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• Components of the immune system
• Evaluation of immunologic response
• Immunodeficiency
• Immune mediated (autoimmune) disease - mechanisms
• Immunodiagnostic tests
• Hemolytic anemia
• Immune mediated thrombocytopenia
• Systemic lupus erythematosus
• Discoid lupus
• Rheumatoid arthritis
• Immunosupressive therapy

The normal immune response of animals is protective and beneficial. It is designed to prevent disease after exposure to infectious and parasitic organisms, cancer cells and other foreign antigenic substances. However, the immune response becomes abnormal and deleterious when it causes autoimmunity, immunodeficiency and allergic disorders."

Components of the immune system

Please review previous class notes or textbooks with regard to the function and interaction of the components of the immune system including:

• B lymphocytes (humoral immunity)
• T lymphocytes (cell mediated immunity)
• Accessory cells - macrophages, neutrophils
• Additional effector systems - complement

Evaluation of immunologic response:
The immune system is dynamic and is difficult to objectively assess. Serial evaluations of immune function are usually required to document an incompetent immune system. Many tests of immune function are not readily available to the practicing veterinarian therefore a diagnosis if immunodeficiency is often speculative and difficult to objectively confirm.

Humoral immunity can be evaluated to some extent with the following tests:

• Measurement of plasma globulins. This is a very crude evaluation of humoral immune competence. Young animals will normally have lower values for total plasma globulins compared to adults.
• Electrophoresis is the separation of plasma proteins by molecular weight in an electric field. Electrophoresis provides a semi- quantitative assessment of the amount of subclasses of globulins.
• The amount of each immunoglobulin class (IgG, IgA, IgM) can be quantitated
• Measurement of serum titers following vaccination can provide some information about the ability of the B lymphocytes to make antibody.
• Lymph node biopsy - B cells are located in follicles in the cortex of the node
• B cell quantitation is the actual measurement of numbers of B lymphocytes. This test is not readily available.

Cell mediated response can be evaluated as follows:

• Lymphocyte counts are a very crude estimate
• T cells are located in the paracortical region of lymph node observed on lymph node biopsy
• Delayed type hypersensitivity (DTH) reactions are mediated by T cells and can be accessed by the size of a skin wheal that develops after second exposure to an injected antigen
• Lymphocyte stimulation tests in vitro measure the ability of cells to undergo DNA synthesis when stimulated with mitogens or foreign antigens. These tests are not readily available.
• T cell quantitation is not readily available

Tests of neutrophil phagocytic function are not readily available

Complement assays are not readily available

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Immunodeficiency
Immunodeficiency can be due to impairment in phagocytic, humoral or CMI and may be congenital or acquired

Signs of immune deficiency states include:

• Development of infections early in life
• Repeat infections despite appropriate therapy
• Infections with low grade pathogens or with organisms rarely observed in immunocompetent patients such as Pneumocystis carinii
• Systemic illness following MLV vaccine administration allowing modified live vaccine virus to revert to virulence
• Profound and persistent neutropenia or lymphopenia

Hereditary (inherited) or congenital (present at birth)  immunodeficiencies are often found in related animals suggesting heritability and generally manifest in young animals suggesting congenital nature. Some reported examples include:

• Canine cyclic hematopoiesis of gray collies. This disorder is characterized by the cyclic release of neutrophils and all other cell types from bone marrow at 11-12 day intervals. Neutropenia persists for 2-4 days. Clinical signs of infection occur during neutropenic states. Clinical findings may include cyclic fever, stunted growth and recurrent infections of increasing severity and duration. The dogs may develop amyloid deposition in the kidneys due to chronic antigenic stimulation. The dogs may be treated with chronic antibiotics but will develop infections with resistant organisms. Irradiation of bone marrow of affected dogs followed by marrow transplantation from normal litter mates has resulted in some dogs remaining normal for up to 2 years after transplant.
• Canine granulocytopathy of Irish setters is a disease characterized by reduced bactericidal function of neutrophils resulting in recurrent infections, fever and neutrophilia.
• Complement deficiency of Brittany spaniels resulting in recurrent infections.

