resistance has developed

S & S of Pulmonary TB
* Chronic disease
* Progressive weight loss
* Night sweats
* Chronic cough
* Hemoptysis
Mycobacterium tuberculosis
* Acid fast bacillus (AFB)
* Resistant to drying
* Aerobic, slow growth
* Airborne transmission
* Inhale airborne droplets
* Ingested by alveolar macrophages
* Multiply in macrophages even with ongoing immune response

TB Response
* Host immune response-delayed type hypersensitivity reaction
* Tissue damage DT Inflammatory response
TB Conversion
* TST skin reaction is positive
* Occurs within 24 – 48 hours after exposure to TB antigens
* Purified protein derivative of bacillus
* Cell mediated immunity
* Sensitized T cells react with proteins
QuantiferonGold
* Blood test
* Detects interferon gamma

How to Confirm Diagnosis
* Sputum cultures for AFB smear & culture
* Chest xray
Pathogenesis
* LTBI (latent TB infection)
Active Disease

* Low resistance

TB Outcomes
* Primary infection- positive skin test
* 10% progressive primary infection-not controlled
Secondary or Reactivation Infection
* Reinfection-2nd exposure or
* Bacteria escape immune system-reactivation
* Activated macrophages release cytokines
* Delayed hypersensitivity reaction

Prevention of Transmission
* Negative pressure rooms
* Respirator masks-fit tested
* Admit staff aware of symptoms of TB
* Yearly TST of staff
* Conversions treated with 6-9 months of INH

Treatment
* INH for LTBI or TB conversion
* TB disease-active TB
* 9- 12 months of treatment
Resistant TB
* MDR TB
* XDR TB
* DT improper treatment

BCG
* Live culture of M. bovis
Latent vs Active
* Latent TB
* Active TB
Leprosy
* Hanson’s disease- discovered in 1873
* Seen in tropics and underserved countries
* U.S.-150 new cases per year
* Infection of nervous system
* Infects the peripheral nerves within skin
* 2 forms of disease dependent upon immune response

M. leprae
* Tuberculoid form
Lepromatous Form
* Weak immune response & microbe spreads
* Skin & nerve cells infected
* Shed large #s in nasal secretions and oozing sores-more infectious
Invasion via Enzymes
Kinases
Enzymes
Invasion via Toxins
Exotoxins
A-B Toxins
Superantigens
Naming of Exotoxins
Endotoxin
S & S
Shock
Staphylococci
S. aureus
Successful Pathogen
Biofilm
Capsule
Skin Infections
Invasion via Toxins
Toxic shock syndrome
S. aureus Intoxication
Treatment
CA-MRSA
Outbreaks in Community
PVL Gene
Preventing Transmission
Clostridium botulinum
Neurotoxin
Botulism-Foodborne Disease
Toxin
Clostridium tetani
Neurotoxin
Lockjaw
Clostridium difficile
Epidemiology
Range of Disease
Pathogenesis of CDI
New Issues
Treatment
Transmission
Environment

* Clean and disinfect surfaces in close proximity of the patient
* Patient care equipment.
* Use bleach for C. difficile
* Privacy drapes

Bacteria Pathogenicity Ability to Cause Infection.ppt

Microbial Interactions with Humans

Microbial Interactions with Humans

Types of Interactions: Symbiosis
* Symbiotic Relationships

Overview of Human-Microbial Interactions
* Pathogens
* Pathogenicity
* Virulence
* Opportunistic Pathogen

Infection Versus Disease
* Infection
* Disease

Opportunistic Pathogens
* Don’t normally cause disease, but may under some circumstances
* 3 circumstances for gaining control/disease

Types of Interactions:
Normal Flora
* Normal Flora
* Factors that influence normal flora

Normal Flora
* Hundreds of different niches associated with human
* Some normal flora are pathogenic
* Resident versus transient flora
* Considered part of the first line of defense!
* Microbial antagonism
* Competitive exclusion

Portals of Entry
* Skin
* Mucous membranes

Preferred Portal
* Many microorganisms have to enter in a specific way and in a certain place to cause disease.
* Skin Portal

Skin
* Epidermis and Keratin
* Hair often deters microbial contact with skin
* Dermis and subcutaneous tissue
* Apocrine and sebaceous glands
* Eccrine glands (sweat)

Skin as a Barrier
Mucous Membranes
* Found in mouth, pharynx, esophagus, GI, respiratory, and urinary tracts
* Epithelial cells coated with protective glycoprotein layer (mucous)
* Less protection than skin
* Cilia and mucous produced by goblet cells

Mucous Membranes
* Respiratory tract Portal
* Respiratory Normal Flora
* Respiratory Barrier Mechanisms
* Gastrointestinal tract portal
* GI Normal Flora

Gastrointestinal Tract
* Large intestine
* GI Barrier
* Genitourinary tract Portal
* Genitourinary Tract Normal Flora
* Genitourinary Tract Barrier

LD50 and ID50
* LD50: Number of microbes in a dose that kill 50% of the organisms infected in a sample
* ID50: Number of microbes in a dose that causes disease in 50% of the organisms infected
* The higher the virulence the lower the ID50 or LD50

Microbial Virulence
Microbe Versus Host
* To cause disease a microbe must…
* Why it is difficult for microbes…
o Skin, antimicrobial sweat
Microorganisms and Mechanisms of Pathogenesis

Line of Defense
* First line: Skin and mucous membranes, normal flora
* Second line: phagocytes, inflammation, fever and antimicrobial substances
* Third line: (specific response) special lymphocytes (B and T cells) and antibodies

Step One: Adherence
* Specific adherence
* Pathogens have attachment structures
* Pathogens have attachment structures

Step 2: Invasion/Colonization
* Increase in numbers beyond the point of attachment.
* Three goals

Step 2: Invasion/Colonization
* Localized versus Systematic infections
* Bacteremia, viremia, toxemia
* Septicemia

Step 3: Cause Damage
* Virulence
* Three Ways to cause damage

Virulence Factors
* Usually help organism colonize and grow
* Coagulase
* Siderophores
* Collagenase
* Protease

Another Way to Classify Exotoxins
* Descriptive classifications
A-B toxin
* Cholera toxin (Vibrio cholera)—cholera

The Action of Chlorea Enterotoxin
More A-B toxin examples
Botulinum Toxin
Tetanus Toxin
Membrane Disrupting Toxins
Superantigens
Endotoxins
* Gram type negatives
* Part of outer portion of cell wall (outer membrane)
* Lipid A portion
* Exert effects when G- microbe lyses
* Same symptoms for different species of microbe
* No antitoxins produced by host
* Very stable—can’t destroy easily
* Rarely fatal
* Disseminated intravascular clotting
* General symptoms

Pyrogenic Response
* Macrophage ingestion
* Release of interleukin-1 in bloodstream
* Interleukin-1 to hypothalamus and production of prostaglandins
* Resetting of bodies thermostat

Susceptibility/Resistance of Host
* Species specificity
* Tissue specificity
* Age
* Stress
* Diet
* Pre-existing disease (Genetic and Infectious)
* Gender
* Behavior
* Weather?
* Your first line of defense—Review this

Microbial Interactions with Humans.ppt