Autoimmunity PDF

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Page | 1II4.) AUTOIMMUNITYIn the early 1900s, Paul Ehrlich described this phenomenon as “horror autotoxicus” literally meaning “fear of self-poisoning.” Autoimmune diseases are conditions in which damage to organs or tissues results from the presence of autoantibody or autoreactive cells. o T-cell m...

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CLINICAL IMMUNOLOGY AND SEROLOGY II4.) AUTOIMMUNITY In the early 1900s, Paul Ehrlich described this phenomenon as “horror autotoxicus” literally meaning “fear of self-poisoning.” Autoimmune diseases are conditions in which damage to organs or tissues results from the presence of autoantibody or autoreactive cells. o T-cell mediated immune responses that are directed against host antigens Cause tissue and organ damage Due to loss of self-tolerance o Ability of the immune system to accept self-antigens and not initiate an immune response against them During the maturation process of T cells as well as B cells, the great majority of undifferentiated lymphocytes that are processed through the thymus or bone marrow do not survive. o It is thought that this is where potentially self-reactive T-cell clones are destroyed. ETIOLOGY OF AUTOIMMUNE DISEASE Self-tolerance is a type of immunologic tolerance, or a state of immune unresponsiveness that is

directed against a specific antigen, in this case, a self-antigen. o Inability of lymphocytes to distinguish self-antigen to nonself antigen Two level of self-tolerance: o Central Tolerance o Peripheral Tolerance Central Tolerance occurs in the central or primary lymphoid organs, thymus and bone marrow. T cell central tolerance: In a process called negative selection, T cells that express T cell receptors (TCRs) with a strong affinity for these self-antigens are deleted by apoptosis. o Occurs in the thymic medulla, and this is where we test if T cells are autoreactive. So we are asking if they bind strongly to then self-antigens presented by mTECs. o Occurs with both immature, double positive T cells in the cortex and with the more mature, single positive T cells in the medulla.  Some of the selfreactive CD4 T cells are not deleted, rather differentiate into T reg to inhibit immune response against selfantigen

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B cell central tolerance: B cells gets multiple chances to ensure they are not autoreactive through receptor editing change their light chains. It comes back and is tested again. Has 4 chances of correction, and will be deleted if they are still autoreactive. o Similar T cell central tolerance o B cells in the bone marrow with receptors having strong affinity for self-antigens are eliminated by apoptosis. o Some self-reactive B cells are not deleted but instead, they are stimulated to rearrange their immunoglobulin genes resulting for B cell receptors not antigen specific (Receptor editing) o Anergy: Specific state of unresponsiveness to the antigen Some of the self-reactive antigens manage to escape to the secondary lymphoid organs such as the lymph nodes and spleen. Peripheral tolerance: Lymphocytes that recognize self-antigens in the secondary lymphoid organs are rendered incapable of reacting with those antigens. o T cell peripheral tolerance from anergy: caused by the absence of a costimulatory

CLINICAL IMMUNOLOGY AND SEROLOGY signal from an antigenpresenting cells (APCs) or binding of inhibitory receptors such as CTLA-4 ( a molecule that prevents T cell activation) o Also result from inhibition by T regs or death by apoptosis. B cells Peripheral Tolerance: Selfreactive B cells in the periphery can be deleted by apoptosis, be rendered anergic after repeated stimulation with self-antigens, or receive inhibitory signals through receptors such as CD22. o They are CD4 B cells which are slightly autoreactive to one self-antigen. The CTLA-4 will seek out those autoreactive cells and will remove the CD28 from the APC. o This is to attack those that escaped central tolerance. The removal of the CD28 will prevent the activation of autoreactive T cells. o Failure in self-tolerance results to Autoimmune disease GENETICS: MAJOR AUTOIMMUNITY

FACTOR

OF

More prevalent among family members than among unrelated individuals

More prevalent among monozygotic ( genetically identical) twins than dizygotic (non-identical) twins or siblings Major histocompatibility complex (MHC) products also seem to influence antigen recognition or nonrecognition by determining the type of peptides that can be presented to the T cells. HLA-B27 has stronger association in ankylosing spondylitis, an autoinflammatory disease that affect the spine. ( 100x chance) In addition, the expression of class II molecules on cells where they are

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not normally found may result in the presentation of self-antigens for which no tolerance has been established. Non-MHC genes can also be associated with the development of autoimmune disease. Many of these genes influence the development and regulation of immune response o PTPN22 gene: Has a role in T and B cell receptor signaling o IL2RA gene: Involved in T cell activation and maintenance of Tregs o CTLA4 gene: Inhibitory effect on T cell activation o BLK gene: Involve in B cell activation and development o AIRE (autoimmune regulator): Promotes the development of T cell tolerance in the thymus OTHER ENDOGENOUS AND ENVIRONMENTAL FACTORS HORMONAL INFLUENCE Women are 2.7 times more likely to acquire an autoimmune disease than men; 78% with autoimmune diseases are of female gender. Females have been found to have higher absolute CD4+ T cell counts and higher levels of circulating antibodies than men.

