Auto Immune Diseases Autoimmune Diseases - PowerPoint Presentation

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Auto Immune Diseases

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Autoimmune Diseases

Autoimmune disease is a group of disorders in which tissue injury is caused by humeral (by auto-antibodies) or cells mediated immune response (by auto reactive T cells) to self antigens.

Normal, the immune system does not attach the self. However, there is a large

g

roup of autoimmune diseases in which the immune system does attack self-cells.

The attack can be directed either against a very specific tissue or to a large no. of tissues.

Once started, autoimmune disease are hard to stop.

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Causes of Autoimmune Diseases

Sequestered or Hidden antigens.

Ag in the secluded places – are not accessible to the immune system.

E.G. Lens Ag, Sperm Ag and Thyroglobulin.

Neo Antigens.

Altered or Modified Antigens –by physical (irradiation), chemical (drugs) or microbial agents (intracellular viruses)

Cessation of Tolerance

It may

result

when tolerance to the self Ag in abrogated

.

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Cross reacting Antigens

A foreign Ag which resembles self a 2nd Ag.

Many Species share organ specific Ags.

E.g. Ag of Human brain and Ag of sheep brain, streptococcal M protein and heart muscles, Nephritogenic strains of streptococci Ags and Renal glomeruli shares similar epitopes.

Loss

of Immunoregulation.

Loss of Self tolerance – caused by over activity or

l

owered activity of T and B-cells.

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Classification of Autoimmune Disease

Hemolytic autoimmune diseases

Localized autoimmune disease.

Systemic autoimmune diseases

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Hemolytic autoimmune diseases

Clinical disorder due to destructions of blood components, auto Ab are formed against one’s own RBCs, Platelets or leucocytes.

E.g.:

Haemolytic

Anemia, Leucopenia,

Throbocytopenia

.

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Localized autoimmune diseases.

Called also Organ specific autoimmune diseases.

A particular organ is affected due to auto Abs.

For Example:

Thyroiditis (attack the Thyroid).

Multiple sclerosis (attacks myelin coating of nerve axons).

Myasthenia gravis (attacks nerve-muscle junction).

Juvenile diabetes or Type I DM (attack insulin-producing cells).

Celiac disease.

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Systemic autoimmune diseases.

Called also Non-organ specific autoimmune diseases.

Immune complexes accumulate in many tissue and cause inflammation and damage.

Affects many organs or the whole body.

For Examples:

Systemic Lupus Erythematous (anti-nuclear Ab); Harms kidneys, heart, brain, lungs, skin,……

Rheumatoid Arthritis (anti-

IgG

antibodies); Joints, hearts, lungs, nervous

Rheumatic fever: cross-reaction between antibodies to streptococcus and auto-antibodies.

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Celiac disease

Celiac disease

 -- also known

as

celiac

 sprue

or

gluten-sensitive enteropathy

-- is a digestive and autoimmune disorder that results in damage to the lining of the small intestine when foods with gluten are eaten.

Gluten

is a form of protein found in some grains.

The

damage to the intestine makes it hard for the body to absorb nutrients, especially fat

, calcium

, iron, and folate

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What Causes Celiac Disease?

When

people with celiac disease eat foods containing gluten, their immune system forms antibodies to gluten which then attack the intestinal lining.

This

causes inflammation in the intestines and damages the villi, the hair-like structures on the lining of the small intestine

.

Nutrients

 from food are normally absorbed by the villi. If the villi are damaged, the person cannot absorb nutrients properly and ends up malnourished, no matter how much he or she eats.

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Symptoms

of Celiac

Disease

Symptoms

of celiac disease vary among sufferers and include:

Digestive problems (abdominal bloating, pain, gas, diarrhea, pale stools, and weight loss)

A severe skin rash called dermatitis

herpetiformis

Iron deficiency anemia (low blood count)

Musculoskeletal problems (muscle cramps, joint and bone pain)

Growth problems and failure to thrive (in children)

Seizures

Tingling sensation in the legs (caused by nerve damage and low calcium)

Aphthous

ulcers (sores in the mouth)

Missed menstrual periods

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Health Problems Accompany Celiac Disease

Celiac

disease can leave a person susceptible to other health problems, including:

Osteoporosis, a disease that weakens bones and leads to fractures. This occurs because the person has trouble absorbing enough calcium and vitamin D.

