Inflammation is an important physiological response for the maintenance of tissue homeostasis, protecting the host against foreign substances, invading microorganisms and host self disturbances, such as the molecules derived from metabolically stressed or even dying cells. After the host has been incited, important microcirculatory events occur in response to local release of pro inflammatory mediators (prostaglandines, histamine, leukotrienes, chemokines, cytokines) leading to increased vascular permeability and leukocyte recruitment. Inflammatory cells, such as neutrophils and macrophages release further inflammatory mediators and acting as effector cells and phagocytes to remove the inflammatory agents and stimuli .
The involvement of the whole body in the acute inflammatory response is known as an acute-phase reaction (APR). During an APR, mechanisms that operate remote from the site of local inflammation can overwhelm the normal homeostasis of the organism. Many organ systems become involved in the host-defense process, primarily through systemically active inflammatory cytokines .
Continuous inflammatory stimuli (longer than three months), also known as chronic inflammation, can be detrimental to the organism and are predisposing factors for many chronic diseases. Chronic inflammation is a predictor of disease and mortality, even in the absence of clinical symptoms. This applies in particular to individuals with low-grade systemic inflammation, who have slightly elevated inflammatory markers such as C-reactive protein compared to the rest of the population. The secretion of mediators of inflammation by white adipose tissue in obesity, for example, results in low-grade inflammation and an increased long-term risk of cardiovascular disease, stroke and type 2 diabetes /, /.
In patients with severe trauma, survival rates depend on whether inflammatory states such as acute lung injury (ALI), adult respiratory distress syndrome (ARDS), and multiple organ dysfunction syndrome (MODS) and their associated complications are present.
Complex organisms have three mechanisms to defend against foreign substances and invading pathogens:
- The skin and mucous membranes act as the first barrier
- The innate immune system provides the second line of defense
- The last line of defense is the antigen-dependent acquired immune system.
Inflammation is the innate immune system’s response to tissue injury. Because the response to a particular foreign agent is always the same (qualitatively and quantitatively) in the same patient, the response to re-exposure is similar to the initial response.
Examples of inflammatory stimuli include bacteria, viruses, parasites, allergens, immune complexes, and mechanical and surgical tissue damage (myocardial infarction, bone fracture, foreign agents, erythrocyte shear stress, hydrostatic pressure, urate crystals, and uremia).
Symptoms of local inflammation
Pain (dolor), heat (calor), redness (rubor), swelling (tumor), and loss of function (functio laesa).
- Microcirculatory events; dilation of arterioles, capillaries, and venules, associated with increased blood flow lead to higher vascular permeability
- Exudation of fluid and plasma proteins into the tissues
- Leukocyte recruitment and migration to the inflammatory focus.
From a pathophysiological perspective, local inflammation comprises a broad range of cellular, intercellular, and intracellular processes that facilitate the phagocytosis of the foreign agent and recruit additional cells to the inflammatory process. The type of inflammation that is triggered by each different foreign agent represents a complex, concerted, finely-tuned process that is focused on the inflammatory stimulus, in which soluble inflammatory mediators, immune cells, and local tissue cells interact with one another. If local inflammation develops into an acute-phase response, the local process is then controlled by the systemic metabolic changes associated with the acute-phase reaction . The inflammatory response is highly individualized. For example, one individual might experience a bacterial peritoneal infection as a well-encapsulated abscess, while another might develop diffuse peritonitis and possibly multi organ failure .
Local inflammatory effects
Various stimuli trigger a cellular, inter cellular, and intracellular inflammatory response at the site of tissue damage via the following mechanisms:
- Activation of the complement system, kinin system, and coagulation cascade. The products of these systems, such as C5a, bradykinin, and thrombin, increase vascular permeability, attract leukocytes, and alter vasomotor tone at the inflammatory site. These soluble mediators induce cellular reactions. Local macrophages are activated and produce pro inflammatory mediators such as TNF-α, IL-1β, and chemokines. Chemokines recruit leukocytes and determine which leukocytes will accumulate at a particular site at a particular time.
- Stimulation of inflammatory cells such as polymorphonuclear neutrophils, eosinophils, monocytes/macrophages, dendritic cells, mast cells, fibroblasts, smooth muscle cells, thrombocytes, and lymphocytes. Intercellular actions occur: inflammatory cells interact with each other and with vascular endothelial cells. Lipid mediators (eicosanoids), reactive oxygen species, heat shock proteins, and selectins are produced.
- Activation of intracellular communication in inflammatory cells. This is a prerequisite for synthesizing new mediators such as cytokines, chemokines, and adhesion molecules, which in turn involve polymorphonuclear neutrophils, monocytes, T-cells, and dendritic cells in the inflammatory process. The intracellular processes for producing inflammatory proteins depend on the transcription of particular DNA sequences under the control of transcription factors. Nuclear factor kappa B (NF-kB) is a pro inflammatory transcription factor that has an important role in regulating the synthesis of RNA and, therefore, proteins . In non-activated cells, NF-kB is present in the cytoplasm bound to the inhibitor kappa B (IkB) and prevents NF-kB from permeating into the nucleus. When an inflammatory cell becomes activated (e.g., by TNF-α) IkB is phosphorylated and degraded. This enables NF-kB to enter the nucleus, where it binds to the promoter region of a target gene and initiates the synthesis of inflammatory mediators (). NF-kB is essential for normal immune function, however, over stimulation can lead to inflammation and tumorigenesis .
