HUMORAL INNATE IMMUNE SYSTEM ACUTE PHASE

PROTEINS TEST
EXCERCIES NO. 1 1
ACUTE PHASE PROTEINS
 The body's initial responses to immune stress encompass innate mechanisms and a non-specific reaction
that precedes the specific immune response.

 The acute phase response represents a significant systemic reaction of the organism to disturbances in
local or systemic homeostasis induced by infection, tissue damage, malignant growth, or immune
disorders.

 At the site of microorganism invasion or tissue injury, a cascade of responses within the tissue itself is
initiated. This includes the release of inflammatory cytokines and activation of the vascular system and
immune cells.
 These reactions trigger the secretion of additional cytokines and other inflammatory mediators, which
traverse intercellular spaces, enter the bloodstream, and circulate throughout the body.
ACUTE PHASE PROTEINS

 Infection

 Tissue damage

 Tumor growth

 Immunological disorders
• Acute phase changes encompass alterations in the concentrations of numerous plasma proteins, referred to as
acute phase proteins (APPs), as well as various modifications in behavior, physiological processes, biochemical
reactions, and nutrition.

• An acute phase protein is characterized as a protein whose concentration changes, either positively or negatively, during
inflammatory diseases.

• The changes in protein concentration primarily hinge on synthesis occurring in the liver. The extent of these changes
varies significantly, ranging from approximately 50% for proteins like ceruloplasmin and complement proteins to
over 1000-fold for proteins such as C-reactive protein and serum amyloid A, which serves as the precursor for
amyloid A, leading to secondary deposits in tissues.
ACUTE PHASE
PROTEINS
POSITIVE APPS
 CONDITIONS ACCORDING TO STRENGTH OF CHANGES

• Significant changes: infection, trauma, surgery, burns, malignant process

• Moderate changes: intense exercise, childbirth, heatstroke
• Minor changes: psychological stress, psychiatric illnesses

The escalation of protein levels during the acute phase varies among
individuals, even among patients afflicted with the same disease.

Therefore, febrile patients may have a normal concentration of C-reactive protein but experience changes
in other components of acute phase proteins.

The individual differences in regulating the concentration of acute phase proteins suggest that each
individual secretes different cytokines in varying concentrations in distinct pathological conditions.
REGULATION OF ACUTE PHASE PROTEINS

 The acute phase response consists of a local reaction at the site of damage characterized by numerous reactions, such as the
aggregation of blood platelets, the formation of a clot, the expansion and permeability of blood vessels, as well as the
accumulation and activation of mononuclear cells (granulocytes, monocytes) that release cytokines.

 In addition, activated fibroblasts and endothelial cells are capable of secreting cytokines. These mediators act on
specific receptors on various target cells, leading to a systemic reaction marked by fever, increased white cell count
(leukocytosis), heightened red cell sedimentation, increased secretion of adrenocorticotropic hormone (ACTH) and
glucocorticoids, activation of the complement and clotting cascade, diminished iron and zinc levels in plasma, negative
nitrogen balance, and significant alterations in the concentration of certain plasma proteins.

 The ultimate goal of acute phase proteins is to alleviate disruptions to homeostasis and establish a robust defense function in
the body, a state defined as a negative feedback loop or complete recovery. The failure to rectify disturbances in
homeostasis results in a positive feedback loop, leading to chronic inflammation.
 INDUCTION OF ACUTE PHASE PROTEINS BY CYTOKINES AND OTHER
SIGNALING MOLECULES

 Cytokines are intracellular signaling polypeptides produced by activated cells. Most cytokines originate from multiple
cells, affect multiple target cells, and have multiple actions.
 Cytokines, which are produced during inflammation and participate in inflammatory processes, are the main
stimulators of acute phase proteins. Inflammatory-associated cytokines include interleukin-6, interleukin-1beta, tumor
necrosis factor-alpha, interferon-gamma, transforming growth factor-beta, and possibly interleukin-8.
 They are produced by different types of cells, but their most important source is macrophages and monocytes at the
site of inflammation.

