1Immunology

Tolerance and Autoimmunity

Chapter 14

Tolerance

zTolerance (immune paralysis, immune unresponsiveness) - the state of unresponsiveness to an immunogen (tolerogen)(tolerogen).zSelf-tolerance - the inability of the body

to respond to autologous antigens

Characteristics of Tolerance

zTolerance is an active antigen-dependent process in response to an antigen.zTolerance is specific and can exist in T-cells,

B cells or both.B cells or both.{Tolerance at the T cell level is longer lasting

than tolerance at the B cell level.{Induction of tolerance in T cells is easier and

requires relatively smaller amounts of tolerogen than tolerance in B cells

zMaintenance of immunological tolerance requires persistence of antigen.z Tolerance can be broken naturally (autoimmune

diseases) or artificially (shown in experimental animals by x irradiation certain drug treatmentsanimals, by x-irradiation, certain drug treatments and by exposure to cross reactive antigens).z Tolerance may be induced to all epitopes or only

some epitopes on an antigen and tolerance to a single antigen may exist at the B cell level or T cell level or at both levels.

Factors Affect Response to Ag

Favor Immune Response Favor Tolerance

Physical form Ag large, aggregated, complex molecules

soluble, aggregate-free, smaller, less complex molecules

Route Ag administration sub-cutaneous or intramuscular

oral or sometimes intravenous

Dose Ag optimal dose very large (or sometime very small) dosevery small) dose

Age animal older and immunologically mature

Newborn (mice), immunologically immature

Differentiation state cells fully differentiated cells; memory T and memory B cells

relatively undifferentiated: B cells with only IgM (no IgD), T cells (e.g. cells in thymic cortex)

Explanations for Tolerance

z Clonal abortion (deletion){ Immature cells encountering self-ag undergo negative

selection-programmed cell deathzT cells that bind to self-ag in thymuszB cells expressing only IgM exposed to self-agzB cells expressing only IgM exposed to self ag

z Clonal anergy (lack of response){T cells exposed to ag without co-stimulatory B7 {B cells exposed large amounts soluble ag

z Clonal ignorance {T cells reactive to sequestered antigen die due to lack of

stimulation{B cells dont find ag or have Th cell so cant react-die

2z T-cell suppression{ Low and high doses of ag may induce suppressor T cells{Suppress T and B cells

z Receptor editing {B cells encounter large amounts soluble ag, bind with low

affinityaffinity{May recombine genes changing specificity

z Balance between Th1 and Th2 cells (see p 213 in book){Th1 cells implicated as primary mediators in autoimmunity

because release proinflammatory cytokinesz Anti-idiotype antibodies{Antibodies to idiotypes (in sIg or TCR){May combine to receptors blocking reaction with ag- inhibits

immune response

Autoimmunity: Definitions

z Autoimmunity{Breakdown of immune system's ability to

discriminate between self and non-self (tolerance){Res lts in generation of self reacti e clones of T or B{Results in generation of self-reactive clones of T or B

cells.

z Autoimmune disease{ Immunoglobulins (autoantibodies) and/or sensitized

T cells displaying specificity for self-antigens cause damage to cells or organs.

Factors Influencing Development

zGenetic{Familial aggregates {More than one disorder {HLA association {More frequent women{More frequent women.

z Age { Increases with age.

z Exogenous Factors {UV radiation, drugs, viruses, chronic infections

Mechanisms of Autoimmunityz Sequestered antigen theory{Tissue antigens are sequestered (hidden) during

neonatal period (no tolerance){Due to injury; viral infection, etc. antigen gets

into circulation, autoantibodies formed.z Altered antigens (body constituents) {Surface antigens on host altered by chemical, g y ,

biological, or physical means.{New antigenic determinant may be recognized

as foreign.z Cross Reactivity/Molecular mimicry{Microorganisms have ag determinants identical or

similar to normal host-cell components{Antibodies produced to determinants on

microorganism cross react with self antigens

Anti-M protein

Cross reactive ag on heart

Strept with M proteins

Rheumatic fever

Inappropriate expression of Class II molecules Class II molecules expressed on the surface of cells

where not normally found Cell can then function as APC, present self-antigens,

which sensitize Th resulting in activation of B or Tc cells.