The innate immunity represents the first barrier in the host immune defense; it identifies pathogens or other harmful triggers inducing an inflammatory process with the aim of blocking their diffusion, and activates adaptive immunity. The effectors are:
- The complement system
- Inflammatory proteins e.g., C-reactive protein
- Effector cells of innate immunity, e.g., macrophages, dendritic cells and other antigen presenting cells (APC).
The innate immune system acts through pattern recognition receptors which bind to highly conserved structures expressed by pathogens (pathogens associated molecular patterns, PAMPs) or by damaged cells (damage associated molecular patterns DAMPs) . More specific recognition (immune recognition) takes place subsequently by cellular (T and B-lymphocytes) and humoral components (antibodies) of the adaptive immune system.
- Toll-like receptors (TLRs)
- Retinoic acid-inducible gene-I (RIG-I-) like receptors (RLRs)
- Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs).
The alternative complement pathway is activated by complex structured molecules and by denatured foreign agents. These include lipopolysaccharides in the cell walls of Gram-negative bacteria (endotoxins), viral envelopes, and denatured erythrocyte membranes. Inflammation is initiated by :
- The formation of membrane attack complex C5b-9, which punches holes in the bacterial and cellular lipid membranes. The membrane attack complex is particularly effective against Gram-negative bacteria. It is formed following activation of the complement cascade through the deposition of complement component C3b on the surface of the target cell. Target cells coated with C3b are recognized by macrophages, which engulf the target cell and digest it.
- Generation of anaphylatoxins C3a and C5a. Both of these induce contraction of smooth muscles in vessel walls, increased vascular permeability, and mast cell degranulation. C5a is a chemoattractant and induces the accumulation of neutrophilic granulocytes and macrophages at the site of tissue damage.
The adaptive immune system recognizes foreign agents using surface receptors on macrophages, T-cells, and B-cells as well as antibodies produced by B-cells. After binding of an antigen to the B-cell receptor the cells differentiate into plasma cells, which synthesize IgM and IgG antibodies against the corresponding antigen. Immune complexes are formed, which activate the classic pathway of the complement system and trigger an inflammatory response.
HSP are usually found in various intracellular compartments and some have chaperone and protease functions . Chaperones are proteins that are involved in the assembly, folding, and stabilization of oligomeric proteins. HSPs are classified according to their molecular weight (e.g., 60 kDa HSP or 70 kDa HSP).
HSPs are released into the extracellular space by necrotic and stressed (but not apoptotic) cells, where they usually constitute 5–10% of the total protein. An increased presence always indicates cellular compromise or unnatural cell death. HSPs have a pro inflammatory effect in the extracellular compartment. They act as intercellular signaling molecules to induce the production of pro inflammatory mediators, and can trigger an inflammatory response. They cause vascular endothelial cells to express E-selectin, adhesion molecules such as ICAM-1 and VCAM-1, and interleukin 6 (IL-6). Bacterial HSPs also induce the production of pro inflammatory mediators.
The stimulation of inflammatory cells by HSP to produce pro inflammatory cytokines occur after their uptake by macrophages and dendritic cells. These cells present HSPs together with MHC class I proteins on their cell surfaces. There they are recognized by CD4+T-cell receptors, which activate the cells to produce inflammatory cytokins. depicts the induction and regulation of HSP expression.
The recognition of foreign molecules is followed by the activation of intracellular signal transduction pathways, which induces the expression of genes for the production of inflammatory mediators. These molecules enhance the inflammatory response. Their role is to recruit other effectors of the host-defense mechanisms into the inflammatory response. Mediators are humoral substances that are produced from endogenous lipids or peptides, are secreted by activated inflammatory cells, or can even be released by invading bacteria. Initially small stimuli can be strongly amplified by mediators.
The main task of inflammatory mediators is to recruit inflammatory cells to the site where the foreign agent is localized or where tissue damage is present. The main mediators of the inflammatory response are presented in .
The eicosanoids are essential mediators of inflammation:
- Their hydrolysis from phospholipids is depicted in
- The production of different eicosanoids is shown in
- The activation of eicosanoid production via receptors in the cell membrane can be seen in .
- The metabolic products of the eicosanoids together with their functions are listed in .
Monocytes are recruited as follows:
- Proteins are released from the granules of activated PMNs. These induce the migration of activated monocytes to the vascular region of the inflammation site. The β2 integrins and formyl peptide receptors play an important role in this.
- PMNs modify the chemokine network and their granular proteins create an environment that facilitates monocyte extravasation.
- Accumulated PMNs at the site of inflammation are subject to rapid apoptosis, which leads to the release of lysophosphatidylcholine. As a result, monocytes are captured by G2A receptors.
Monocytes are extravasated as follows:
- Selectins on the vascular endothelium (P-selectins) and on monocytes (L-selectins) allow the monocyte to attach to the vascular wall and roll along its surface
- When it slows down, the monocyte can detect endothelium-bound chemokines and chemoattractants such as PAF and leukotriene B4, which activate the monocyte
- Monocyte activation leads to the release of integrins, which interact with adhesion molecules of the vessel wall (ICAM-1, VCAM-1) to fix the monocyte to the endothelium
- Chemokines than induce cytoskeletal changes in the monocyte, which lead to trans epithelial migration and extravasation.