CLINICAL IMMUNOLOGY AND SEROLOGY Estrogen tend to direct the immune system in favor of a type 2 helper cell response, resulting in more B cell production and antibody production Androgen favor the type 1 helper cell response with activation of CD8+ T cells. Prolactin stimulates production of breast milk in pregnant and nursing women Stimulate both humoral and cellmediated immune response TISSUE TRAUMA AND RELEASE OF CRYPTIC ANTIGEN Some self-antigen may be cryptic, or hidden within the tissues of the host Cryptic antigens are released during the inflammation or tissue trauma and be accessible to the uneducated lymphocytes, triggering an immune response. Immunologic Ignorance may be responsible for the production of autoantibodies to the lens of the eye following an ocular injury, autoantibodies to sperm after a vasectomy and autoantibodies to DNA following damage to skin cells by overexposure to UV rays from the sun

MICROBIAL INFECTION Molecular mimicry refers to the fact that many individual viral or bacterial agents contain antigens that closely resemble self-antigens. Exposure to such foreign antigens may trigger antibody production that in turn reacts with similar self-antigens. Bystander effect: Microbial organism does not have to share structurally similar antigens with the host. Instead, the microorganism can induce a local inflammatory response that recruits leukocytes and stimulates APCs to release cytokines that nonspecifically activate T cells. o Epitope spreading: an expansion of the immune response to unrelated antigens Superantigens: Proteins that are produced by various microbes that have the ability to bind to both class II MHC molecules and TCRs regardless of their antigen specificity. o Act as potent T cell mitogens by activating a large number of T cells with different antigen specificities in cases of Food poisoning and Toxic Shock syndrome

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Microbiota or normal flora can induce innate immune response through interactions with pattern recognition receptors such as TLRs o Triggers cell signaling pathways that result tot the production of cytokines such as IFN (oo), which can stimulate cells of the adaptive immune system Decrease number and function of Treg can perpetuate the activity of the autoreactive cytotoxic T cells and hyperactive B cells that produce autoantibodies EPIGENETICS AND MODIFICATION OF SELFANTIGENS Epigenetics refers to modification in gene expression that are not caused by changes in the original DNA sequence. Over and underexpression of certain genes in the immune system may result in homeostatic imbalances and a breakdown of self-tolerance, leading to autoimmunity Exposure to environmental factors can lead to changes at the protein level (Post-Translational Modification) and may involve biochemical processes such as acetylation, lipidation, citrullination, and glycosylation.

CLINICAL IMMUNOLOGY AND SEROLOGY Citrullination of collagen might play a role in the pathogenesis of Rheumatoid Arthritis o Glycosylation of myelin may involved in the pathology of multiple sclerosis Such alterations of self-antigens can make them more immunogenic, leading to autoimmune responses. o

Lesions from damaged tissue & autoantibodies are not confined to any organs ORGAN SPECIFIC Lesions from damaged tissue & autoantibodies are directed towards a single target organ SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) Chronic systemic inflammatory disease that affect between 40 and more than 200 persons per 100,000 depending on the population. Women are much more like affected than men, by a ratio of about 9:1 ETIOLOGY

TWO CLINICAL TYPES OF AUTOIMMUNE DISEASE SYSTEMIC / NON-ORGAN SPECIFIC: Sometimes called vascular disease”

“Collagen

Environmental factors thought to play a role in SLE include UV light, certain medications, and possibly infectious agents Exposure to sunlight is a well-known trigger of the photosensitive skin rashes seen in many lupus patients. Certain drugs such as procainamide (used to treat abnormal heart rhythms), hydralazine, (used for high

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blood pressure), and the tuberculosis drug isoniazid can induce a transient lupus-like syndrome that resolves once the drug is stopped. More than 20 genetic loci associated with lupus in humans have been reported HLA-A1, B8, and DR3- Have an increased chance of developing lupus. Type III Hypersensitivity: The immune complex generated in SLE activate complement, inducing the generation of chemotaxins such as C5a. Other lupus-associated genes include polymorphisms in genes associated with immune function, genes coding for various cytokines,