 Interleukin 6 (IL-6) is the main stimulator of the creation of most of the acute phase proteins, while other cytokines
have an influence on separate subsets of the acute phase proteins. In addition to its effect on liver cells, IL-6 also acts
on several other target cells. Cytokines act as both a ladder (cascade) and a network in the inducing effect of acute
phase protein production.
 Many cytokines have the ability to regulate the production of other cytokines and cytokine receptors.
Tumor necrosis factor-alpha is the primary inducer in the generation of interleukin-1 in patients with rheumatoid arthritis
(inflammation of the joints). Interleukin-1beta, in turn, can either increase or decrease the production of its own receptors.
 Cytokines are integral components of a complex signaling network. Cells are seldom exposed to just one cytokine; more
commonly, they encounter a combination of cytokines with diverse biological actions.

 The impacts of cytokines on target cells can be either inhibited or stimulated by other cytokines, hormones, cytokine receptor
antagonists, and circulating receptors. Combinations of cytokines can yield additive, inhibitory, or synergistic effects.

 The expression of genes for acute phase proteins is primarily regulated at the transcriptional level, although post-transcriptional
mechanisms also play a role.

 Post-translational changes in plasma protein glycosylation during the inflammatory response encompass damage to
oligosaccharide branches, heightened sialylation of orosomucoid, and reduced galactosylation of IgG.

 Changes in oligosaccharide branches are prompted by inflammatory cytokines, independent of their impact on acute phase
protein generation.
 IMMUNOTURBIDIMETRY

• Acute phase proteins in bodily fluids are assessed using various methods,
one of which involves immunoturbidimetry utilizing a turbidimeter.

• The turbidimetry measurement system operates on the principles of kinetic

turbidimetry.
• The turbidimeter concurrently measures two parameters: the maximum
reaction rate (Vmax) of the formed precipitate and the time required to
reach this maximum rate (tVmax).

• Plotting these values against concentration yields a graphical

representation.
 IMMUNOTURBIDIMETRY

Nephelometry and turbidometry entail measuring the light scattering and light absorption properties, respectively,
of antigen-antibody complexes. These techniques are utilized to ascertain protein concentrations in serum or
cerebrospinal fluid. The tests are rapid and highly sensitive.

The composition of the reagents ensures that the two

curves complement each other. Measurements are
conducted at a wavelength of 340 nm (nanometers).

The measurement of short wavelengths facilitates the

detection of minute concentrations of antigen/antibody
complexes, which form within a few seconds after mixing
the reagent and the sample.

The light beam is generated from a xenon source and

transmitted through two-channel quartz fiber optics. The
light beam entering the cuvette is recorded every 0.1
second and analyzed by a precise combination of software
and hardware.
 PROCEDURE

The Turbiquant bottle is inserted into the reagent compartment, initiating the
temperature compensation process.
A sample dilution is then prepared based on the specific acute phase protein or
immunoglobulin being measured.
Next, pipette 50 microliters of the pre-diluted sample into the cuvette of the Turbittimer,
followed by insertion of the cuvette into the instrument.
Subsequently, pipette 50 microliters of the reagent.
The mixing of the reagent and sample, as well as the measurement and printing of
results, is carried out automatically.
While the measurement is underway, preparation for the next sample takes place.
The measured results are then recorded in a table.
 TEMPERATURE COMPENSATION (+18°C+32°C)

• The light beam is sourced from a xenon emitter and transmitted through dual-channel quartz fiber optics.
• Measurements are conducted at a wavelength of 340 nm.
• The light beam is recorded every 0.1 second and subsequently analyzed by software.
 PRINCIPLE NEPHELOMETRIC OR TURBIDIMETRIC IMMUNOANALYSIS

The method operates with an excess of antibodies in a zone where the antibody concentration remains constant, and the quantity of
antigen-antibody complexes formed is directly proportional to the antigen concentration in the mixture.
This facilitates the formation of complexes of consistent size, ensuring a reproducible, stoichiometric relationship between the
number of complexes formed at a specific antigen concentration.
Polymers or hydrophilic agents are introduced to expedite immunoprecipitation in both nephelometry and turbidimetry. The most
desirable polymer attributes include a high molecular weight, a high degree of linearity, and substantial aqueous solubility.
The influence of polymers on enhancing immunoprecipitation is likely attributed to steric exclusion of water, resulting in several
effects:
(a) Reduced solubility of protein molecules.
(b) Promotion of antigen-antibody interaction, leading to the formation of an immunoprecipitin.
(c) Increased slope of the excess antibody surface in the immunoprecipitation curve.
(d) Shift of equivalence towards higher antigen concentration.