Other important effector cells in inflammation (in addition to PMNs and monocytes) are:
- Platelets, which are recruited to the damaged vessel wall by the interaction of platelet glycoprotein (GP) Ib with von Willebrand factor and the subendothelial matrix
- Dendritic cells, which are important in chronic inflammation
- Tissue-specific mast cells and basophils, which have a role in the immediate hypersensitivity response.
The acute phase response is a highly orchestrated defense mechanism against infectious and inflammatory insults. The initiation of this response is predominantly driven by the endogenous cytokines interleukin-1β, interleukin-6 and tumor necrosis factor (TNF)-α, induced in macrophages and other leukocytes by exogenous pathogens binding to toll-like receptors. Besides systemic and metabolic changes, such as fever and anorexia, these cytokines also provide the induction of acute phase proteins in the liver .
- Fever: increased temperature increases enzymatic reactions in inflammatory cells that do not occur optimally at normal temperature due to their potentially damaging effects
- Leukocytosis: a large number of phagocytic cells become available
- Increased hormone secretion to mobilize energy from glucose, free fatty acids, and amino acids
- Synthesis of acute phase proteins (APP) and restricted synthesis of albumin, transferrin, and lipoproteins (negative acute phase proteins) to supply tissues with inflammatory proteins
- Production of hepcidin to reduce functional iron by increasing its storage in macrophages and inhibiting enteral iron absorption and hemoglobin synthesis; iron withdrawal restricts the growth of microorganisms
- Initiation of counter-regulatory mechanisms. Thus, the potentially destructive effects of the inflammation are modulated and reduced through an ACTH induced increased release of cortisol.
The APR is an integrated reaction between pro inflammatory and anti inflammatory stimuli. The systemic changes signaled in the tissues in an APR are mediated by inflammatory cytokines that are produced in inflammatory cells. Distinctions are made between (i) pro inflammatory cytokines (TNF-α, IL-1β, IL-6, transforming growth factor β, interferon-γ) and (ii)anti-inflammatory cytokines (IL-4, IL-10, IL-13).
APRs can be different. For example, comparable degrees of tissue damage caused by different agents can trigger APRs with different degrees of severity. The same injury (e.g., infection) can lead to a normal inflammatory response in one patient and an inappropriate response in another. Genetic polymorphism of cytokine production is an important cause of this phenomenon.
The goal of the acute phase reaction (APR) is to achieve an appropriate balance between activating and down-regulating mechanisms that benefits the organism as a whole . An inappropriate inflammatory response to harmless localized inflammation can prevent healing and has negative consequences for the organism that can culminate in SIRS, sepsis, or MODS, for example. The nature and dose of the antigen that is presented by the antigen-presenting cell and the genetic polymorphism of cytokine production determine the course of the APR. The extent to which the response of the T-helper cell system (also known as the T-helper cell (Th1/Th2) paradigm) is influenced is also important. The Th1 system has pro inflammatory effects while the Th2 system down-regulates inflammation (). If these systems are not in balance, this can have lethal consequences for the patient.
An appropriate balance between APR and CARS is the goal of acute inflammation. Systemically mechanisms of CARS that counteract the APR are:
- Dilution of pro inflammatory cytokines when they escape from the area of injury; they are diluted in the large volumes of the interstitial fluid and the circulation
- Production of soluble receptors (e.g., for TNF-α and IL-1β). These circulating receptors bind their ligands and thereby inhibit the binding at the receptors on target cells.
APPs are proteins whose concentration rises (positive APPs) or falls (negative APPs) by more than 25% during the course of an inflammatory disease . IL-6 is the main stimulator of APP synthesis and can induce exactly the same pattern of APP synthesis in cell cultures as in inflammatory reactions. APPs are synthesized in the hepatocytes. At the peak of the acute phase response (APR), around 20% of the protein synthesis capacity of the liver is used to produce APPs.
The positive APPs consist of a family of around 30 proteins. The peak increase fluctuates depending on the protein, from a 0.5-fold increase for ceruloplasmin to a 1,000 fold increase for C-reactive protein (CRP) . Although the APPs usually rise together in concert during an APR, there are significant differences between the response time, maximum rise, and half-life of the decline since the individual proteins are regulated separately. Refer to:
- Fibrinogen: its concentration can be normal or reduced in an APR if disseminated intravascular coagulation is also present
- Haptoglobin (Hp) with a combination of APR and intravascular hemolysis. Furthermore, 20% of African Americans have reduced Hp α-chain synthesis, which means that the normal increase in Hp during an APR is moderate.
- α1-antitrypsin: if a polymorphism with reduced synthesis is present, the protein does not increase, or does not increase in proportion to the APR. Prevalences of the genetic polymorphism: Type ZZ, 1 : 2080; type SZ, 1 : 1160; both types combined, 1 : 750.
- Complement protein C3: this does not increase appropriately with a combination of APR and immune complex disease.
During an APR, the serum concentration of most positive APPs, with the exception of serum amyloid A, increases much less than that of CRP. The concentration of the negative APPs declines. Directly or indirectly, all positive and negative APPs have an inflammatory or anti-inflammatory effect.