Miscarriage or infertility.

Birth defects, such as neural tube defects (improper formation of the spine) caused by poor absorption of such nutrients as folic acid.

Seizures.

Growth problems in children because they don't absorb enough nutrients.

Cancer of the intestine (very rare).

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Diagnosis

The two major steps leading to diagnosis of celiac disease are:

Lab. tests

for gluten autoantibodies (These are IgA based tests accurate only while on a gluten-containing diet)

A

small bowel biopsy to assess gut damage. For those with suspected dermatitis

herpetiformis

, skin biopsies will be taken of the skin near the lesion.

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Lab diagnosis

Anti-tissue transglutaminase antibody (anti-

tTG

), IgA:

Detects

antibodies to tissue transglutaminase, an enzyme that causes the crosslinking of certain proteins.

Anti-

tTG

, IgA is the most sensitive and specific blood test for celiac disease.

The

IgG class of anti-

tTG

may be ordered for people who have a deficiency of IgA

.

Deamidated

gliadin peptide (DGP) antibodies, IgA:

Detects anti-DGP

IgA antibodies; like anti-

tTG

, the IgG class may be performed for a person with an IgA deficiency

.

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Other tests less commonly performed include:

Anti-

endomysial

antibodies (EMA), IgA class:

Detects antibodies to endomysium, the thin connective tissue layer that covers individual muscle fibers

Anti-

reticulin

antibodies (ARA), IgA class: not as specific or sensitive as the other autoantibodi

es

Lab diagnosis

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Diagnosis of Auto-Immune Disease

Diagnosed by Clinical symptoms

Confirmed by detecting the auto Ab in the serum of the patients.

Autoantibodies are demonstrated by immunoflurescent Ab test , Haemagglutination, Complement fixation, immundodiffusion, Radio immuno assay, etc.

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Immunofluorescence assay

Immunofluorescence is a technique allowing the visualization of a specific protein or antigen in tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye such as fluorescein isothiocyanante (FITC)

The specific antibodies are labeled with a compound (FITC) that makes them glow an apple-green color when observed microscopically under ultraviolet light.

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Fluorescence

is the property of certain molecules of fluorophores to absorb light at one wave length and emit light at longer wave length (emission wavelength) when it is illuminated by light of a different wave length (excitation wavelength).

The incident light excites the molecule to a higher level of vibration energy. As the molecules return to the ground state, the excited fluorophores emits a photon (=fluorescence emission).

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Type of Immunofluorescence

Direct immunofluorescence : Staining in which the primary antibody is labeled with fluorescence dye.

Indirect immunofluorescence: staining in which a secondary antibody labeled with fluorochome is used to recognize a primary antibody.

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Direct Immunofluorescence:

It is a simple procedure.

Ag is fixed on the slide

Fluorescein labeled

Ab’s

are layered over it.

Slide is washed to remove unattached

Ab’s

.

Examined under UV light using fluorescent microscope .

The site where the Ab attached to its specific Ag will show apple green fluorescence.

Use: direct detection of pathogens or their Ag’s tissues or in pathological samples.

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Indirect Immunofluorescence

Indirect test is a double-layer technique.

The unlabeled antibody is applied directly to the tissue substrate.

Treated with a fluorochome-conjugated anti-immunoglobulin serum

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Advangate

of indirect

Gives an amplification effect – more tag or label (signal) per molecule of target protein.

Requires only one labeled antibody to identify many protein– same labeled secondary antibody can be used to bind to (light up) many different proteins ( preparation of labeled antibody is difficult and expensive.

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Systemic Lupus

Erythematosus

(SLE)

It is a skin disease due to the production of anti-nuclear factor (ANF) or anti-nuclear auto Ab.

ANF reacts with the breakdown products of nuclei in the normal wear and tear of cells and form immune complexes which cause the tissue damage.

In these patients, LE cell ( a mature neutrophil) appears in blood and bone marrow.

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Characteristics of SLE

Appearance of blood red spots over the bridge of nose and cheeks. The lesion take the shape of a butterfly.