The most effective agent for accelerating complex formation, significantly reducing reaction time, and increasing the peak rate up to
tenfold, is polyethylene glycol (PEG) with a molecular weight of 8000 Da.
 CALIBRATION

The calibration curve does not need to be performed for the Turbitimer system. Instead, Vmax and tVmax for a specific
concentration are measured at various temperatures (+18°C to +32°C) during the calibration procedure. These Vmax and
tVmax parameters, along with concentration, are displayed in a single graph, creating a three-dimensional curve.

Calibration curves are established for each reagent and production lot (identified by a production number). These curves are
then translated into a line code (barcode), printed, and recorded for each reagent. They are only read once at the outset of
using the reagent.
ERYTHROCYTE SEDIMENTATION RATE AND C-REACTIVE PROTEIN

 The significance of CRP lies in its prognostic rather than diagnostic value, as it indicates a wide
range of changes in a short time.

 Amyloid is more specific but not routinely used. It is employed for monitoring confirmed
pathological conditions and assessing the effect of therapy, yet it is not disease-specific.

 Sedimentation of erythrocytes is also a good marker of inflammation or pathological processes;

however, it lacks specificity.
 CONDITIONS OF CRP PRESENCE

Regularly present Occasionally present Absent

Rheumatic fever Local infections
Acute myocardial Rheumatoid arthritis Skin diseases
infarction
Стрептококна инфекција перикардитис Горно респираторни
инфекции
пневмонии Пиелонефрит Бубрежни заболувања
 THE MOST COMMONLY USED PROTEIN IN ACUTE PHASE

•Inflammation is a complex, highly orchestrated process involving many types of cells and molecules. Some of
these elements initiate, amplify, or maintain processes, while others prevent them, and still others allow the process to
resume.

•Many acute phase proteins have the potential to influence one or more of the inflammatory processes.

•The main function of C-reactive protein, a component of the innate humoral immune system, is its ability to bind
phosphocholine, thereby recognizing certain foreign agents as well as phospholipid structures from damaged cells.

•C-reactive protein can activate the complement system by binding to one of its ligands. Additionally, it binds to
phagocytic cells, which is explained by its simultaneous effect on the removal of target cells by both the humoral and
cellular immune systems.
 THE MOST COMMONLY USED PROTEIN IN ACUTE PHASE

• The most commonly used indicators of the acute phase response are the sedimentation of erythrocytes and the plasma
concentration of CRP. The sedimentation of erythrocytes mainly depends on the concentration of fibrinogen in
plasma.

• Measuring CRP has several advantages over erythrocyte sedimentation rate. Sedimentation of erythrocytes is an indirect
measure of the concentration of proteins from the acute phase and can be greatly influenced by the size, shape, number
of red cells (erythrocytes), as well as the concentration of immunoglobulins. Therefore, the results of erythrocyte
sedimentation are imprecise and sometimes meaningless.

• Sedimentation of erythrocytes is considered an outdated method for the indirect examination of fibrinogen in plasma, as
today there are direct and precise methods available for its measurement.
 SEDIMENTATION IN RELATION TO CRP

• As the patient's condition changes (improvement or deterioration), the sedimentation of erythrocytes changes slightly,
while the concentration of CRP in plasma changes sharply.
• The range of pathological values for CRP is much wider than that for erythrocyte sedimentation, with diagnostic
implications: in patients with a plasma CRP concentration greater than 100 mg/L, 80 to 85% have a bacterial
infection.

• Sedimentation of erythrocytes constantly increases with age, while the concentration of CRP in plasma does
not change.

• However, many patients with active systemic lupus erythematosus do not have high plasma CRP concentrations , but
they do during bacterial infection. The application of this knowledge for the differential diagnosis of fever in
patients with systemic lupus erythematosus is limited by findings indicating that the concentration of CRP in
plasma is increased in patients with active lupus serositis or chronic synovitis.
 CRP VALUES- MEANING

• Healthy individuals typically have plasma CRP concentrations of 2 mg/L or less, though some may have
concentrations as high as 10 mg/L.

• More recent studies indicate that values below 10 mg/L have no clinical significance.

• However, a CRP concentration below 10 mg/L, but significantly above the values observed in the healthy population,
shows a significant association with osteoarthritis and may also predict later coronary syndrome.

• Therefore, it is considered that slightly increased concentrations (from 2 to 10 mg/L) could indicate
a mild chronic inflammatory reaction in the body.