3z Cytokine imbalance {Unregulated cytokine production may initiate

autoimmunity by various mechanisms{Ex. Increasing MHC expression

z Polyclonal B-cell activation {Certain viruses and bacteria can induce

nonspecific polyclonal B-cell activation (ex. EBV, CMV).{If B cells reactive to self-antigens are activated,

autoantibodies can appear.z Disturbance of Th1/Th2 balance{Th1 cells implicated in autoimmunity{Th2 protect against induction of disease and

progression of established disease.

zLoss of immunoregulatory function by T-cell subsets {T suppressor cellszMay prevent immune response to self-antigen.zIf Ts cells removed response to antigen can occurzIf Ts cells removed, response to antigen can occur.

{T helper cellszDeletion of Th cells may prevent response to self-

antigen. zAlternate signal may be provided which stimulates B

cell to respond

zForbidden clone theory{A clone of changed or altered lymphocytes

arises through mutation.{These cells lack foreign surface antigens and

are not destroyed by host.{Because of alteration may recognize host as

foreign.

Spectrum of Diseases

Hashimotos Thyroiditis

Graves Disease

Pernicious Anemia

Addisons Disease

Organ Specific

Myasthenia Gravis

Goodpastures Syndrome

Rheumatoid Arthritis

Scleroderma

Systemic Lupus ErythematosusSystemic

Organ-Specific Disorders

z Antibodies and lesions specific to an organz Clinical and serological overlap seen.z Ags only available to lymphoid system in low

concentrations.z A k ifi b i l i lz Ags evoke organ-specific ab in normal animals

with Freunds adjuvant.z Familial tendency to developz Lymphoid invasion, parenchymal destruction by

cell-mediated hypersensitivity or absz Tendency to develop cancer in organ.

Organ-nonspecific Systemic Disorders

z Antibodies and lesions not specific to organ.z Overlap of SLE, RA, and other connective tissue

disorders.z Ags accessible at higher concentrations.z No abs produced in animals with comparable stimulation.p pz Familial tendency to develop connective tissue diseasez Abnormalities in immunoglobulin syntheses in relatives.z Lesions caused by deposition of ag-ab (immune)

complexes (immune complex hypersensitivity)..z Tendency to develop lymphoreticular neoplasia.

4Similarities in Disorders

zCirculating autoantibodies react with normal body constituents.z Increased immunoglobulin

concentration in serum often found.zAntibodies may appear in each of the

main immunoglobulin classes.zDisease process not always

progressive, exacerbations and remissions occur.zAutoantibody tests of diagnostic value.

Laboratory Tests

z Tests for Specific Autoantibodies{Methods- IFA, TRCA, PHA, Ouchterlony, ELISA{Examples- RF, ANA, etc

z Complement Assays {CH50, C4, C3{Complement levels usually decreased during acute phase{Complement levels usually decreased during acute phase

of non-organ specific disorders.zTissue damage due to immune complex hypersensitivity-

complement is used up-levels decreasezComplement levels may be used to monitor therapy.

z Tests for Ig levels {Methods- Nephelometry, RID{ Ig levels often increased in autoimmune disorders due to

polyclonal activation of B cells

zTests for Circulating Immune Complexes (CIC){CIC occur when antigen combines with

antibody and complexes circulate in blood. zComplexes may settle in certain areas (ex skinzComplexes may settle in certain areas (ex. skin,

blood vessels, kidney), activate complement and cause tissue damage. zImmune Complex Hypersensitivity.

{CICs are commonly found in non-organ specific autoimmune disorders.

{Methods for detecting CICs zRaji cell assay

Raji cells are B cells found in Burkitts lymphoma

Have receptors for C3bCan be labeled in order to detect ICs

ICs in serum activate complement C3b attaches

Labeled AHG

complement, C3b attaches Mix serum with labeled Raji

cells- cells combine with C3b, can measure label.

zC1q assay C1q attached to solid support Add serum, ICs attach (via

antibody) Add labeled AHG Measure label.