- Binding of exogenous and endogenous ligands and promotion of their removal from the circulation by facilitation of opsonization
- Because substances that contain serum amyloid A (SAA) increase the phagocytic activity of macrophages, SAA has a pro inflammatory effect
- Haptoglobin and hemopexin are positive APPs and protect against reactive oxygen species; haptoglobin also promotes wound healing by stimulating angiogenesis
- α1-antichymotrypsin and α1-proteinase inhibitors are positive APPs that inhibit the activity of proteolytic enzymes; α1-antichymotrypsin also inhibits the production of superoxide anions
- Transthyretin, a positive APP, inhibits the production of IL-1 by monocytes and endothelial cells
- Fibrinogen, a positive APP, mediates the binding of inflammatory cells, in particular platelets, to the vascular endothelium and plays an important role in wound healing
- Transferrin, a negative APP, reduces the functional iron so that less iron is supplied to the tissues and invading microorganisms
- Lipoproteins are negative APPs as well as the substrate for eicosanoid production via arachidonic acid metabolism and the cyclooxygenase and lipoxygenase pathways
- Albumin is a negative APP whose synthesis is inhibited by pro inflammatory cytokines. The pathophysiological effect of reduced albumin during an APR is not known.
The usual result of inflammation is protection from the spread of infection, followed by resolution (the restoration of affected tissues to their normal structural and functional state). Non resolving inflammation is a major driver of disease. Perpetuation of inflammation is an inherent risk because inflammation can damage tissue and necrosis can provoke inflammation .
Inflammation sometimes progresses from acute to chronic and then stalls for a prolonged period, although signs of acute inflammation, such as accumulation of neutrophils, may reappear later. Classic examples involve persistent infections. The most straightforward cause of non resolving inflammation is the persistence of inflammatory stimuli of exogeneous origin e.g., persistent infections by M. tuberculosis, H. pylori, schistosomes and hepatitis viruses .
Often the host inflammatory response, not the pathogen, is chiefly responsible for the damage of the host. Some chronic inflammatory diseases appear to begin with repeated exposure to a pathogen, leading to tissue injury that prevokes an autoimmune reaction. The autoimmune reaction may then perpetuate the inflammation. This is the view of chronic obstructive pulmonary disease .
Nondegradable particles of asbestos and silica that trigger inflammation promote chronic inflammation.
Acute and chronic inflammation can coexist over long periods, implying continual reinitiation. Examples are found in rheumatoid arthritis, asthma, chronic obstructive pulmonary disease, multiple sclerosis, Crohn’s disease, ulcerative colitis and cancers whose stroma is infiltrated both by macrophages and immature myeloid cells .
Non resolving inflammation is not a primary cause of atherosclerosis, obesity, cancer, chronic obstructive pulmonary disease, asthma, inflammatory bowel disease, neurodegenerative disease, multiple sclerosis, or rheumatoid arthritis, but it contributes significantly to their pathogenesis .
- In rheumatoid arthritis, psoriatic arthropathy, and Reiter’s syndrome, CRP elevation indicates a persistent inflammatory process and reflects the mass of inflamed tissue. In degenerative inflammatory processes such as osteoarthritis, CRP is not usually elevated.
- Poly myalgia rheumatica is associated with a significant elevation in CRP at diagnosis, whereas fibromyalgia is not
- While CRP can be increased in immune complex vasculitides such as Wegener’s granulomatosis and polyarteritis nodosa, it is not a direct biomarker of inflammatory activity. It is only useful for adjusting corticosteroid therapy to minimize side effects.
- In inflammatory small bowel disease, CRP is significantly increased in active ulcerative colitis but only slightly increased in Crohn’s disease. Because of the significant overlap in values, CRP is not suitable for differentiating between these two diseases. However, it can be used to differentiate inflammatory bowel disease from irritable bowel syndrome, in which CRP is always normal.
Successful pro inflammatory tissue repair requires the coordinated restitution of different cell types and structures, including epithelial and mesenchymal cells but also extracellular matrix and vasculature. Without appropriate restitution of vasculature, reduced tissue oxygenation may preclude normal tissue repair resulting in atrophy or fibrosis. Atrophy is often accompanied by expansion of extracellular tissue elements, particularly collagen, resulting in fibrosis, the deposition of excess connective tissue. Fibrosis sufficient to interfere with organ function can arise without known preceding inflammation .
Auto inflammatory diseases (AIDs) refer to a group of rare, hereditary recurrent unprovoked inflammatory disorders which occur in the absence of infection. What is auto inflammation from the pathogenetic point of view ?
- The pathological process is directed against self but the patients do not have autoantibodies or auto reactive antigen-specific T cells and B cells driving the disease process
- Monocytes and macrophages rather than T and B cells are drivers for inflammation and damage
- The chronic activation of the immune system, which eventually leads to tissue inflammation occurs in genetically predisposed individuals
- Auto inflammation includes monogenic and polygenic diseases
- Frequently the skin and musculoskeletal systems are involved
- The innate immune system directly causes tissue inflammation
- Patients affected with AID do not have associations with the major histocompatibility (MHC) class II haplotypes.
Mutations in inflammasome-related proteins, particularly in NOD-like receptor (NLR) genes are associated with the occurrence of AIDs. Inflammasomes are large protein complexes serving as a molecular platform which mediates the activation of pro-caspase-1, which cleaves the pro form of IL-1β to the active form. Activation of NLR proteins (NLRPs) results in the formation of inflammasomes. Inflammasome activation is crucial for host defense of pathogens.