• IgA deficiency has been reported in Beagles, Shepherds and Sharpei. Serum IgA levels do not always correlate with immunoglobulin levels on mucosal surfaces, eg respiratory tract and therefore the significance of reduced serum levels of IgA is unknown and may not correlate with impaired immunocompetence.  Signs that may be associated with low serum IgA include respiratory disease (pneumonia), and chronic skin disease. Some animals with low serum IgA levels have no clinical signs again casting doubt over the significance of serum IgA levels as a predictor of mucosal IgA.

Acquired Immunodeficiencies are more common than congenital immunodeficiencies

• Failure to ingest colostrum can result in an immune compromised animal.  ~ 80% of neonatal antibody protection is via colostrum which must be ingested within the first 24-48 hours of life to provide protection against infectious diseases.
• Infectious agents that may suppress immune function include:

Canine distemper   
Canine parvovirus
Ehrlichiosis
Demodex
Feline panleukopenia
Feline leukemia virus
Feline immunodeficiency virus

• Age extremes may result in abnormal immune function.  Very young and very old animals may have abnormal immune function
• Dietary imbalances - protein malnutrition, insufficient calories, vitamin or mineral insufficiencies can impair immune function
• Hormonal fluctuations of estrogen or testosterone, and pregnancy have variable effects on the immune system. Immune mediated diseases may vary in severity in conjunction with hormonal changes, for example, immune mediated diseases may exacerbate during estrus.
• Drugs

Corticosteroids cause dose dependent suppressive effects on immune function.
Cytotoxic drugs for example, cyclophosphamide, (cytoxan) are potent suppressers of immune function.

• Systemic diseases may depress immune function

  Diabetes mellitus
  Renal failure
  Systemic lupus erythematosus
  Neoplasia

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Immune Mediated (Autoimmune) Disease

Autoimmune is defined as an immune system that no longer recognizes "self" and forms antibodies against "self" antigens.

Immune mediated describes a situation in which foreign antigens (viruses, drugs) act as haptens, adhere to or alter the surface of cells so antibodies form against the hapten - cell complex or against cell antigens that become exposed. Often it is not possible to differentiate between autoimmunity and immune mediated diseases.

There are 4 basic mechanisms of immune mediated tissue injury all of which may incite inflammation. One or more mechanisms may be involved in immune mediated diseases.

• Type I injury includes anaphylactic reactions and atopy

IgE binds to mast cells or basophils causing a release of inflammatory mediators. The organs most often affected by the inflammatory mediators vary between species. Gastrointestinal signs of vomiting or diarrhea and cutaneous signs predominate in dogs. Respiratory signs including pulmonary edema, laryngeal spasm and bronchial constriction predominate in cats.

• Type II injury occurs when antibodies and complement bind antigens on the surface of a cell resulting in the destruction of the cell or phagocytosis of the cells by the mononuclear-phagocytic system.

Examples include:

Immune mediated (hemolytic) anemia (IMA) (IMHA)
Immune mediated thrombocytopenia (IMT)
Pemphigus skin diseases

• Type III injury occurs when antigen, antibody and complement are deposited the walls of blood vessels with subsequent organ malfunction due to the associated inflammatory response. Examples include:

GN (glomerulonephropathy)
RA (rheumatoid arthritis)
Immune mediated uveitis

• Type IV injury is cell mediated immunity in which sensitized T-lymphocytes react with antigen resulting in a delayed reaction peaking in 24 hours: Example - lymphocytic thyroiditis

Immunodiagnostic tests - READ ASSIGNED READING - See Course Objectives and please be familiar with:

Coombs' test (Also called DAT, direct antibody test)
Lupus Erythematosus test (LE test)
Antinuclear antibody test (ANA)
Direct immunofluorescence
Rheumatoid Factor - Rose-Waaler test
Anti platelet and anti megakaryocyte tests

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Immune mediated diseases

Immune mediated hemolytic anemia (IMA, IMHA) is the destruction of normal RBC (primary= autoimmune) or destruction of RBC with foreign antigens on surface (viral, drug) (secondary= immune mediated). IMA = IMHA can occur in any age patient but young adults are most commonly affected. Females are affected more often than males. Cocker spaniels appear to be over represented. IMA may occur alone, or with other immune disorders. When both RBC and platelets are destroyed the disease is called Evans syndrome.  

