Immunology

·         PRIMARY IMMUNODEFICIENCY’S:

1.       Antibody/B cell deficiencies [Bruton's X-linked agammaglobulinemia, AR agammaglobulinemia, CVID (ICOS and TACI deficiencies), HIM (AR), ICF syndrome]

2.       Cellular deficiencies (IFN-γ/IL-12 axis, APS type 1, defective NK function)

3.       Combined [SCID, Wiskott-Aldrich, DiGeorge, ataxia telangiectasia, HIM (AR), XLP syndrome, WHIM syndrome, caspase 8 deficiency]

4.       Complement

5.       Phagocytic (CGD, Chediak-Higashi syndrome, LAD, specific granule deficiency, cyclic neutropenia, X-linked neutropenia)

·         ASSAYS FOR CELLS:

T cells: Flow cytometry for CD3, CD4, CD8, CD45, T-cell receptor

B cells: CD19, CD20, CD21, Ig-associated

NK cells: CD16/CD56

·         B Cell-Signal-Transduction Complex: Ig-a;, Ig-l3,CD19, CD20, CD21

·         T-Cell Receptor (TCR): CD3 complex (gamma, delta, epsilon and zeta chain dimers)

·         IgG has highest Affinity.

IgM has highest Avidity.

·         ROUTINE HLA TYPING:

Done for HLA-A, HLA-B, HLA-DR

The micro cytotoxicity assay is used to identify class I MHC molecules. This test is performed using antisera against specific class I antigens, plus complement. If the test cell has class I antigen to match the antibody, then complement will be bound and the cell will be killed. The entry of the dye into the interior of the cell illustrates cell death.

The mixed lymphocyte assay is used to test reactions between donor and recipient cells.T cells are incubated with a B cell to check the response to class II (DR) antigens. The B cells act as the stimulator cell, and they are irradiated to prevent proliferation. The T cells respond because they recognize MHC molecules on the irradiated (stimulator) cellthat are different from their own MHC class II molecules. Proliferation is measured by uptake of titrated thymidine in cellular DNA.

·         Because the B cells divide rapidly in germinal centers of the lymph nodes, there are many opportunities for mutations. Although these mutations can be detrimental to individual B cells (these cells are then eliminated), mutations that increase antibody affinity are preferentially selected, and mutations thus playa role in affinity maturation of B-cell clones. Somatic mutation, however, plays essentially no role in T-cell receptor diversity.

·         TAP-2 deficiency (also known as bare lymphocyte syndrome I) is caused by defects in the transporter associated with the antigen presentation (TAP; either TAP1 or TAP2) system. These defects ultimately impair the loading of peptide fragments into nascent MHC class I molecules in all nucleated cells and reduce the number of MHC class I molecules that successfully reach the cell surface. This reduced MHC I expression decreases the number of functional CD8+ T cells and can also affect the functions of NK cells monitoring MHC class I expression on body cells (although the NK cells appear not to attack uninfected host cells). Likewise, defects inhibiting expression of MHC class II molecules reduce the number of functional CD4+ T cells.

·         Some IgA-deficient patients with anti-IgA antibodies are at increased risk of a severe reaction to trace IgA from IVIg and from blood transfusions (washed packed cells should be given if transfusion is needed).

·         Persons with IgG deficiencies, As a rule are not susceptible to viral infections, However, IgG-deficient patients are susceptible to polioviruses (and should not receive live virus vaccine) and to hepatitis B and C.

In patients with XLA (but not CVID or HIM), a chronic meningoencephalitis, which is ultimately fatal, can develop with enteroviruses (echovirus or coxsackie) infection.

·         Different types of SCID:

1.       ADA deficiency

2.       PNP deficiency

3.       RAG1, RAG2 (Omenn syndrome)

4.       Defective cytokine signaling

5.       X-linked (common gamma chain) this one is MC

6.       Autosomal recessive (IL2, IL7 receptor)

·         Idiopathic CD4 lymphocytopenia (Nezelof syndrome) isT cell immunodeficiency; just like DiGeorge syndrome without the associated congenital anomalies; resembles HIV (failure to thrive, candidiasis, anemia, thrombocytopenia, cancer).

·         Chronic mucocutaneous candidiasis (autoimmune polyglandular syndrome type I) is T cell immunodeficiency; superficial candidiasis (not systemic) associated with single or multiple endocrinopathies, iron deficiency, and anergy; molecular defect in AIRE

·         Hyperimmunoglobulin E is a/w Uncertain immunodeficiency, with increased serum levels of IgE (up to 10 times normal); recurrent infections of the skin and sinopulmonary tract with S. aureus, H. influenzae, and Aspergillus; coarse facial features; delayed shedding of primary teeth; and chronic eczematous rashes.