AIDs are strongly linked to mutations in inflammasome-forming NLRs. The majority of patients with AIDs have mutations in either pyrin, cryopyrin (NLRP3) or tumor necrosis factor receptor super family genes .
IL-1β is not only triggered by infectious signals but also by signals released from metabolically stressed or even dying cells. NLRP3 containing inflammasomes are intracellular receptors that are triggered not only in response to exogenous pathogens but also to endogenous stress molecules and coordinate processing and IL-1 secretion through caspase-1 activation. Metabolic substrates that accumulate in target tissues can stimulate the NLRP3 inflammasome to release IL-1β. Metabolic substrates that accumulate in target tissues are urate in gout, islet amyloid polypeptide and oxidized low-density lipoprotein (oxLDL) in diabetes, ceramide and others in obesity, and cholesterol crystals in atherosclerosis .
The majority of monogenic AIDs clinically occur in neonatal period or early infancy. Polygenic AIDs usually develop during adolescence. Rarely AIDs occur in the elderly.
Reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Antiinflammatory therapy targeting the IL-1β innate immunity pathway with canakinumab leads to a significantly lower rate of recurrent cardiovascular events than placebo .
Acute graft-versus-host disease (GVHD) occurs when immunocompetent T cells in the donated tissue (the graft) recognize the recipient (the host) as foreign. The resulting immune response activates donor T cells to gain cytolytic capacity and to the attack the recipient to eliminate foreign antigen-bearing cells. Important target organs are the skin, the gastrointestinal tract, and the liver. In the early phase of acute GVHD inflammation triggers can drive both innate and adaptive immune responses .
- Sterile damage associated molecular pattern (DAMP) molecules. These molecules are released into the extracellular space only when there is tissue damage that causes immune activation.
- Pathogen associated molecular pattern (PAMP) molecules. Bacterial components engage immune system pattern recognition receptors, such as toll-like receptors and NOD receptors to activate antigen presenting cells and promote acute GVHD.
Risk factors for acute GVHD include the degree of HLA mismatch, receipt of a transplant from an unrelated donor, a female donor for a male recipient, the use of peripheral blood stem-cell grafts, and the intensity of the conditioning regimen .
- Immediate allergic reaction mediated by IgE (type 1 in Gell-Coombs classification). Re exposure to an antigen or hapten weeks after initial exposure results in the release of vasoactive substances from IgE-containing mast cells within minutes. The granules in these cells are filled with histamine and tryptase. Each cell has more than 100,000 IgE receptors. The binding of IgE to the receptors stimulates degranulation and, since mast cells are located perivascularly in the skin, lung, and intestine, the surrounding tissue is involved immediately in the reaction. In addition to the release of vasoactive substances such as histamine that cause increased capillary permeability, vasodilatation, and bronchoconstriction, lymphocytes, monocytes, and polymorphonuclear neutrophils are also attracted by chemoattractants to act as amplifiers of the inflammatory reaction. An acute phase reaction is also triggered.
- Complement-mediated anaphylaxis (Gell-Coombs type III). The complement system is activated by immune complexes and activated factors C3a and C5a cause vasoactive mediators of inflammation to be released from mast cells.
- Chemicals such as water-soluble X-ray contrast medium can provoke an anaphylactoid reaction mediated by IgE or the complement system.
A 1991 consensus conference developed initial definitions that focused on the then-prevailing view that sepsis resulted from a host’s systemic inflammatory response syndrome to infection. Sepsis complicated by organ dysfunction was termed severe sepsis, which could progress to septic shock, defined as sepsis induced hypotension persisting despite adequate fluid resuscitation . Components of SIRS include tachycardia, tachypnea, hyperthermia or hypothermia and abnormalities in peripheral blood cell count. However, the presence of SIRS is nearly ubiquitous in hospitalized patients and occurs in many benign conditions, both related to and not related to infection, and thus is not adequately specific for the diagnosis of sepsis. According to the new consensus definition of sepsis no longer includes SIRS . SIRS is defined as two or more of the variables presented in .
A fundamental component of the new definitions for sepsis and septic shock remains the presence of infection. Yet negative microbiologic cultures from blood or relevant anatomic sites are frequent in patients clinically identified as being septic . However, new techniques thus as those using matrix-associated laser de sorption ionization-time of flight (MALDI-TOF) , metabolites like myristic acid in blood or circulating microRNAs are likely to enhance the current ability to diagnose infection.
Criteria for organ dysfunction in sepsis represented by the SOFA score are listed in . The score grades abnormality of six organs in five degrees of severity (0–4) including laboratory tests, namely PaO2, platelet count, creatinine concentration and bilirubin level are needed for full computation. A severely limited function is rated with four points, normal function is rated with zero points. In total 0–24 points are possible. A score of 2 points or more is associated with an in-hospital mortality greater than 10% .
According to the new definitions, sepsis is now defined as evidence of a life threatening organ dysfunction caused by deregulated host response to infection plus life-threatening organ dysfunction, clinically characterized by an acute change of 2 points or greater in the SOFA score. The consensus document introduced a new index, called qSOFA, which helps to identify patients with suspected infection without using laboratory tests. The quick SOFA (qSOFA) requires at least ≥ 2 of the following 3 risk variables :
- Respiratory rate of 22 or more breaths per minute
- Systolic blood pressure of 100 mmHg or less
- Altered mental status (Glasgow coma scale < 15).