There are several types of IMA. The prognosis may be influenced by the type of hemolysis.

• The most common type of hemolytic anemia is the extravascular removal of RBC that have antibodies on their surface. The antibody-coated RBCs are removed by the mononuclear phagocytic system in the spleen, bone marrow and liver. The antibody on the RBC may be directed against normal components of the RBC in which the anemia is called primary or the antibody may be directed against a non-RBC antigen "stuck" on the cell surface. The antigen may be a virus, part of a drug or part of a neoplastic cell. This form of hemolytic anemia is called secondary. Most often the antigen on the RBC surface can not be identified. The involvement of foreign antigens is often speculative based on history.  Animals with extravascular hemolysis DO NOT have hemoglobinemia or hemoglobinuria but icterus and bilirubinuria can be present depending upon the rate of hemolysis and ability of the liver to handle the bilirubin generated from degraded RBC. Some of these patients will have intravascular agglutination of RBC. The patients with grossly visible clumping of RBC as soon as the sample is drawn, tend to have a less favorable prognosis than those without obvious gross agglutination. Most patients with IHA will demonstrate some degree of gross RBC agglutination as the blood sample cools to room temperature. A Coombs test is unnecessary if gross agglutination is present. Roleaux is a microscopic event; agglutination is microscopic and macroscopic. Roleaux can be dispersed by diluting the blood sample with an equal volume of saline. Agglutinated cells will not disperse. Roleaux occurs due to fibrinogen or globulins on the cell surface. Agglutination occurs due to immunoglobulin on the cell surface. This subclass of hemolytic anemia is sometimes called "in-saline" agglutination as saline will not disperse the agglutination of RBC.

• Intravascular hemolysis occurs when complement-fixing antibodies on RBC cause RBC lysis in the blood stream and release of free hemoglobin into the blood stream. The released hemoglobin is bound to a carrier protein, haptoglobin. When all the haptoglobin is bound to hemoglobin, any free (unbound) hemoglobin will cross the glomerulus and lead to hemoglobinuria. Hemoglobinemia (red plasma) and hemoglobinuria (red urine) are observed in patients with intravascular hemolysis. Patients may be icteric as well. Intravascular hemolysis carries a poorer prognosis than extravascular hemolysis. These patients are at high risk for development of DIC, thrombosis and renal failure from cell products released from lysed RBC.

• There are two rare conditions in which antibodies against RBC are only active at reduced temperatures. One type results in anemia and skin lesions (ischemia); only in cold weather as the RBC agglutinate in the cool parts of the body (extremities) and impair blood flow. The other form is characterized by anemia and hemoglobinuria in cold weather as the RBC lyse intravascular during cold weather.

Clinical and Laboratory Findings of IMA
The clinical signs and laboratory findings of patients with IMA are determined by the type and severity. Pale mucous membranes are usually observed. Anemia may cause weakness or depression. Fever is variable. Icterus can be present in patients with either intravascular or extravascular hemolysis. Splenomegaly/hepatomegaly are variably present. The anemia is usually regenerative anemia although occasionally the immune destruction is directed toward RBC precursors in the bone marrow resulting in nonregenerative anemia. Spherocytes are diagnostic when they are observed. Hemoglobinuria and hemoglobinemia are only observed in patients with intravascular hemolysis. Leukocytosis is often present due to the bone marrow' s attempt to respond to the anemia, the bone marrow increases output of all cell types. IMA can lead to development of DIC, thrombosis or acute renal failure.

Diagnosis of IMA
IMA is suspected when a patient has a regenerative anemia with normal total protein. If spherocytes or agglutination are present a diagnosis of IMA is highly probable. A Coombs test is often positive in patients with IMA but can be falsely positive or negative. Because IMA can be secondary to other diseases (infections, neoplasia, drug reactions) or may be a part of SLE, a complete of evaluation of the patient is indicated. A complete history may yield clues as to precipitating factors such as drug administration or travel to areas where infectious agents such as Babesia or Ehrlichia are endemic. Evaluation might include CBC, UA, biochemical screen, thoracic radiographs and abdominal radiographs or ultrasound and serology to assess exposure to infectious agents. If multiple organs appear to be affected , an ANA test is indicated to evaluate for SLE.