CLINICAL IMMUNOLOGY AND SEROLOGY and genes involved in signaling of innate immune responses. IMMUNOPATHOLOGY Typical patient has an average of 3 circulating autoantibodies Abnormal apoptosis of certain types of cells may occur, releasing excess amounts of cellular constituents such as DNA and RNA. Dysfunction removal of cellular debris by phagocytes may allow these cellular components to persist, increasing the chances for autoantibody production Antibodies to dsDNA are present in 70% of patients with lupus and are highly specific. o Accumulation of IgG to dsDNA seems to be the most pathogenic because it forms complexes of an intermediate size that become deposited in the GBM Possible deficiencies in lupus patients: o Defect in complement receptors on phagocytic cells o Defect in receptors for the Fc portion of immunoglobulins o (Rarely) Deficiency of early complement components such as C1q, C2 or C4.

The immune complexes activate complement and initiate an inflammatory response. Leukocytes are attracted to the sites of inflammation and release cytokines that perpetuate the response, resulting in tissue damage Autoantibodies to nuclear and nonnuclear antigens can also cause cellular destruction by other mechanism. o Ab to RBCs can cause hemolytic anemia and antibodies to platelets can cause thrombocytopenia by antibody-mediated Type II Hypersensitivity Antibody to endothelial cells can cause inflammation of the blood vessels and vascular damage in lupus. Phospholipid antibodies are associated with miscarriage, stillbirth and preterm delivery

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Summary of the pathogenesis of systemic lupus erythematosus (SLE) The immune dysregulation caused by environmental triggers and genetic predisposition leads to increased apoptosis. Decreased clearance causes recognition of self-antigens by the immune system, activation of B and T cells, and the production of selfantibodies. Those antibodies, mainly IgG, cause the formation of immune complexes that can lead to renal impairment in kidneys and vasculopathy and atheromatous plaque formation in blood vessels Atheroma plaques are caused by the action of anti-oxidized low-density lipoprotein (LDL), which is very common in SLE, which leads to the decrease of

CLINICAL IMMUNOLOGY AND SEROLOGY ABCA1 and ABCG1 by a yet unknown mechanism. Resveratrol acts as an SIRT1 activator, inhibiting proliferation of B and T cells and antibody production and also increasing ABCA1 and ABCG1 levels. ABCA1: ATPbinding cassette transporter A1; ABCG1: ATPbinding cassette transporter G1; APC: antigen presenting cell; IL: interleukin; MHC: major histocompatibility complex; SIRT1: sirtuin 1; TCR: T-cell receptor; TNF-α: tumor necrosis factor alpha.

CLINICAL SIGNS AND SYMPTOMS Nonspecific Symptoms: Fatigue, Weight loss, Malaise, Fever and Anorexia Marked by alternating relapses or flares and periods of remission. Joint involvement seems to be most frequently reported manifestation because over 90% of patients with SLE are subject to polyarthralgias or arthritis Skin Manifestation: 80% patients with lupus o Erythematous rash may appear on any area of the body exposed to UV light, Classic butterfly rash: Less common but more dramatic symptoms o Responsible for the name lupus, derived from the Latin term meaning “wolf-like”.

Skin lesions have central atrophy and scarring Evidence of renal involvement is present in about half of all patients with lupus o Nephritis is a major cause of illness and death o Diffuse proliferative glomerulonephritis: Most dangerous Cardiac involvement with pericarditis, tachycardia, or ventricular enlargement o Pleuritis with chest pain o Neuropsychiatric manifestation such as seizures, mild cognitive dysfunction , psychoses, or depression Hematologic abnormalities such as anemia, leukopenia, thrombocytopenia, or lymphopenia can also be present Drug-induced lupus differs from the more chronic form of the disease, in that symptoms usually disappear once the drug is discontinued. 11 clinical criteria and 6 immunologic criteria according to Systemic Lupus International Collaborating Clinics o Immunologic Criteria o 1. Elevated antinuclear antibody titer o

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o o o o o

2. Elevated anti-dsDNA titer 3. Presence of antibody to the Sm nuclear antigen 4. Presence of antiphospholipid antibody 5. Low Complement Level 6. Positive direct Coombs’ test

TREATMENT Mild symptoms: High dose of aspirin or other anti-inflammatory drug may bring relief For skin manifestation: Hydroxychloroquine or chloroquine and topical steroids in antimalarials are prescribed o Inhibit signaling of TLR 7, 8 and 9 Systemic corticosteroids are used for fulminant LABORATORY DIAGNOSIS OF SYSTEMIC LUPUS ERYTHEMATOSUS Initial diagnosis: CBC, Urinalysis, ESR o Leukopenia o Possible anemia o Thrombocytopenia o Low to Normal CRP levels Quantitation: Complement proteins o C3 levels can be decrease during inflammation Antinuclear antibodies (ANAs) o Present in majority of the patients