Septic shock should be defined as a subset of sepsis in which particularly profound circulatory, cellular, and metabolic abnormalities are associated with a greater risk of mortality than with sepsis alone . Patients with septic shock can be clinically identified by a vasopressor requirement to maintain a mean arterial pressure of 65 mmHg or greater and serum lactate levels greater than 18 mg/dL (2 mmol/L) in the absence of hypovolemia. This combination is associated with hospital mortality rates greater than 40%.
Nonsteroidal anti-inflammatory drugs (NSAIDs) are valuable agents in the treatment of arthritis and other musculoskeletal disorders, and as analgesics in a variety of clinical scenarios. It is generally accepted that NSAIDs exert their anti-inflammatory effects by inhibiting prostaglandin synthesis. Unfortunately the use of COX-2 inhibitors has been limited by their association with mucosal injury to the upper gastrointestinal tract, including the development of peptic ulcer disease and its complications, most notably upper gastrointestinal hemorrhage, and perforation .
However, individual NSAIDs have different targets:
- Indomethacin, fenoprofen, and ibuprofen inhibit the enzyme cyclooxygenase (COX) through competitive displacement of arachidonic acid from the substrate binding site
- Imidazole and dazoxiben inhibit COX leading to a significant decrease in prostaglandin production. COX exist as two isoenzymes, COX-1 and COX-2. COX-1 is a constitutive enzyme and exists in many tissues. COX-2 is an inducible enzyme and is associated with inflammation in the joints. The selective inhibition of COX-2 leads to decreased inflammation in musculoskeletal tissues.
- Acetylsalicylic acid (ASA) acts by irreversibly acetylating a serine residue within the active center of cyclooxygenase. This irreversible inhibition of cyclooxygenase is the basis for using low-dose ASA for atherosclerosis prophylaxis. Cyclooxygenase is the enzyme responsible for TXA2 synthesis in platelets and for PGI2 synthesis in the vascular endothelium. Studies have shown that the effect of ASA on vascular endothelial cells is only short-term and therefore incomplete while its effects on platelets last 8–10 days. This is because vascular endothelial cells have a nucleus and can quickly regenerate new cyclooxygenase whereas platelets do not have a nucleus and cannot generate the enzyme, which means that the effect of ASA lasts for the duration of the life of the platelet. TXA2 has a proaggregatory effect on thrombocytes and has a vasoconstrictor effect on the vascular system, while PGI2 has an effect in the opposite direction, so that ASA has an anti thrombotic effect.
- Treatment of inflammatory diseases
- Suppression of inflammation and immune responses associated with organ transplantation
- Any disease that is associated with increased expression of pro inflammatory cytokines, chemokines, and adhesion molecules.
- Inhibit the release of pro inflammatory cytokines and stimulate the production of antiinflammatory cytokines such as IL-10. Overall, inflammation is down-regulated through inhibition of the Th1 immune response and stimulation of the Th2 immune response ()
- Inhibit inflammatory mediators. GCs decrease the production of eicosanoids by inhibiting COX-2; leukotriene catabolism is accelerated. Nitric oxide (NO) synthesis is reduced through the inhibition of inducible NO synthase (iNOS) in macrophages, polymorphonuclear neutrophils, and endothelial cells.
- Inhibit cell surface markers. GCs down-regulate the expression of surface markers such as the CD14 endotoxin receptor and adhesion molecules ELAM-1 and ICAM-1 on endothelial cells. This prevents polymorphonuclear neutrophils from binding to the endothelium and migrating through the vascular wall to the inflammatory focus. The migration of lymphocytes is also slowed down due to the inhibition of the expression of LFA-1 and CD2.
GCs exert their effects on the cells via the glucocorticoid receptor, which forms a complex with a heat shock protein. The receptor becomes dimerized and permeates the nuclear membrane (). An overview of the effects of glucocorticoids is shown in .
- Pro inflammatory cytokines (TNF-α, IL-1, IL-6) stimulate the production of GCs via the hypothalamic-pituitary-adrenal axis, which in turn promotes the synthesis of acute phase proteins in the liver
- GCs increase the expression of cytokine receptors on their target cells (e.g., T-cells) which makes the target cells more sensitive to cytokines
- GCs have a stimulatory effect on the release of macrophage migration inhibitory factor (MIF). It is released by lipopolysaccharide-stimulated macrophages and has an inflammatory effect.
- GCs induce apoptosis in lymphocytes and eosinophils, to a lesser extent in monocytes, and do not induce apoptosis in polymorphonuclear neutrophils.
The effect of physiological concentrations of GCs with an initial increase in receptors for pro inflammatory cytokines followed by inhibition of their synthesis serves to trigger an inflammatory response quickly but also end it again quickly.
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In the body, the oxidation-reduction reactions which effect the transfer of one species with reducing character to another with oxidizing character are particularly important because they activate or inactivate some biologically active compounds. The entirety of redox reactions may create particularly reactive secondary products, oxygen or nitrogen species of radical or non radical type, commonly referred to as reactive oxygen species (ROS) and reactive nitrogen species (RNS) /, /.
The body can counteract the high reactivity of ROS and RNS by synthesizing antioxidants. Antioxidants are species that prevent ROS and RNS formation and their interaction with biostructures.