Connective tissue of the skin, kidney, heart, spleen and blood vessels are severely damaged resulting in joint pain, fever and anemia.

Glomerulonephritis due to deposition of immune complex in the glomerulus region.

It is a systemic disease affecting the whole body.

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Malar Rash (SLE)

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LE cells

the

LE cell reaction is positive in 50%-75% of individuals with acute disseminated lupus.

Positive reactions are also

seen in rheumatoid arthritis, chronic hepatitis, acquired hemolytic anemia, and Hodgkin disease.

It may also be positive in persons taking

phenylbutazone

and hydralazine.

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LE cells

An LE cell is either a neutrophil or a macrophage that has engulfed (phagocytized) degraded nuclear material from another cell. The engulfed nuclear material takes up

Haematoxylin

stain strongly; this strongly-stained engulfed nuclear material is called LE body

Detection of LE cell is made through microscopic examination.

The

test was commonly used in the past to diagnose systemic lupus erythematosus. But currently, SLE is diagnosed by more sensitive and specific tests such as anti-nuclear antibody (ANA) blood test

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Diagnosis of SLE

The

American

College of Rheumatology (ACR) criteria summarized features necessary to diagnose

SLE at 1982.

 

These

criteria were last updated in

1997.

 

The

presence of 4 of the 11 criteria yields a sensitivity of 85% and a specificity of 95% for SLE

Patients

with SLE may present with any combination of clinical features and serologic evidence of lupus

.

The Systemic Lupus International Collaborating Clinics (SLICC) group revised and validated the ACR SLE classification criteria in 2012

.

According to the revision, a patient is classified as having SLE if the patient has biopsy-proven lupus nephritis with ANA or anti-

dsDNA

antibodies or if the patient satisfies 4 of the diagnostic criteria, including at least 1 clinical and 1 immunologic criterion.

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ACR

diagnostic

criteria in SLE

Serositis

- Pleurisy, pericarditis on examination or diagnostic electrocardiogram (ECG) or imaging

Oral ulcers - Oral or nasopharyngeal, usually painless; palate is most specific

Arthritis -

Nonerosive

, 2 or more peripheral joints with tenderness or swelling

Photosensitivity - Unusual skin reaction to light exposure

Blood disorders - Leukopenia (< 4 × 10 

3

 cells/µL on >1 occasion),

lymphopenia

(< 1500 cells/µL on >1 occasion), thrombocytopenia (< 100 × 10 

3

 cells/µL in the absence of offending medications), hemolytic

anemia

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ACR

diagnostic

criteria in SLE

Renal

involvement – Based on presence of proteinuria (>0.5 g/day or 3+ positive on dipstick testing) or cellular casts (including red blood cells [RBCs], hemoglobin, granular, tubular, or mixed) 

 

or based on the opinion of a rheumatologist or nephrologist 

Neurologic disorder - Seizures or psychosis in the absence of other

causes

Malar rash - Fixed erythema over the cheeks and nasal bridge, flat or raised

Discoid rash - Erythematous raised-rimmed lesions with

keratotic

scaling and follicular plugging, often scarring

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Antinuclear

antibodies (ANAs) - Higher titers generally more specific (>1:160); must be in the absence of medications associated with drug-induced lupus

Immunologic

phenomena -

dsDNA

; anti-Smith (Sm) antibodies;

antiphospholipid

antibodies (

anticardiolipin

immunoglobulin G [

IgG

] or immunoglobulin M [

IgM

] or lupus anticoagulant); biologic false-positive serologic test results for syphilis, lupus erythematosus (LE) cells (omitted in 1997 revised criteria

)

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In

patients with high clinical suspicion and/or high ANA titers, additional testing is indicated.

This

commonly includes evaluation of antibodies to

dsDNA

, complement, and ANA subtypes such as Sm, SSA, SSB, and

ribonucleoprotein

(RNP) (often called the ENA panel), as well as screening

anticardiolipin

antibodies, lupus anticoagulant, and +/- beta-2 glycoprotein antibodies.