Treatment of IMA
Treatment involves removal of the inciting cause if known, supportive care and the administration of immune suppressive drugs. Supportive care should include fluid therapy if the animal is not eating and drinking. Blood transfusions may be necessary if the animal is showing signs of hypoxia (respiratory distress) at rest. Cross match should be performed. The transfused cells may be destroyed as fast as the animals own RBC, necessitating multiple transfusions. Only transfuse if really needed..not just to achieve an arbitrary PCV but do not with hold transfusion if the animal is showing signs of hypoxia. Organ failure can be a consequence of hypoxia.

Corticosteroids , usually prednisone or prednisolone are most commonly used in the treatment of hemolytic anemia.  Immunosuppresive doses of prednisone are ~ 1 to 2 mg/kg every 12 hours. Corticosteroids suppress erythrophagocytosis and may have an effect on antibody production. If the patient responds favorably,  the dose of prednisone is gradually tapered when the PCV stabilizes. The entire course of therapy is often several months. Some patients will relapse as the dose of prednisone is reduced and may need to be maintained on the lowest dose required to maintain an acceptable PCV. Failure to respond to prednisone or unacceptable side effects of prednisone may necessitate the use of other immune suppressive drugs such as cyclophosphamide, azathioprine or cyclosporine.

Some clinicians will be more aggressive early in the treatment of patients with marked agglutination or intravascular hemolysis and initiate potent immunosuppressives such as cytoxin at the same time as glucocorticoids. Patients with marked agglutination or intravascular hemolysis tend to be less responsive to treatment and mose likely to develop complications such as thrombosis compared to patients with non-agglutinating extravascular hemolysis.  Less common treatments that may be given to patients with refractory disease include danazol or intravenous administration of human globulins. Some animals may benefit from splenectomy if relapse is a problem. Blood transfusions should be considered if the patient is symptomatic at rest. Transfused cells are often rapidly destroyed. Intact females should be neutered as signs tend to reoccur during estrus.

Prognosis of IMA
The prognosis is variable. Those animals with intravascular hemolysis and gross agglutination of RBC have a less favorable prognosis than animals with extravascular hemolysis without marked agglutination. Secondary disorders may develop including DIC, pulmonary thrombosis and acute renal failure.

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Immune mediate thrombocytopenia (IMT) occurs when antibody attaches to platelets promoting their destruction by the mononuclear-phagocytic system. Like IMA this disease can be primary or secondary to drug, viral, or neoplastic antigens on the platelet surface. IMT can occur alone or with other immune diseases. Female dogs are most commonly affected. Clinical signs usually those of superficial bleeding: petechia or ecchymoses, melena, hematuria, gastrointestinal bleeding and epistaxis. Deep bleeding occurs less commonly. Anemia may develop secondary to blood loss. Fever may be present. Splenomegaly is infrequently present.

Laboratory findings of IMT
Platelet counts are very low (< 10,000/mm³ ). Large, young, platelets are often present. Young platelets are more functional than mature platelets so some patients with IMT may not show signs of bleeding. Regenerative anemia may be present due to blood loss or concurrent IMA. A prolonged bleeding time and clot retraction may be present but PT, PTT are normal. Bone marrow examination is not necessary to make a diagnosis but if performed reveals large numbers of megakaryocytes. Bone marrow samples can be evaluated for the presence of antimegakaryocytic antibodies that are speculated to reflect the presence of circulating anti platelet antibodies. Blood test to measure anti platelet antibodies are now available. PF3 tests are unreliable and not recommended.