·         CVID; chronic mucocutaneous candidiasis and hyperimmunoglobulin E are disorders compatible with living to adulthood; the other T-cell or combined immunodeficiencies are severe and generally present early in life; without bone marrow transplantation, they are generally fatal.

·         Hyper IgM Syndrome: X linked mutation in CD40 ligand on T cells. So no class switching. IgM level 10 times normal. Deficiency of IgA, IgG, IgE. Fails to make Germinal Center. Recurrent Respiratory Infections.

·         Anaphylactoid reaction is clinically similar to anaphylaxis but is not IgE-mediated. Agents such as radiocontrast media, NSAIDs, muscle relaxants, and paclitaxel cause direct stimulation of mast cells and basophils. Anaphylactoid reactions can often be prevented with glucocorticoids and antihistamines.

·         If the C4 level is normal during an episode of angioedema, there is no problem with C1INH because C4 is used up in this process. If the C4 level is low or if a person is seen in an asymptomatic period, C1INH level and functional activity should be measured (to determine if it is HAE I or II), as should the C1q level.

C1q levels are normal in hereditary angioedema and decreased in acquired C1INH deficiency.

C2 is reduced during acute episodes.

 Acquired C1INH is associated with malignancy, particularly B-cell lymphomas.

·         C1INH deficiency is treated with attenuated androgenic steroids, which increase the production of C1INH. This is effective in patients with deficient production, deficient activity, and increased catabolism of C1INH. Epinephrine may not work in a crisis, and a tracheostomy is indicated for laryngeal edema. Antifibrinolytics (epsilon-aminocaproic acid or tranexamic acid) may be helpful.

·         DIAGNOSIS OF MASTOCYTOSIS:

One major diagnostic criterion + one minor criterion or three minor criteria

Major criteria: Bone marrow biopsy and aspirate with dense infiltrates of mast cells

Minor criteria:

1         Spindle-shaped mast cells

2         Detection of 816 c-kit mutation

3         Flow cytometry with CD2, CD25, and CD117 expression

4         Serum tryptase >20 µg/mL

·         TRANSPLANT MATCH :

Although matching for intrafamilial transplants of all types is performed, nonfamilial cardiac, lung, and liver grafts are not MHC-matched because other factors such as size, location, and availability limit the transplants much more. Kidney transplantation, for which there is the potential for living related and unrelated donors, allows for matching. Bone marrow transplants must be matched, whereas matching in liver transplants may actually decrease survival.

·         Graft-versus-host disease is an immune response of the donor T cells against the recipient usually 6 or more days after transplant. It is a problem only when transplanting hematopoietic tissue (bone marrow, nonirradiated blood transfusions).

·         Pneumococcal polysaccharide vaccine (PPV) protect against 23 pneumococcal bacteria.

·         Of the many methods available for antigen and antibody detection, LA, ELISA, EMIT, CIE, and COA are the most widely used.

Latex agglutination (LA) employs latex polystyrene particles sensitized by either antibody or antigen. LA is more sensitive than CIE and COA but slightly less sensitive than either RIA or EIA. LA has been used to detect Haemophilusinfluenzae, Neisseria meningitidis, and Streptococcus pneumoniae antigens in cerebrospinal fluid. LA has also been used for detection of cryptococcal antigen. Most recently, LA has been widely used for rapid detection of group A streptococcal antigen directly from the pharynx. The test is rapid (5 minutes), sensitive (approximately 90%), and specific (99%).

Coagglutination (COA), also an agglutination test, is slightly less sensitive than LA but less susceptible to changes in environment (e.g., temperature). Most strains of coagulase-positive staphylococci have protein A in their cell wall. Protein A binds the Fc fragment of microbial antigens in body fluids. COA has also been used to rapidly type or group bacterial isolates.

Enzyme immunoassays (EIAs) can be either homogeneous (EMIT) or heterogeneous (ELISA). EMIT has been used primarily for assays of low-molecular-weight drugs. Its primary use in microbiology has been for assays of aminoglycoside antibiotics. EIAs vary as to the solid support used. A variety of supports can be used, such as polystyrene microdilution plates, paddles, plastic beads, and tubes. The number of layers in the antibody-antigen sandwich varies; usually as additional layers are added, detection sensitivity is increased. The two most common enzymes are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Beta galactosidase has also been employed. Orthophenylene diamine is the most common substrate for HRP and p-nitrophenyl phosphate for AP. Because EIAs are usually read in the visible color range, the tests can be read qualitatively by eye or quantitatively by machine.