Excess ROS and RNS causes oxidative stress, a pathogen condition when the antioxidant defense can no longer compensate for the production of ROS and RNS. Oxidative stress can generate multiple diseases e.g., degradation of cell membranes, DNA, lipids, proteins, and the distortion of cell proliferation, mitochondrial structure and function.
Free radicals are compounds that have an unpaired (and therefore, highly reactive) electron on their outer shell . This definition includes: the hydrogen atom, most metal ions (which can exist in various states), and the oxygen molecule, which is a biradical, since its two external electrons are on different shells.
Free radicals can be positively or negatively charged. A dot is placed after the chemical symbol to represent the free electron and to indicate that a compound is a radical. For example: A·, A+·, A–·.
Free radicals are found in lipids, amino acids, nucleotides, and especially in oxygen compounds.
- Homolysis of covalent bonds:
A:B → A· + B·; this is very unusual in biological systems
- Addition of a single electron (e) to a neutral atom:
A + e → A–· a common biological event
- Loss of a single electron from a neutral atom:
A → A+· + e; rare in biological systems.
Radicals, especially those of low molecular weight, are extremely reactive and are, therefore, short-lived; having an unpaired electron, they are usually highly reactive electrophilic species. As a result, they attack sites of increased electron density such as the nitrogen atom (nitrogen contains two unpaired 2s electrons) carbon-carbon double bonds present in polyunsaturated fatty acids and phospholipids to produce additional free radical intermediates .
The main endogenous source of free radical generation is cellular metabolism, while exogenous sources are pollution, smoking, some exogenous drugs, irradiation, the presence of some transition metals such as iron and manganese in the body /, /.
The main reactive oxygen species (ROS) include:
1. free radicals
- Superoxide anion; O2-
- Peroxide anion; O22-
- Hydoxyl radical; OH.
2. Non radical molecules
- Hydrogen peroxide; H2O2
- Hydroxyl anion; OH-
ROS are produced as a result of intracellular catabolism that requires oxygen as terminal electron acceptor (oxidant). During this process, ROS such as super oxide anion radical
In addition to ROS, radicals that contain a nitrogen atom exist, for example, nitrogen monoxide (NO·).
- The super oxide anion radical (O2–·)
- The hydoxyl radical (HO·).
In biological systems, oxygen is involved primarily in the intracellular production of free radicals by various reactions, but are most frequently initiated by the addition of an electron to molecular oxygen to produce the super oxide anion radical .
O2 + e → O2–·
This reaction in cells results from electron leakage from the electron transport chains in mitochondria and from endoplasmic reticulum.
- Non catalytic oxidation of oxyhemoglobin to methemoglobin
Hb-Fe2+ + O2 → Hb-Fe3+ + O2–·
The methemoglobin produced is then reduced to oxyhemoglobin catalyzed by methemoglobin reductase.
- The autoxidation of reduced transition metals also generates the super oxide anion radical
Fe2+ + O2 → Fe3+ + O2–·
Free hydroxyl radicals are produced by the homolytic dissociation of water by ionizing radiation to form hydrogen atom and hydroxyl radical
H2O → H· + HO·
The respiratory burst as an example of ROS formation
The respiratory burst is a reaction that takes place in phagocytic cells such as polymorphonuclear neutrophils and monocytes/macrophages. Phagocytes ingest bacteria and destroy them intracellularly by producing HO· and HOCl. The process starts with the uptake of O2 and activation of NADPH oxidase to produce O2–·.
Subsequent dis mutation, either spontaneous or catalyzed by super oxide dis mutase (SOD), leads to the formation of H2O2:
Potent oxidizing and antimicrobial substances such as HOCl and HO· are produced from H2O2 or O2–· as follows:
- In the presence of Cl–, myeloperoxidase (MPO) from polymorphonuclear neutrophils generates HOCl
- HO· is formed independently of MPO in the Fenton or Haber-Weiss reaction:
ROS are produced physiologically:
- During mitochondrial respiration, for which a cell consumes 1012 molecules of O2 per day, of which around 2% (2 × 1010 ROS) reach the circulation as intermediate stages in the form of O2–·, HO·, and H2O2
- During granulocyte and macrophage activation, resulting in the production of O2–· and HO·
- By the catalytic activity of oxidases such as xanthine oxidase, monoamine oxidase, L-amino acid oxidase, tyrosine hydroxylase, and NO synthase resulting in the production of O2–· and H2O2
- By the Fenton reaction (radical production catalyzed by iron).
The main reactive nitrogen species are:
1. Radical molecules
- Nitric oxide; NO.
2. Non radical anions
- Peroxinitrite anion; ONOO-
Nitrosative stress results from an increase in reactive nitrogen species (RNS). Though the RNS, the radical molecule nitric oxide (NO·) and the non radical anion peroxynitrite (ONOO-) play pivotal physiological roles. At elevated concentrations, both moieties can be poisonous to cells due to their capacity to disrupt a variety of essential biological processes.
Nitric oxide (NO)
- Neuronal NOS (nNOS), which is linked to intracellular signaling
- Inducible NOS (iNOS) which has a variety of situational functions. The enzyme is activated in response to various endotoxin or cytokine signals, which can lead to a rapid production of large fluxes of NO·. The expression of iNOS is regulated by well characterized signal pathways suggesting that the inducible production of NO· must be tightly controlled.
- Endothelial NOS (eNOS). This isoenzyme is present in all tissues and produces low concentrations of NO· following activation. The NO· formed by eNOS has a vasoprotective effect.