C1q

zTests for cryoglobulins{Cryoglobulins are immunoglobulins which

precipitate in the cold.{Circulating immune complexes produced in{Circulating immune complexes produced in

response to autologous antigens may function as cryoglobulins.{Will give procedure later

Specific Autoantibodies- Rheumatoid Factor (RF)

zFound in the majority of patients with rheumatoid arthritis (RA).zMost are IgM immunoglobulins{Some are IgG, IgA, or IgE.

zReacts with the Fc portion of human and animal IgG

5Lab Tests for Rheumatoid Factor

zPassive hemagglutination (Rose-Waaler)zPassive (latex) agglutination (Singer

and Plotz)and Plotz)zNephelometryzEnzyme linked immunosorbent assay

(ELISA)

Passive hemagglutination (Rose-Waaler)

zPrinciple- sheep blood cells coated with rabbit IgG (anti-sheep rbcs) (ag) are mixed with serum. If RF (ab) is present hemagglutination occurshemagglutination occurs.zSpecificity = 90%zSensitivity = 50%

Latex Agglutination (Singer and Plotz)

zPrinciple- latex particles coated with human IgG (antigen) is mixed with serum. If RF (antibody) is present in serum, agglutination occurs. zSpecificity = 75%zSensitivity = 75%zCan do tube test to determine titerzSlide test is positive if concentration of RF

is >20 IU/mL

Singer Plotz

Rh t id F t

Latex particles

Human IgG

Rheumatoid Factor

Nephelometry

z Principle- IgG (ag) (in excess) is mixed with serum. If RF (ab) is present, it will combine with IgG forming immune complexes. The immune complexes scatter light. The rate of light scatter is determined by the nephelometer. z Specificity = 96%z Sensitivity = ~82%z Advantages- automated, better sensitivity and

specificity, newer tests can detect all classes z Disadvantage- detects mostly IgM RFs

ELISAz Principle- IgG (or Fc portion) (Ag) is attached to

solid phase. Serum is added. If RF (ab) is present it will attach to IgG. After washing, ab to RF (anti-IgM, IgG, IgA, etc) labeled with enzyme is added. After additional washing, the substrate is added. A colored reaction occurs in proportion to the amount of RF in the serum. Color is read on a spectrophotometer.z Specificity = 94%z Sensitivity = ~85%z Advantages- more sensitive and specific

traditional tests, automated, can be modified to detect other classes of RF.

6Nephelometry/ELISA: Results

zReported in international units.zCorrelation between RF agglutination

titers and International Units is imperfect{Serum RF level of ~50 IU/mL is equivalent to{Serum RF level of 50 IU/mL is equivalent to

Singer-Plotz latex agglutination titer of 160. {The lowest value of clinical significance is ~

12.5 IU/mL. (~ titer of 40){Usually 20 IU or above is considered

significant.

Clinical Significance of RF

z RF is not specific for RA. {May be found in other conditions. zSjogrens syndrome, SLE, hepatitis, cirrhosis,

Waldenstroms macroglobulinemia, non-Hodgkin lymphoma, chronic lymphocytic leukemia, etc.

{May be found in normal persons{May be found in normal personsz Incidence increases with age.

z RF is not found in all patients with RA. {10-30% of patients dont produce RF.{Many tests detect IgM ab, patients may produce RFs

of other classes.

Autoantibodies- Thyroid Antibodies

z Thyroid consists of spherical units called follicles, which are lined with cuboidalepithelial cells and filled with colloid. z Components of thyroid which may function as

antigens to which antibodies are produced:antigens to which antibodies are produced:{ThyroglobulinzPrimary constituent of colloid. zMade up of hormones T3 and T4.

{Thyroid peroxidase (TPO) (microsomes)z In cytoplasm of epithelial cells.

{Second Colloid Ag (CA 2)zColloid protein in follicles

Tests to Detect Thyroid Ab

zPassive hemagglutination test (tanned red cell agglutination or chromic chloride hemagglutination test)z Indirect immunofluorescence assayz Indirect immunofluorescence assayzELISA

Tanned Red Cell Agglutination (TRCA) for anti-thyroglobulin

zPrinciple {Passive hemagglutination test.{Antigen= thyroglobulinzTh roglob lin e tracted from h man th roid glands iszThyroglobulin extracted from human thyroid glands is

absorbed to tanned turkey red cells (cells treated with tannic acid). zCells treated with choline chloride may also be used

{Antibody= anti-thyroglobulin.{If ab are present in the patients serum, they will

react with ag on rbcs resulting in hemagglutination.

z Procedure (microtiter method) (Thymune-T){Serially dilute patients serum, add TRC coated with

thyroglobulin, incubate ~30 minutes{Observe for hemagglutination

7TRCA Test: Interpretationz Titers of 20 or more are considered

significant (interpretations vary).z Ab to thyroglobulin are found in patients with

Hashimotos thyroiditis, myxedema, Graves disease and thyroid tumors.z~80% of patients with Hashimotos disease have

titers of 1,000 or more.zPatients with myxedema show elevated titers, but

not as high.zHigh titers are found in only about 10% of patients

with Graves disease.

