Basic Principles and Concepts II: Immunology and Pathology

Website Readings:Evasion of Immunity http://www.aber.ac.uk/parasitology/Edu/EvasImmu/ImmuTxt1.htm Parasite Diagnosis - http://www.aber.ac.uk/parasitology/Edu/Diag/DiagTxt.html CDC Diagnostic Procedures -

http://www.dpd.cdc.gov/dpdx/HTML/DiagnosticProcedures.htm

A basic tenet in parasitology is that as a symbiont becomes more specialized, it increases its host specificity (i.e. has fewer hosts it can colonize).

The host reacts to the presence of the symbiont by mounting an immune response to the symbiont; a successful symbiont then mounts an effective defense or evasive strategy.

This amounts to an “arms race”.Much of the disease caused by parasitic infections is actually due to the host’s

defense mechanisms, not caused directly by something the parasite does.

SUSCEPTIBILITY AND RESISTANCE

susceptible - cannot eliminate a parasite before it become establishedresistant - the physiological status of the host prevents establishment and survival

of the parasiteThese terms are not absolute; some hosts may be more or less susceptible than

another.

immunity - synonymous with resistance --> implies a sensitive and specific immune response exhibited best by vertebrates, but also seen in invertebrates

innate immunity- mechanism of defense that does not depend upon prior exposure to the invader

acquired immunity - immunity specific for nonself; takes time to develop; does require exposure to invader; second exposure is often quicker and more vigorous; basis behind vaccines

premunition - resistance to reinfection or superinfection conferred by an existing infection that does not destroy the organisms already established; prevents any further infectionalso known as concomitant immunity

INNATE DEFENSE MECHANISMS

PHYSICAL AND CHEMICAL BARRIERS

Unbroken skin of most hosts provides a very effective barrier to invading organisms.May be tough and keratinized or protected by a layer of mucus.Some organisms contain substances present in the skin that are parasiticidal

magainins - antibacterial peptide present in skin of African clawed frog squalamine - antimicrobial steroid in skin of sharks defensins - produced by neutrophils and macrophages; increase membrane

permeability of invader lysisThere are hydrolytic enzymes in stomach of most organisms.Other molecules that may help prevent invasion by parasites includes:

Mucus - contains IgA (class of antibody found associated with intestine) Lysozyme (enzyme that degrades bacterial cell walls)

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Interferons - low molecular weight molecules released by eukaryotic cells in response to invasion.

Tumor necrosis factor (TNF) released by macrophages and activated T cells and natural killer cells; major mediator of inflammation, including fever.

Complement pathway is an innate defense mechanism triggered by bacteria and some fungi --> small holes are poked into invaders --> cell lysis

CELLULAR DEFENSES: PHAGOCYTOSIS

Almost all organisms engage in phagocytosis, engulfing an invader (nonself); also a feeding mechanism in some protozoa.

Fate of phagocytosis is digestion by lysosomes (contain hydrolytic enzymes)Lysosomes also contain reactive oxygen intermediates (ROIs) and reactive

nitrogen intermediates (RNIs) --> potentially toxic to invaders.Most organisms have specialized cells that engage in phagocytosis.Invertebrates contain amebocytes, coelomocytes, hemocytes, etc. that engage in

phagocytosis.If a particle is small, it is phagocytized; if large, it is encapsulated.Arthropods can wall off invading organisms using melanin to create an

encapsulation.Vertebrates have monocytes (macrophages in lymph nodes, Kupffer cells in liver,

or microglial cells in CNS).Circulating phagocytes are called polymorphonuclear leukocytes (PMNs).

neutrophils - most abundant granulocytes or PMNseosinophils - high in [ ] in allergic reactions and parasitic infectionsbasophils - involved in immediate hypersensitivity

Mast cells are found in dermis and are basophil-like; release substances that affect nearby cells.

Lymphocytes - critical to immune response; are B-cells and T-cellsNatural killer cells - kill viruses and tumor cells in the body by lysing them

ACQUIRED IMMUNE RESPONSE IN VERTEBRATES

Defined as the ability to discriminate between self and nonself.antigen - foreign substance that elicits an immune response; usually protein

greater than 3000 daltonsThere are two arms to the immune response:

1) humoral immunity - based on antibodies, both cell-bound and soluble in blood and lymph fluids2) cellular immunity - associated with cell surfaces

Much communication and interaction between humoral and cellular immunity.

BASIS OF SELF AND NONSELF RECOGNITION

Molecular basis of nonself recognition involves proteins embedded on cell surfaces; known as major histocompatibility complex or MHC molecules.

There are two types of MHC molecules: 1) class I - found on almost all cells2) class II - found only on cells such as lymphocytes and macrophages

The two different types of molecules that are recognize foreign molecules are1) antibodies2) T-cell receptors

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Antibodies or immunoglobulins are either displayed on surface of B lymphocytes or B cells or secreted by plasma cells (derived from B cells).

Antibody structure: Contains 4 polypeptide chains: 2 heavy and 2 light held together by disulfide

bonds and hydrogen bonds. Contain on ends of heavy and light chains (end of arms of Y) a variable

region known as the Fab region or antigen binding site. Constant end (end of Y) is Fc region; binds to cell surface or other molecules. Heavy chains may be one of five types: mu, gamma, alpha, delta, epsilon --->

IgM, IgG, IgA, IgD, or IgE.Antibodies mediate destruction of an invader by one of the following ways:

invader can become coated in antibodies (known as opsonization) and then engulfed by macrophages

antibodies coated on invader may activate complement pathway --> lysis antibodies coated on invader may trigger antibody-dependent cell-

mediated cytotoxicity (ADCC)Can happen in T. cruzi infections, T. spiralis, and S. mansoni infections.