Counterimmunoelectrophoresis (CIE) was originally used for "Australia antigen" (HBsAg) but was soon replaced by RIA. For a decade, CIE was used to detect antigens in body fluids, CIE is not an easy technique. Its success depends on the control of many variables, including solid support, voltage, current, buffer, affinity and avidity of antibodies, charge on the antigen, and time of electrophoresing.

• While acute stress tends to suppress the immune system, chronic stress tends to enhance the immune system, especially over time.

• If the immune system is weakened so that thelatent virus becomes more active, the antibody titers to the virus will increase. Relaxation training produces decreased antibody titers to latent viruses by generating an increase in natural killer cell activity.

• Consumption of alcohol has been shown to affect the immune system, apparently in relation to dose. Macrophages are inhibited and move more slowly toward chemical signals released by the body. Proliferation and cytotoxicity of T cells are also inhibited. Increased doses of alcohol can damage certain bodily tissues, such as the thymus. However, no inhibition of production of antibodies has been observed.

·         The brain and the endocrine and immune systems communicate with and influence each other. Studies of the hypothalamus have demonstrated that stimulation of the anterior hypothalamus enhances immune responsiveness, and stimulation of the posterior lobe inhibits immune reactivity. Removal of the thymus gland will result in the atrophy of these hypothalamic neurons. Also, the limbic system contains high concentrations of neuropeptide receptors, as do the lining of the intestines and lymphocytes. It has also been found that immune cells produce the same neuropeptides that are produced in the brain. Some of these peptide hormones that are produced in the brain stimulate T cells to produce lymphokines, such as interleukins and interferon.

·         R. Ader and N. Cohen discovered that the immune system could be conditioned by neutral  stimuli.

Robert Ader, also discovered that the immune system can be suppressed through classical conditioning.

·         A battery of immunologic assessments found that the caregivers had suppressed immune systems; cellular immune system control of latent viruses was poorer than that of a matched control group, the percentage of T lymphocytes was lower, and the helper/suppressor ratio was smaller.

·         99% Proteins are antigens. Cardiolipin (seen in SLE) is the ONLY FAT that is an antigen.

·         Electrophoresis of human serum proteins identifies five distinct types: albumin, 1-proteins, 2-proteins, -proteins, and globulins.

Many human diseases can be diagnosed, at least in part, on the basis of abnormal electrophoresis profiles. For example, absence of the second peak) is compatible with a diagnosis of Alpha 1-antitrypsin deficiency in symptomatic persons. A sharp and high peak indicates the presence of a monoclonal gammopathy, such as multiple myeloma; on the other hand, a peak that is diffusely elevated points to polyclonal hypergammaglobulinemia. Complete absence of the peak is associated with Swiss-type agammaglobulinemia.

• The complement-fixation (CF) test is a two-stage test. The first stage involves the union of antigen with its specific antibody, followed by the fixation of complement to the antigen-antibody structure. In order to determine whether complement has been "fixed," an indicator system must be employed to determine the presence of free complement. Free complement binds to the complexes formed when red blood cells (RBCs) are mixed with anti-RBC antibody; this binding causes lysis of the cells. Complement that has been "fixed" before addition of red blood cells and anti-RBC antibody cannot cause lysis.
• A DNA probe is available. It is an 125I probe for the 16S ribosomal RNA of M. pneumoniae.
• Falsely positive ELISAs and Western blots is an influenza vaccination within the past few months.

• COMPLEMENT AND DISEASE:

Patients with deficiencies of the middle complement components (C3 and C5) are at risk for recurrent pyogenic infections,. Individuals with C3 deficiency have recurrent serious pyogenic bacterial infections that can be fatal. The absence of C3 leads to the inability to generate the opsonin, C3b, which, when deposited on the surface of the bacteria, promotes phagocytosis. Isolated C3 deficiency typically presents at a very early age, most often shortly after birth.

Individuals with C2 deficiency have a predisposition for immune complex disease such as systemic lupus erythematosus.

while those lacking terminal complement components (C6, C7, or C8, but not C9) are prone to developing recurrent infections with Neisseria species.

A deficiency of decay-accelerating factor (DAF), which breaks down the C3 convertase complex, is seen in paroxysmal nocturnal hemoglobinuria (PNH), a disorder that is characterized by recurrent episodes of hemolysis of red cells because of the excessive intravascular activation of complement.