TRCA Test: Interpretation

z Thyroid ab have been observed in sera of patients with other conditions{of the thyroid such as hyperthyroidism and tumors.{such as pernicious anemia, diabetes mellitus, and

adrenal insufficiency.

z Lower titers of ab may be found in normalz Lower titers of ab may be found in normal individuals{ Incidence higher in women{ Incidence increases with age.

z Absence of anti-thyroglobulin antibodies does not exclude a diagnosis of Hashimotos thyroiditis.

TRCA Test for anti-thyroid peroxidase

z TRCA (TCH) or CCH test is also available for detecting anti-TPO ab.z 90-95% of patients with Hashimotos thyroiditis

have anti-TPOz T t f ti th l b li d ti TPO h ldz Tests for anti-thyroglobulin and anti-TPO should

be done in suspected Hashimotos thyroiditis. {Combination will detect practically all cases {Some produce ab to one, but not other

z 75% of patients with Graves disease produce anti-TPO.

Indirect IFA for Thyroid Ab

z For anti-thyroglobulin{Ag = methanol - or acetone - fixed human or monkey

thyroid gland. {Ag (on slide) + Serum (Ab) + AHG labeled FITC

floccular puffy fluorescence.{Test is less sensitive than TRCA, but can detect{Test is less sensitive than TRCA, but can detect

nonagglutinating antibodies missed by the hemagglutination tests.

z For anti-TPO antibodies{Ag = unfixed monkey or human thyroid gland.{Positive = fluorescence of cytoplasm of thyroid

epithelial cells.

Positive Test for anti-microsomal (TPO) ab- fluorescence in cytoplasm of cells only ELISA Tests for Thyroid AB

zCommercial kits for detecting thyroglobulin antibodies are available. zTests are very sensitive.

T t d t t b th l ti ti dzTests can detect both agglutinating and nonagglutinating antibodies.

8Antinuclear Antibodies (ANAs): Characteristics

zHeterogeneous group of circulating immunoglobulins that react with antigenic sites on nuclear components {Most are IgG, but can be of any class.

zNo organ or species specificity. {Cross react with nuclear materials fromzOther humans (ex. human epithelial cells)zVarious animal tissue (ex.rat liver, mouse kidney).

Autoantibodies- Antinuclear Antibodies (ANAs): Characteristics

zFound in serum of patients with various systemic autoimmune disorders. {Antibodies that react with specific nuclear

components are characteristic of certain pdiseases.

zAlso may be found in {Other conditions- autoimmune liver disease,

certain viral infections, persons on certain drugs, etc{Healthy elderly and occasionally nonelderly

persons

Methods to Screen for ANAs: Indirect IFA or FANA

zMost common methodzPrinciple:{Antibody- antinuclear antibodies (ANAs).{Antigen nuclear antigens{Antigen- nuclear antigens zprovided by tissue substrate such as mouse or rat

kidney or liver, or human epithelial cells (hep-2).{Serum is added to tissue substrate on a

microscopic slide. After washing, FITC-labeled anti-human immunoglobulin (AHG) is added. The slide is observed using a fluorescent microscope.

FANA: ResultszPositive test{Fluorescence seen in nuclei of cells of tissue

substrate. {Nuclear staining patterns depend on location

of antigen to which ANAs are reacting. zResults reported with titer and pattern p p{Original serum dilutions tested (ex. 1:40 and

1:80). {Positive specimens serially diluted zTiter of antibodies is reciprocal of highest dilution

showing the pattern.{Patient can have more than one pattern

(different antibodies) with different titers.

FANA: Results

zLow titers (40-80) are considered suspicious (borderline){Often are false positive reactions.{Some labs consider 40 or less negative and 80{Some labs consider 40 or less negative and 80

borderline. zTiters of > 160 are considered positive.{Strongly suggestive of SLE. {ANAs may also be found in other diseases.

