Transcript lecture8-Autoimmunity-and-Fluoresence

AUTO IMMUNE
DISEASES
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 attack the self. However,
there is a large group 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.
Causes of Autoimmune Diseases
Sequestered or Hidden antigens.
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Ag in the secluded places – are not accessible to the immune
system.
E.G. Lens Ag, Sperm Ag and Thyroglobulin.
Neo Antigens.
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Altered or Modified Antigens –by physical (irradiation),
chemical (drugs) or microbial agents (intracellular viruses)
Cessation of Tolerance
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It may result when tolerance to the self Ag in abrogated.
Cross reacting Antigens
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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.
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Loss of Self tolerance – caused by over activity or lowered
activity of T and B-cells.
Classification of Autoimmune Disease
1. Hemolytic autoimmune diseases
2. Localized autoimmune disease.
3. Systemic autoimmune diseases
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.
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.
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.
Autoimmune Hemolytic anemia.
• Lysis of RBCs is due the production of autoantibodies against
the RBC –antigens
• RBC half life=100 days, HA. Anemia < 7 days.
• Caused by -20 to infections or drug therapy (penicillin, anti-
hypertensive agent like methylodpa results in destruction of
RBCs).
• Antibody mediated autoimmune disease.
• 2 classes of autoantibodies involved are:
• IgM or cold Agglutins – active at 4o C but not at 37oC
• IgG or warm Agglutins – active at 37o C but not at 4o C>
Thrombocytopenia
• Characterized by low platelet count due to the production
of antiplatelet Ab. (IgG type)
• Mechanism:
• An interaction of Ab with bound drug or new Ag. Causes
intravascular agglutination of platelets and can be eliminated by
phagocytic cells.
• Antibody mediated autoimmune diseases.
Thyrotoxicosis or Grave’s disease
• The Ab (IgG type) is directed against the receptor for
thyroid stimulatory hormone (TSH).
• This Ab is called as Long Acting thyroid stimulator (LATS)
or thyroid stimulating Ab (TSab).
• Primary causes: stimulation of Thyroid gland to secrete
more TSH (Hyperthyroidism) resulting in Exophthalmos,
bulging eyes and Goiter.
Graves’ disease
(anti-thyroid stimulating hormone; anti-TSH)
• In Graves’ disease, the
antibodies do not
destroy the thyroid but
act as if they are TSH
(i.e., they bind and
activate the TSH
receptor)
Hashimoto’s thyroiditis
• Atrophy of thyroid gland, which results in hypothyroidism
and destruction of thyroid fun.
• Characterized by
• Goitre, enlarged thyroid gland, deficiency of TH (Thyroxin)
• Type IV hypersensitive Rxns.
• Caused by auto Ab of IgG and IgM type against the
constituents of thyroid gland (Thyroid Epithelial cells,
colloid and nuclear components)
• It is a T-cell associated auto immune disease.
Rheumatoid Arthritis
• It is a chronic systemic disease of the joints.
• Caused by the auto antibody of IgM type, called as
rheumatoid factors.
• Characteristics:
• The synovial fluid of these patients contain increased no of T cells
and macrophages.
• Marked by inflammatory changes in the synovial membrane and by
atrophy of bones .
• In later stage, deformity and ankylosis develops
Rheumatoid Arithritis
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
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.
Symptoms of Celiac Disease
• Symptoms of celiac disease vary among sufferers and include:
• Digestive problems (abdominal swelling, pain, gas, diarrhea, pale stools,
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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)
Tingling sensation in the legs (caused by nerve damage and low calcium)
Aphthous ulcers (sores in the mouth)
Missed menstrual periods
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
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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.
Growth problems in children because they don't absorb enough nutrients.
Cancer of the intestine (very rare).
Diagnosis
• The two major steps leading to diagnosis of celiac disease are:
1. 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.
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.
Lab diagnosis
• 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 autoantibodies
Addison’s Disease
• Is due to adrenocortical damage and hence insufficient
secretion of adrenal hormones.
• Tissue damage is caused by auto Ab against zona
glomerulosa cells of adrenal cortex.
Myasthemia Gravis
• It is caused by auto antibody against muscle antigen and
acetylcholine receptor antigen.
• Characterized by increased muscular weakness that
make one fatigue. Speaking, eating and walking become
tired some.
• Eventually death from respiratory failure.
• Acetylcholine can’t be produced, thus nerve impulse can’t
be transmitted form the nerves to the muscles, hence the
neuromuscular junction is severely affected
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.
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.
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
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.
Malar Rash (SLE)
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.
Treatment
• Some autoimmune diseases are treated with medications
that alleviate specific symptoms.
• Hemolytic anemia treated with Vit-B12
• Thyrotoxicosis treated with anti-thyroid drugs.
• Myasthenia Gravis treated with choline esterase inhibitors.
• Rheumatoid Arthritis treated with anti-inflammatory drugs.
• SLE treated with Immuno-suppressive or anti-mitiotic drugs such as
Corticosteroid, cyclophosphamide and azothioprine.
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.
• 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).
Type of Immunofluorescence
1) Direct immunofluorescence : Staining in which the
primary antibody is labeled with fluorescence dye.
2) Indirect immunofluorescence: staining in which a
secondary antibody labeled with fluorochome is used to
recognize a primary antibody.
Direct Immunofluorescence:
• It is a simple procedure.
• Ag is fixed on the slide
• Fluorescein labeled Ab’s are layered over
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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.
Indirect Immunofluorescence
• Indirect test is a double-layer technique.
• The unlabeled antibody is applied directly to the tissue
substrate.
• Treated with a fluorochome-conjugated antiimmunoglobulin serum
Subtypes of ANAs
• There are many subtypes of ANAs such as,
• Anti-Ro Abs
• Ant-La
• Anti-Sm
• Anti-nRNP
• Anti-Scl-70
• Anit-histone
• Antibodies to nuclear pore complexes
• Anti-centromere antibodies
• Anti-sp100 antibodies.
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
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
Advantage of Hep2 cells over rodent tissue
1. Higher sensitivity (greater Ag expression)
2. Human origin ensure better specificity.
3. Cell division rates are higher so cell cycle dependent Ab
are easily identified.
4. Nucleus are much larger, visible and complete nucleolar
detail can be seen.
5. Ags distribution are uniform not obscuring intercellular
matrix.
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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