T-cell receptors are very similar in structure to antibodies; have other molecules associated with these receptors: CD4 and CD8 molecules.

There are a great many different T cells with receptors for specific antigens.Lymphocytes are activated after they bind to antigens --> proliferation and

differentiation with the goal of eliminating the antigen.There are many different subsets of T cells that are distinguished by the types of proteins displayed on their surface:

CD4+ - T- helper cells; activate either humoral or cell-mediated immunity

CD8+ - cytotoxic T cells - bind to target cell and secretes a protein that causes pores to form in cell membrane --> lysis of

target cell

Humoral ImmunityAntigens bind to a specific antibody on the surface of a B cell; some antigens are

taken up by antigen-presenting cells (APCs), which partially digest the protein, then display a portion of the antigen on their surface along with class II MHC molecules.

That portion of antigen displayed on cell surface is an epitope or determinant. --> binds to T cell receptor on T helper cells --> secrete cytokines.

Some of these cytokines activate the previous B cell to proliferate, and produce plasma cells, which secrete antibodies --> accumulation of antibodies in blood ---> titer ([antibody]) rises.

If we challenge or give another dose of antigen, secondary or anamnestic response is much faster and causes further rise in antibody titer.

This is due to the previous generation of memory B cells --> don’t have to go through antigen presenting, cytokine stimulation , etc. ---> faster rise in antibody levels.

Cell-Mediated Immunity

Some antigens cause generation of cell-mediated immunity rather than antibody production; some do both, with one arm of the immune system stronger than the other.

There is still antigen-presenting, but macrophages, PMNs, cytotoxic T cells, natural killer cells are activated instead of B cells.

Inflammation occurs when body’s defenses are mobilized by an invader or tissue

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damage.This can be illustrated by delayed type hypersensitivity (DTH) or immediate

hypersensitivity.DTH is caused by activated macrophages at least 24 hours after exposure.Tumor necrosis factor causes endothelial cells around blood vessels to display a

protein (selectin) to which leukocytes adhere; also makes endothelial cells “leaky” so that leukocytes can pass from the blood to the tissues.

Monocytes, drawn to area by above mechanisms, become activated macrophages in tissue --> phagocytize antigen, secrete cytokines and growth factors to repair damage.

If antigen is not destroyed and removed, its presence causes deposition of fibrous connective tissue --> fibrosis --> nodules of granulomas accumulate, particularly around persistent antigen (live parasites).

Immediate hypersensitivity involves degranulation of mast cells in area --> histamine released --> causes vascular dilation and increased vascular permeability.

Escape of plasma into surrounding tissue causes swelling (wheal),Systematic immediate hypersensitivity can cause anaphylaxis.Some cells always die (necrosis) in inflammation; if confined to a particular area the

leukocytes and tissue fluid create pus --> increase in pressure --> abscess.If this opens out onto skin or mucous surface --> ulcer.

IMMUNODIAGNOSISMost often parasitic infections diagnosed by presence of eggs or adult organisms. Not always possible.Tests have been developed to do this.Six additional techniques are described:

indirect hemoagglutination (IHA) testo RBCs are incubated with antigen, then incubated with patient serum

containing antibodies.o RBCs then agglutinate in presence of antibody --> positive

indirect fluorescent antibody (IFA) testo Parasite are fixed on a microscope slide, then incubated with test

serum, washed, treated with antibodies against IgG labeled with fluorescein --> fluorescence means that person had antibodies to parasite.

complement fixation (CF) testo Test serum is incubated with parasite antigen in presence of guinea

pig complement.o Antibody binds to antigen to form antigen-antibody complex.o Sheep RBCs added along with antibody to sheep RBCs.o If lysis occurs, complement did not fix earlier --> no antibody present

in test serum.o If lysis does not occur, complement was fixed earlier --> antibodies

were present in serum. enzyme-linked immunosorbent assay (ELISA)

o Antigen is applied to wells of a microplate - serum added to wells and allowed to incubate.

o Wells are washed to remove unbound antibody.o Antibody to IgG is added (anti-Ig) that is covalently coupled to an

enzyme.o Wells are washed again to remove unbound anti-Ig.o Substrate is added to wells.

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o If serum contained antibodies, anti-Ig will have bound --> color.o If serum did not --> no color change.

“sandwich” ELISAo Used to detect parasite antigen rather than antibodies to

parasite.o Antibody to the antigen is applied to wells of a microplate

and allowed to incubate.o Test serum is added to wells and allowed to incubate.o Enzyme-labeled antibody to antigen is added to wells.o Color reaction occurs if parasite antigens detected.

“dipstick” ELISAo Modification of sandwich ELISA using dipstick of acetate

plastic.o Antibody to antigen is adsorbed onto nitrocellulose.o Used to detect parasite antigens in feces in the field.

o Also used as ParaSight®-F test to detect Plasmodium falciparum.

o Use a drop of blood that is hemolyzed by detergent.o End of dipstick is immersed in drop of blood.o Blood is absorbed by nitrocellulose and travels up dipstick.o A solution containing antibody coupled to colored reagent

is added.

Immunoblot Technique

Often more specific and accurate than enzyme immunoassay (EIA) because there is little chance of cross-reactivity (i.e. false positive).Used to diagnose T. cruzi infection in humans suspected of cysticercosis.

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