9Staining Patterns (on Hep-2 cells)

zHomogeneous (Diffuse){Uniform fluorescence of entire nucleus with or without

masking of nucleoli.{Chromosome region of metaphase mitotic cells{Chromosome region of metaphase mitotic cells

positive with smooth or peripheral staining intensity greater than or equal to interphase nuclei.

zPeripheral (Rim, outline, halo){Fluorescence circling around outer edge of

nucleus{Chromosome region of metaphase mitotic cells

clearly positive with smooth or peripheralclearly positive with smooth or peripheral staining intensity greater than, or equal to, interphase nuclei{Often seen with homogeneous pattern in which

case entire nucleus may fluoresce with brighter ring around edge.

PeripheralHomogeneous

z Speckled{Discrete fluorescent speckles ranging in size from small

to large seen throughout nucleus generally without staining of the nucleoli.{Nonchromosome region of metaphase mitotic cells

demonstrates staining while chromosome region isdemonstrates staining, while chromosome region is negative.

z Nucleolar{Large coarse speckled staining within nucleus, generally

less than 6 in number per cell, with or without occasional fine speckles. (5-10).{Nonchromosome region of metaphase mitotic cells

demonstrates strong staining, while chromosome region may demonstrate faint staining.

Speckled Nucleolar

zAnti-centromere{Fluorescence appears in a discrete speckled

pattern on interphase and metaphase chromatin of cells.{Mitotic cells will show the same speckling

reaction in chromosome region

10

Anti-centromere

Immunoenzyme Test for ANAs

Pattern AutoAb Antigen Disease

Homogeneous Anti-dsDNAAnti-ssDNAAnti-histone

Anti-DNP

Native or ds-DNADenatured DNANucleoprotein, major constituent chromatinDeoxynucleoprotein (DNA-histone complex)

SLE (high titers)SLE, drug-induced, RASLE, drug-induced, RA

SLE, drug-induced , other

Peripheral Anti-dsDNAAnti-ssDNA

See above SLE predominantly

Speckled Anti-Sm RNA component of SLEp

Anti-RNPAnti-SS-A (Ro)

Anti-SS-B (La)

Anti-Scl-70

pextractable nuclear agRibonuclear proteinProteins complexed RNA

Phosphoprotein-RNA polymerase DNA topoisomerase I

MCTD, SLE Sjogrens syndrome (SS), SLESS, SLE

Scleroderma

Nucleolar Anti-nucleolar Nucleolar protein or RNA, 4-6s RNP or RNA

SLE, scleroderma, SS

11

Advantages of ANA Test

zVery sensitive {Used as screening test for SLE and other

systemic rheumatic diseases.{Typically a negative test rules out SLE{Typically a negative test rules out SLE

zRelative ease of performancezDetects a wide range of ANAs.zLarge number of commercially prepared

kits are available.

Disadvantages of ANA Test

zLacks specificity (other tests have to be done to define antibody specificity).zFalse negatives may occur with certain

rheumatic diseases zFalse positives may be seen {in other conditions{with cell culture substrates due to the presence

of passenger viruses.

Follow-Up Testing for Positive ANAs

zTests for anti-DNA{Done when homogeneous or peripheral

pattern is seen, or high titer (>160) of any pattern is obtained (SLE is suspected).{Methods{MethodszCrithidia luciliae Immunofluorescent TestzFarr RIA ProcedurezELISA TestszAthena Multi-Lyte Test System (Wampole)

{Note: Tests for DNP are also availablezLatex agglutination test kitszDetected by old LE cell prep

Crithidia luciliae Test

z Principle {Type of test- indirect immunofluorescence {Ag- kinetoplast (composed of ds or native DNA) of

nonpathogenic hemoflagellate Crithidia luciliae. {Ab- anti-dsDNA{Patients serum added to organism on slide. If anti-

dsDNA ab are present in serum, ab will attach to kinetoplast. After washing, FITC-labeled AHG added. Kinetoplast will fluoresce.

z Procedure can also be done by the indirect immunoenzyme method.

12

RIA (Farr)Procedure

ELISA Assay

substrate

Uses of Anti-DNA Tests

zDiagnosis of SLE (ab to ds DNA are highly specific for SLE).z Monitor disease progression and

response to therapy in SLE patientsresponse to therapy in SLE patients.