ENA :Extractable

Nuclear Antigen Antibodies

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Serological tests for SLE

Test

Description

ANA

Screening test; sensitivity 95%; not diagnostic without clinical features

Anti-dsDNA

High specificity; sensitivity only 70%; level is variable based on disease activity

Anti-Sm

Most specific antibody for SLE; only 30-40% sensitivity

Anti-SSA (Ro) or Anti-SSB (La)

Present in 15% of patients with SLE and other connective-tissue diseases such as 

Sjögren

syndrome

; associated with 

neonatal lupus

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Serological tests for SLE

Anti-ribosomal P

Uncommon antibodies that may correlate with risk for CNS disease, including increased hazards of psychosis in a large inception cohort, although the exact role in clinical diagnosis is

debated

Anti-RNP

Included with anti-Sm, SSA, and SSB in the ENA profile; may indicate mixed connective-tissue disease with overlap SLE, 

scleroderma

, and myositis

Anticardiolipin

IgG

/

IgM

variants measured with ELISA are among the

antiphospholipid

antibodies used to screen for

antiphospholipid

antibody syndrome and pertinent in SLE diagnosis

Lupus anticoagulant

Multiple tests (

eg

, direct Russell viper venom test) to screen for inhibitors in the clotting cascade in

antiphospholipid

antibody syndrome

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Serological tests for SLE

Direct Coombs test

Coombs test–positive anemia to denote antibodies on RBCs

Anti-histone

Drug-induced lupus ANA antibodies are often of this type (

eg

, with procainamide or hydralazine; p-ANCA–positive in minocycline-induced drug-induced lupus)

ANA

= antinuclear antibody; CNS = central nervous system; ds-DNA = double-stranded DNA; ELISA = enzyme-linked immunoassay; ENA = extractable nuclear antigen; Ig = immunoglobulin; p-ANCA =

perinuclear

antineutrophil

cytoplasmic antibody; RBCs = red blood cells; RNP = ribonucleic protein; SLE = systemic lupus erythematosus; Sm = Smith; SSA =

Sjögren

syndrome A; SSB =

Sjögren

syndrome B.

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Other useful tests in suspected SLE

Standard laboratory studies that are diagnostically useful when systemic lupus erythematosus (SLE) is suspected should include the following:

Complete blood count (CBC) with differential

Serum creatinine

Urinalysis with microscopy

The CBC count may help screen for leukopenia,

lymphopenia

, anemia, and thrombocytopenia. Urinalysis and creatinine studies may be useful to screen for kidney disease.

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Levels of inflammatory markers, including the ESR and CRP, may be elevated in any inflammatory condition, including SLE.

Measurement

of complement may be useful, because C3 and C4 levels are often depressed in patients with active SLE as a result of consumption by immune complex–induced inflammation. In addition, some patients have congenital complement deficiency that predisposes them to SLE

.

Other useful tests in suspected SLE

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Indirect immunofluorescence assay:

A laboratory test used to detect antibodies in serum or other body fluid.

Example of autoantibodies

Anti dsDNA Abs.

ANA

APA

ASMA

AMA

Anti LKM

ANCA

Antithyroid Abs

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Substrate

Autoantibodies are detected on specific substrates:

Autoantibodies

Substrate

Anti-dsDNA

on

Crithedia

Lucilae

substrate

ANA

on Hep-2

substrate

or on Mouse

stomach kidney substrate

APA

ASMA

AMA

on mouse stomach kidney

substrate

Anti LKM

on mouse Liver stomach kidney

substrate

ANCA

on

neutrophil substrate

Anti Thyroid Abs

on

thyroid tissue substrate

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Advantage of Hep2 cells over rodent tissue

Higher sensitivity (greater Ag expression)

Human origin ensure better specificity.

Cell division rates are higher so cell cycle dependent Ab are easily identified.

Nucleus are much larger, visible and complete

nucleolar

detail can be seen.

Ags distribution are uniform not obscuring intercellular matrix.

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Staining Patterns:

Diffuse ? Homogeneous: antibodies to histone

Rim: antibodies to nuclear envelope proteins and to dsDNA.

Speckled: antibodies to SM,RNP, Ro/SS-A, LA/SS-B, and other antigens.

Nucleolar: associated with diffuse scleroderma

Contromeric : highly specific to the CREST Syndrome.

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Thank you