AUTO IMMUNE DISEASES Autoimmune Diseases Autoimmune disease is

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AUTO IMMUNE DISEASES

AUTO IMMUNE DISEASES

Autoimmune Diseases • Autoimmune disease is a group of disorders in which tissue injury

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 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 1. Sequestered or Hidden antigens. § Ag in the secluded

Causes of Autoimmune Diseases 1. Sequestered or Hidden antigens. § Ag in the secluded places – are not accessible to the immune system. § E. G. Lens Ag, Sperm Ag and Thyroglobulin. 2. Neo Antigens. § Altered or Modified Antigens –by physical (irradiation), chemical (drugs) or microbial agents (intracellular viruses) 3. Cessation of Tolerance § It may result when tolerance to the self Ag in abrogated.

4. Cross reacting Antigens § A foreign Ag which resembles self a 2 nd

4. Cross reacting Antigens § A foreign Ag which resembles self a 2 nd 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. 5. Loss of Immunoregulation. § 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

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

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

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

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-Ig. G antibodies); Joints, hearts, lungs, nervous • Rheumatic fever: cross-reaction between antibodies to streptococcus and auto-antibodies.

Celiac disease • Celiac disease -- also known as celiac sprue or gluten-sensitive enteropathy

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,

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:

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

Health Problems Accompany Celiac Disease • Celiac disease can leave a person susceptible to

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).

Diagnosis • The two major steps leading to diagnosis of celiac disease are: 1.

Diagnosis • The two major steps leading to diagnosis of celiac disease are: 1. Lab. tests for gluten autoantibodies (These are Ig. A based tests accurate only while on a gluten -containing diet) 2. 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-t. TG), Ig. A: • Detects antibodies to

Lab diagnosis • Anti-tissue transglutaminase antibody (anti-t. TG), Ig. A: • Detects antibodies to tissue transglutaminase, an enzyme that causes the crosslinking of certain proteins. • Anti-t. TG, Ig. A is the most sensitive and specific blood test for celiac disease. • The Ig. G class of anti-t. TG may be ordered for people who have a deficiency of Ig. A. • Deamidated gliadin peptide (DGP) antibodies, Ig. A: Detects anti-DGP Ig. A antibodies; like anti-t. TG, the Ig. G class may be performed for a person with an Ig. A deficiency.

Lab diagnosis • Other tests less commonly performed include: • Anti-endomysial antibodies (EMA), Ig.

Lab diagnosis • Other tests less commonly performed include: • Anti-endomysial antibodies (EMA), Ig. A class: Detects antibodies to endomysium, the thin connective tissue layer that covers individual muscle fibers • Anti-reticulin antibodies (ARA), Ig. A class: not as specific or sensitive as the other autoantibodies

Diagnosis of Auto-Immune Disease • Diagnosed by Clinical symptoms • Confirmed by detecting the

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.

Immunofluorescence assay • Immunofluorescence is a technique allowing the visualization of a specific protein

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

• 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

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

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.

Indirect Immunofluorescence • Indirect test is a double-layer technique. • The unlabeled antibody is

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

Advangate of indirect 1. Gives an amplification effect – more tag or label (signal)

Advangate of indirect 1. Gives an amplification effect – more tag or label (signal) per molecule of target protein. 2. 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.

Systemic Lupus Erythematosus (SLE) • It is a skin disease due to the production

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.

Characteristics of SLE • Appearance of blood red spots over the bridge of nose

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)

Malar Rash (SLE)

LE cells • the LE cell reaction is positive in 50%-75% of individuals with

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

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

Diagnosis of SLE • The American College of Rheumatology (ACR) criteria summarized features necessary

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

ACR diagnostic criteria in SLE 1. Serositis - Pleurisy, pericarditis on examination or 2.

ACR diagnostic criteria in SLE 1. Serositis - Pleurisy, pericarditis on examination or 2. 3. 4. 5. 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

ACR diagnostic criteria in SLE 5. Renal involvement – Based on presence of proteinuria

ACR diagnostic criteria in SLE 5. 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 6. Neurologic disorder - Seizures or psychosis in the absence of other causes 7. Malar rash - Fixed erythema over the cheeks and nasal bridge, flat or raised 8. Discoid rash - Erythematous raised-rimmed lesions with keratotic scaling and follicular plugging, often scarring

10. Antinuclear antibodies (ANAs) - Higher titers generally more specific (>1: 160); must be

10. Antinuclear antibodies (ANAs) - Higher titers generally more specific (>1: 160); must be in the absence of medications associated with drug-induced lupus 11. Immunologic phenomena - ds. DNA; anti-Smith (Sm) antibodies; antiphospholipid antibodies (anticardiolipin immunoglobulin G [Ig. G] or immunoglobulin M [Ig. M] or lupus anticoagulant); biologic false-positive serologic test results for syphilis, lupus erythematosus (LE) cells (omitted in 1997 revised criteria)

 • In patients with high clinical suspicion and/or high ANA titers, additional testing

• In patients with high clinical suspicion and/or high ANA titers, additional testing is indicated. • This commonly includes evaluation of antibodies to ds. DNA, 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

Serological tests for SLE Test Description ANA Screening test; sensitivity 95%; not diagnostic without

Serological tests for SLE Test Description ANA Screening test; sensitivity 95%; not diagnostic without clinical features Anti-ds. DNA High specificity; sensitivity only 70%; level is variable based on disease activity Anti-Sm Most specific antibody for SLE; only 3040% 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

Serological tests for SLE Anti-ribosomal P Uncommon antibodies that may correlate with risk for

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 Ig. G/Ig. M 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

Serological tests for SLE Direct Coombs test–positive anemia to denote antibodies on RBCs Anti-histone

Serological tests for SLE Direct 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 = doublestranded 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.

Other useful tests in suspected SLE • Standard laboratory studies that are diagnostically useful

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.

Other useful tests in suspected SLE • Levels of inflammatory markers, including the ESR

Other useful tests in suspected SLE • 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 C 3 and C 4 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.

Indirect immunofluorescence assay: • A laboratory test used to detect antibodies in serum or

Indirect immunofluorescence assay: • A laboratory test used to detect antibodies in serum or other body fluid. • Example of autoantibodies • Anti ds. DNA Abs. • ANA • APA • ASMA • Anti LKM • ANCA • Antithyroid Abs

Substrate • Autoantibodies are detected on specific substrates: Autoantibodies Substrate Anti-ds. DNA on Crithedia

Substrate • Autoantibodies are detected on specific substrates: Autoantibodies Substrate Anti-ds. DNA 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 Hep 2 cells over rodent tissue 1. Higher sensitivity (greater Ag expression)

Advantage of Hep 2 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

Staining Patterns: • Diffuse ? Homogeneous: antibodies to histone • Rim: antibodies to nuclear envelope proteins and to ds. DNA. • 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.

THANK YOU

THANK YOU