CLINICAL IMMUNOLOGY AND SEROLOGY ANTINUCLEAR ANTIBODIES (ANAs) Directed against antigens in cell nuclei Present in more than 95% of lupus patients used as a major marker However, they are not specific for SLE because they can also be detected in a significant percentage of patients with other connective tissue diseases such as Sjogren’s syndrome, scleroderma, etc. Extractable nuclear antigens are a group of nuclear antigens that were named because they were isolated in saline extracts of mammalian tissues. Double stranded DNA (dsDNA) antibodies are the most specific for SLE because they are mainly seen in patients with lupus and their levels correlate with disease activity. o Typically produce a peripheral or a homogenous staining pattern Antihistone antibodies can also be found in lupus patients. o Histone are nucleoproteins that are essential components of chromatin. o H1, H2A, H2B H3, and H4 are the major classes of Histone

o

o

H2A and H2B can be detected in almost patients with drug-induced lupus Typically produce a homogenous pattern in the IF assay

First describe in a patient named Smith Anti-RNP antibody is directed against RNP, which consists of several nonhistone proteins complexed to a small nuclear called U1-nRNP SS-A/Ro and SS-B/La antigens also belong to the family of extractable nuclear antigens. o

Anti SS-A/Ro- 24-60% patients with SLE Anti SS-B/Ro- 9-35% patients with SLE Antibodies to the Anti SS-A/Ro antigens are best detected on IIF if a special cell line, HEp2000, is used

Nucleosome antibodies are stimulated by DNA-histone complexes, known as nucleosomes or deoxyribonucleoprotein (DNP) o Directly against complexes and not against the DNA or histones Sm antigens are specific for lupus because it is not found in other autoimmune diseases.

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Nucleolus is a prominent structure with nucleus where transcription and processing of rRNA and the assembly of the ribosomes take place. Three nucleolar components: Fibrillarin, RNA polymerase 1, and PM-1. Anticentromere antibodies: bind to proteins in the middle region of a chromosome where the sister chromatids are joined.

CLINICAL IMMUNOLOGY AND SEROLOGY METHOD OF DETECTION

ANTINUCLEAR

ANTIBODY

FLUORESCENT ANTINUCLEAR (FANA) TESTING

ANTIBODY

The screening test is commonly performed with a 1:40 or 1:80 dilution of patient serum in order to avoid detecting low positive titers that may be seen in healthy persons A titer >= 160 is generally considered to be clinically significant PATTERNS OF FANA Homogenous (Diffuse): Characterized by uniform staining of the entire nucleus in interphase cells and condensed chromosomal region in metaphase cells

Peripheral (Rim or outline): Diffuse staining is seen throughout the nucleus, but there is a greater staining intensity around the outer circle surrounding the nucleus in interphase. Speckled: Characterized by discrete, fluorescent specks throughout the nuclei of interphase cells Nucleolar: Prominent staining of the nucleoli within the nuclei of interphase cells Centromere: Numerous discrete speckles are seen in the nuclei of interphase cells and chromatin of dividing cells. Mixed can be observed; in some cases. (For instance, homogenous pattern may conceal the speckled IIF method is considered the gold standard but with limitations OTHER ASSAY FOR ANA ELISA AND CLIA ELISA and Chemiluminescence immunoassay for ANA: For high volume testing laboratories, automated and easy to perform, and yield objective results. However, they may yield a false positive results thus, IFF is still the gold standard for ANA Testing

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MICROSPHERE MULTIPLEX IMMUNOASSAY Patient serum is incubated in a microtiter plate well containing a suspension of polystyrene microsphere that are coated with individual nuclear antigens or with HEp-2 extract More efficient than ELISA because they allow testing for multiple antibody specificities to be performed in a single-stranded DNA IMMUNOFLUOROSCENCE USING Crithidia luciliae An IIF using C.luciliae as the substrate Contains organelle called kinetoplast (rich in ds DNA) Used to detect antibodies to dsDNA A positive test is indicated by a brightly stained kinetoplast with patient serum and an antibody conjugate. High degree of specificity, although it is less sensitive than other FANA tests ENA OUCHTERLONY TEST Immunodiffusion test detects antibodies to specific ENA Patient serum or controls in outer wells react with ENA antigens in center wel...