Treatment of IMT should include removal of the inciting cause if known and the administration of corticosteroids. Like IMA the most commonly used glucocorticoid is prednisone/prednisolone. Dexamethasone can be used. IT IS MORE POTENT THAN PREDNISONE. Dose and duration of therapy are similar to treatment of IMA. Alternative drugs include vincristine, cytoxan or azathioprine. Drugs used in refractory cases include, human intravenous immunoglobulin, danazol and cyclosporine. Splenectomy can be performed in patients with relapsing disease but results are variable. Administration of platelet rich plasma or whole blood is very ineffective in increasing platelet numbers. Intact females should be neutered. The prognosis is variable. Some animals will respond to treatment and be weaned off of immunosupressive drugs whereas others will relapse when drugs are discontinued.

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Systemic lupus erythematosus (SLE) is a disease that affects multiple organs and involves both Type II and III immune injury. SLE is caused by circulating auto-antibodies against nuclear component including DNA, RNA and nuclear proteins. Immune injury may also be directed against specific cells such as RBC and platelets. Although any organ can be affected the most common organs involved are:

• joints resulting in a nonerosive polyarthritis,
• IMT (platelets)
• IMA (RBC)
• ulcerative dermatologic disease
• glomerulonephropathy

Females are more often affected. There appears to be a genetic factor as SLE tends to occur in family groups. Drugs including procainamide, hydralazine, and phenytoin can precipitate a disease that mimics SLE

Clinical signs of SLE: Because SLE can involve so many different organs the signs are very diverse and can be acute or chronic or cyclic. SLE is sometimes called "The great imitator".

Diagnosis of SLE: Several diagnostic tests can be performed, the results of which may support a diagnosis of SLE. LE preparations are positive in ~ 60-80% of cases of SLE. ANA is positive ~ 96% of the time. Coombs tests and antiplatelet antibody tests are variably positive. Direct immunofluorescence of skin biopsies may be positive in animals with skin lesions.

The procedure for performing an LE test is as follows:

10 ml of non anticoagulated blood are drawn

the sample is allowed to clot and the serum separated from the clot

The clot is forced through a fine screen to disrupt cell membranes and release nuclear material

The serum is added to the preparation and incubated

If the serum contains antibodies against nuclear proteins the antibodies will attach to nuclear proteins released by the cell damage

Neutrophils that were not ruptured will phagocytize the antigen antibody complex

The resulting cell is the LE cell

The cytologist must see several LE cells to call the test positive

The round pink cell in the center of the filed is an LE cell. A LE cell is a neutrophil that has phagocytosed nuclear material that is coated with antibody.  An LE cell differs from a tart cell which is a neutrophil which has phagocytosed another cell.

The diagnosis of systemic lupus may be difficult to make. The following "recipe" is suggested in order to make a diagnosis:

Major signs include

• Polyarthritis
• Polymyositis
• Dermatitis
• Proteinuria
• Anemia
• Thrombocytopenia
• Leukopenia

Minor signs include

• Fever
• Oral ulcers
• Pruritis
• Myocarditis
• Pericarditis
• Lymphadenopathy
• Seizures

A diagnosis of systemic lupus can be made if the animal has two major signs and a positive serologic test (LE or ANA) or one major sign plus 2 minor signs plus a positive serologic test.

Treatment of SLE is supportive and immunosupressive using agents similar to those discussed with IMA and IMT.

Prognosis of SLE: is guarded, 40% die within one year of dx.

Discoid lupus (DLE) is a cutaneous form of lupus involving the face. The lesions are distributed in a pattern similar to the mask on a wolf (lupus means wolf). Differentials for DLE include the phemphigoid diseases. The prognosis for DLE is better than SLE. DLE can be treated with: oral or topical glucocorticoids, vitamin E, sunscreens, niacinamide & tetracycline.

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Rheumatoid arthritis (RA) is a Type III immune injury that occurs most often in small breeds of dogs. It is progressive and involves multiple joints. Small distal joints (tarsal, metatarsal, carpal, metacarpal and phalangeal) are involved more often than more proximal joints. It is proposed that rheumatoid factor is an auto-antibody against altered IgG. The cause of IgG alteration is unknown. Antibody is developed against rheumatoid factor and results in deposition of immune complexes (antigen and antibody) in the joints.

Clinical signs of RA include a shifting leg lameness that occurs most often in the smaller distal joints. Joint pain and swelling are present and the joints may become deformed. Fever may be present.