Tests for anti-ENAs

z Tests for anti-ENAs are performed when speckled patterns are seen on ANA tests.z ENAs are extractible nuclear antigens{Ags present in saline solution extracts of mammalian

tissue (ex. Calf thymus).{Most common ENAs: Sm (Smith) and RNP{Most common ENAs: Sm (Smith) and RNP

(ribonucleoprotein).{Other ENAs: SS-A, SS-B, Scl-70, and Jo-1

zMethods {Passive Hemagglutination{Ouchterlony Double Diffusion{ELISA{Athena

Passive Hemagglutination Test

zPrinciple - Tanned sheep erythrocytes (SRBC) are coated with extract of rabbit thymus containing Sm and RNP antigens. A portion of the cells are incubated with RNAse to selectivelyincubated with RNAse to selectively remove RNP antigen. A passive hemagglutination test is performed in a microtiter system, using parallel dilutions of the patients serum against both untreated and RNAse treated cells.

Passive Hemagglutination Test-Results

Antibody present Treated/ Sm antigen only

Untreated/Sm and RNP

Anti-Sm HA HA

Anti-RNP No reaction

HA

Anti-RNP/Anti-Sm HA HA at a higher titer

13

Ouchterlony Double Diffusion

z Principle - A solution of extractible nuclear antigens is placed in central well of an agarose plate. Patient samples and controls are placed in surrounding wells (such that each patient is next to a control). Antigens and antibodies diffuse through the agarose and form a visible line of precipitate (line of identity with control) if sample contains antibodies to any of antigens present. No precipitate forms in absence of specific antibody.

Ouchterlony Patterns

Pattern of IdentityPattern of non-identity

Pattern of partial identity

Summary of Ouchterlony

Anti-Sm Line of identity with anti-Sm control/ line of partial identity with anti-RNP control

Anti-RNP Line of identity with anti-RNP control

Anti-RNP/ anti-Sm Two lines of precipitation are formed/ one with each positive control

Unknown X

undiluted

Sm + control

Unknown X

diluted

RNP + control

Sm + control

Unknown X

undiluted

Unknown X

diluted

RNP + control

A B

Spur

ENAs

RNP + control

Sm + control

Unknown X

undiluted

Unknown X

diluted

C

Spur

Clinical Significance of anti-ENAsz Anti-Sm- marker ab for SLEz Anti-RNP- high titers seen in MCTD or SLE,

increased titers alone suspect MCTDz Anti-SS-A- marker for Sjogrens syndrome,

found in ~40-50% cases of SLE (usually negative ANA)z Anti-SS-B- marker for Sjogren s syndrome,

found in ~15% cases of SLEz Anti-Scl-70- marker for sclerodermaz Anti-Jo-1- marker for

polymyositis/dermatomyositis (negative ANA)

14

Athena Multi-lyte Test System

z Multiplexed, microsphere-based immunoassay

z Polystyrene micro-particles of uniform size used as the solid phasep{ Beads are dyed using a defined combination

of two dyes.{ Results in sets of beads

z Unique bead sets can be conjugated with various, unique target molecules of interest (antigens- ssDNA, RNP, etc).

Athena multi-lyte test system

AHG-FITC

Athena Multi-lyteTest System

z Beads flow through the flow cellz Light scatter will determine

discrete events.{One laser identifies the amount of

reporter (FITC) on the surface.p ( ){The other determines the amount of

classification dye (red), which identifies the bead set (ie. Ag)

z Analysis of the beads occurs at a rate of up to 20,000 beads/per second.

z http://www.invernessmedicalpd.com/clinical/AtheNA-How-It-Works.asp

Specific Autoimmune Diseasesz Read about the following diseases in the textbook,

answer related review questions{Systemic lupus erythematosus{Rheumatoid arthritis{Hashimotos thyroiditis{Graves disease{Type 1 Diabetes Mellitus{Myasthenia gravis{Myasthenia gravis{Goodpastures syndrome{Multiple sclerosis{Addisons disease (just ab involved, chart on p 224){Pernicious anemia (chart){Scleroderma (chart){Sjogrens syndrome (chart){Chronic active hepatitis- know ab is anti-smooth muscle{Primary biliary cirrhosis- know ab is anti-mitochondria

Additional Information

zReview Questions textbook{Chapt 14 (p 232) #1-10,12

zC St di ( 228)zCase Studies (page 228)