Deficiencies of C1 esterase inhibitor result in recurrent angioedema, which refers to episodic nonpitting edema of soft tissue, such as the face. Severe abdominal pain and cramps, occasionally accompanied by vomiting, may be caused by edema of the gastrointestinal tract. To understand how a deficiency of C1 inhibitor can cause vascularly produced edema (angioedema), note that not only does C1 inhibitor inactivate C1, but it also inhibits other pathways, such as the conversion of prekallikrein to kallikrein and kininogen to bradykinin. A deficiency of C1 inhibitor also leads to excess production of C2, a product of C2 called C2 kinin, and bradykinin. It is the uncontrolled activation of bradykinin that produces the angioedema, as bradykinin increases vascular permeability, stimulates smooth muscle contraction, dilates blood vessels, and causes pain.

·         CLASS-SWITCHING: Changes in the constant-region germline, to change isotypes (from IgM to IgG, etc.)

Antigen binding to a BCR (IgM) causes switching from IgM or IgD ------> IgG, IgA or IgE.

The V region remains the same, such that the cell retains its same specificity, but the constant regions change to a different class.

-          SWITCH SITES: This occurs by a process called switch recombination, in which all constant-region genes (DNA) are deleted in progeny, except the ones being expressed.

Switch sites are located in the introns, proximal (5') to each CH locus.

They contain tandem repeats that are highly conserved across species.

-          REVERSIBILITY: Switching is for the most part irreversible, because intervening C regions are deleted. However, a C-region that remains downstream remains intact and can thus still be switched to.

-          Double Class-Switching is possible with help of IL-4, which induces class two class switches in a row in the process of making IgE:

mu () ------> gamma-1 (gamma1)

gamma-1 (gamma1) ------> epsilon ()

·         T-cell markers (e.g., CD4 and CD8) have functional significance, whereas B-cell markers (e.g., CD19 and CD20) are primarily of maturational significance.

·         Opsonization: Facilitating phagocytosis. Neutrophils have lots of opsonic receptors.

Neutrophils have Fc-Receptors, to recognize the Fc (common) portion of an IgG molecule bound to antigen. This extra affinity facilitates phagocytosis of the antigenic material.

Neutrophils have CR1, CR2, CR3 receptors, all of which bind C3b, a Complement degradation product. Thus, the complement pathway facilitates opsonization of antigen.

·         AGRETOPE: The region of the antigen that binds to the MHC complex. A given MHC molecule can bind a variety of different antigens.

·         EPITOPE: The region of the antigen that binds to the T-Cell Receptor (TCR).

·         IDIOTOPE: The individual antigenic determinant region of an immunoglobulin.

The idiotope may or may not be the same as the antigen-binding site, or it may overlap with it.

Each antibody has multiple idiotopes within it -- those regions capable of acting as antigen.

·         IDIOTYPE: The sum of the idiotopes of an immunoglobulin.

Monoclonal antibodies, all with same structure, all have the same idiotypes.

IDIOTYPIC DETERMINANTS: Common idiotopes that tend to show up in immunoglobulins even if they are polyclonal. This is due to the common germline structure of immunoglobulins.

·         ISOTYPE: Constant-region antigenic determinants that are species-specific. They are contained with the constant region of an Ig and are thus endemic to the species.

·         ALLOTYPES: Antigenic determinants that are inherited, and thus represent allelic variation within the constant region.

Only some of the constant region domains have allotypes (i.e. multiple alleles). Others do not, at least not known.

·         ANTIGENIC SHIFT: Making major variations to the viral protein coat, by means of Natural Selection and mutation. This makes it difficult to continually defend against the virus.

ANTIGENIC DRIFT: Minor variations to the viral protein coat. graDually, due to point mutation

·         MITOGEN: Substances that are non-specific polyclonal activators of lymphocytes. They induce polyclonal lymphocyte proliferation.

Lectins: Polyclonal activators that bind to common sugar-residues found on many cells. They can induce agglutination and then proliferation of lymphocytes.

CONCANAVALIN-A (ConA): A polyclonal T-Cell mitogen.

PHYTOHEMAGGLUTININ (PHA): A polyclonal T-Cell mitogen.

Pokeweed Mitogen: A polyclonal activator of both B and T cells.

·         PAPAIN: Protease cuts right above the joining region, generating three parts.

PEPSIN: Protease cuts right below the joining region, generating single main fragment