Diagnostic tests for RA Joint fluid contains increased numbers of WBC, mostly PMN's. Joint fluid is reduced in viscosity. Radiographs of affected joints show joint swelling and destruction of subchondral bone. The joints become unstable resulting in bony proliferation. A synovial biopsy discloses villous hyperplasia and infiltration of plasma cells and lymphocytes. A Rose-Waaler test for rheumatoid factor may be positive.

Treatment of RA Because of the progressive nature of RA, aggressive therapy is indicated as soon as a diagnosis is made. Drugs used include corticosteroids and nonsteroidal anti-inflammatory agents such as aspirin. Gold salts have also been used with variable success. Surgery can be performed to fuse severely affected joints to reduce pain.

Prognosis of RA The prognosis is poor and the disease is progressive.

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Feline progressive polyarthritis is an erosive joint disease like RA. It affects young (1 1/2 - 4 1/2 years of age) male cats most often resulting in fever and joint pain and swelling. 50-70% are FeLV +. ANA and RF are negative. Joint fluid and synovial biopsy appear similar to dogs with RA. Treatment is with immunosuppressive agents but the prognosis is poor.

Idiopathic nondeforming arthritis is the most common immune mediated joint disorder of the dog. It occurs primarily in large breeds of dogs. Signs include cyclic fever, lameness, stiffness, and swollen joints. There are no radiographic changes other than soft tissue swelling. Joint fluid is similar in appearance to RA and SLE. The dogs are negative for LE, ANA, RF. Treatment is with corticosteroids. The recurrence rate is 30-50%.

Other Immunologic diseases will be discussed during lectures of the appropriate organ system

• dermatologic diseases (dermatology)
• Immune complex glomerular disease (nephrology)
• Myasthenia gravis (neuromuscular diseases)
• Polyradiculoneuritis (coonhound paralysis) (neuromuscular diseases)
• Lymphocytic thyroiditis (Type II and IV) (Hashimoto's disease) (endocrinology)
• Sjogren's syndrome (ophthalmology)
• Chronic superficial keratitis (pannus) (ophthalmology)

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Immunosuppressive Drug Therapy

The potential side effects of immunosuppressive doses of glucocorticoids include:

• Infection-especially urinary tract. Dogs with steroid induced UTI may not have pyuria and may not show clinical signs of lower UTI.
• Outward signs of hyperadrenocorticism: thinned haircoat, potbellied appearance, thin skin
• Gastrointestinal ulcers
• Polyphagia, polydipsia, polyuria
• Pancreatitis may be predisposed to in patients receiving glucocorticoids

MONITOR YOUR PATIENT FOR ADVERSE DRUG EFFECTS

Side effects include: 

• urinary tract infection
• sepsis
• signs of hyperadrenocorticism including thin hair coat, potbelly appearance, thin skin
• gastrointestinal ulcers
• polyphagia
• polyuria, polydipsia
• pancreatitis 

Patients receiving high doses of immunosuppressive drugs to treat immune mediated diseases are at risk for developing secondary bacterial infections. Animals receiving immunosuppressive doses of steroids will have neutrophilia and sometimes left shift so these parameters cannot be used to identify infection. Watch the morphology of the white blood cells for evidence of toxic neutrophils indicating the patient is septic.

Animals that are  immunosuppressed  most often develop infections from their own endogenous flora including E. coli and anerobes from the intestinal tract and staphylococcus from the skin.

An attempt to should be made to identify the source of the infection and and the bacterial agent by culturing blood or urine if there's evidence for urinary  tract infection. 

Don't wait on the results of the cultures; rather initiate broad spectrum antibiotics or combinations of antibiotics to provide coverage against coliforms, anerobes and staph.

Animals on immunosuppressive agents commonly develop urinary tract infections but they may not show signs because of the anti-inflammatory effects of the drug. Periodic urine cultures should be performed in animals receiving long term immunosuppressive drugs. 

Relative Potencies of Glucocorticoids

See Textbook of Veterinary Internal Medicine (2000, p 312) for a complete table of relative potencies of the corticosteroids listed below.

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