The activity of eNOS and iNOS is controlled by the concentration of Ca2+. This enables endothelial cells to respond immediately to an extracellular signal by increasing NO· production, for example. Under physiological conditions, small quantities of NO· are released by eNOS and nNOS only, to regulate the blood pressure, for neurological processes, and to counter regulate pathological processes .
NO fulfills important signaling and protective functions . NO produced by eNOS has vasoprotective effects. It inhibits platelet aggregation and the proliferation of smooth muscle cells and promotes the dilatation of blood vessels, which reduces the blood pressure. NO prevents endothelial dysfunction in general. This includes functional injury to the vascular endothelium caused by disruption of the protective signaling pathway. Functional disruption can be caused by ROS, for example, since they can oxidize cofactor BH4, thereby disrupting the function of eNOS.
NO· can permeate freely through the cell membrane and intervene directly in signaling processes. Small quantities are sufficient to activate guanylate cyclase intracellularly. Cyclic GMP produced by guanylate cyclase activates protein kinases, phosphodiesterases, and ion channels, which then mediate the physiological functions of NO·
Nitrite oxide mediated post-translational modification through the reaction with super oxide produces the peroxynitrite anion.
- In aqueous environment, the protonated peroxynitrous acid forms that rapidly decompose into nitrogen dioxide (NO2) and an extremely reactive hydroxyl radical (OH.)
- The hydroxyl radical is reactive to remove electrons from nearly any biological molecule e.g., the reaction with tyrosine creates a tyrosyl radical which can than react with NO2 to form nitro tyrosine
- Peroxynitrite anions directly react with CO2 yielding nitrosoperoxycarbonate anion (ONOOCO2-) which decompensates into NO2 and the carbonate radical (CO3-) that has a similar reactivity to the hydroxyl radical.
- Peroxynitrite anion modifies proteins and other macromolecules. The ability is used by macrophages and polymorphonuclear neutrophils to kill invading bacteria. It is important to recognize that peroxynitrite anion is used to protect the organism from bacteria by releasing oxidative species in the respiratory burst . The example demonstrates that overproduction or deregulation of these pathways is not only damaging but also beneficial for the organism /, /.
- The concentration of NO· declines and the blood vessels cannot dilate adequately
- Peroxynitrit anions play a role in neurodegenerative diseases, rheumatoid arthritis, and diseases of the organs. Enzymes such as manganese super oxide dis mutase (MnSOD) are inactivated by the irreversible conversion of tyrosine into nitro tyrosine. MnSOD neutralizes the superoxide anion radical· through its binding to H2O to form H2O2.
- Under conditions of oxidative stress, production of α-methylated L-arginine metabolite (asymmetrical dimethylarginine, ADMA) in the cells is increased. ADMA competitively inhibits eNOS by binding to it instead of L-arginine .
Antioxidants are species that prevent both ROS/RNS formation and their interaction with biostructures. Free radicals are produced in all cells of the body. It is very important for the body to preserve the balance between the oxidant and antioxidant systems at the cellular level. Living cells manage to maintain a balance between the production and elimination of ROS so that, the concentration of ROS/RNS species is neither too high to produce stress nor too low to deprive the body from these species . However, if the cell function is altered by various factors higher amounts of ROS/RNS are produced in the presence of insufficient amounts of antioxidants and the affected cells will develop pathologies by the per oxidation of proteins, lipids and DNA, leading to a variety of diseases like atherosclerosis, heart disease, neurodegenerative disorders, diabetes, autoimmune diseases, and cancer /, , /.
The first line of defense against oxidative stress are endogenous antioxidants:
A mitochondrial manganese-dependent super oxide dis mutase (SOD) and a cytosolic copper and zinc-dependent SOD that convert super oxide anion· radical to H2O2 which is not toxic to cells. However, in the presence of Fe2+ and Cu+ H2O2 is converted to toxic hydroxyl radical·. SOD is associated with manganese and other transition metals (MnSOD, CuZnSOD) and localized in the mitochondria to control the production of ROS. Around 70% of SOD activity is localized to the heart, of which 90% is contained in myocytes.
- Glutathione peroxidase (GP): before H2O2 can oxidize metal ions, it is converted by GP or catalase into H2O. The activity of GP is selenium-dependent and GP have a major function in the protection of cells against peroxides and free radicals, thus against oxidative stress. GP and catalase reduce hydrogen peroxide to water and alcohol.
GP, glutathione peroxidase; GSH, reduced glutathione; GSSG, oxidized glutathione
- Metal-binding proteins: these proteins bind transition metals such as iron and copper and prevent them from reacting with H2O2 in the Fenton or Haber-Weiss reaction and generation of free radicals. Metal-binding proteins include transferrin, lactoferrin, ferritin, hemoglobin, myoglobin, metallothionein, and the cytochrome oxidases.
- The usual antioxidant free radical scavengers include bilirubin, uric acid, carotinoids, vitamins A, C, and E, thioles (R-SH), copper, glutathione (GSH), manganese, selenium, and zinc.
Along with super oxide dismutase and catalase and together with vitamins E (α-tocopherol) and C (ascorbic acid), glutathione per oxidases protect cells against the effects of ROS, improving the resistance and recovery of tissues, slowing the aging process and participating in detoxification processes in the body. Vitamin E acts as a trap for free radicals and prevents the formation of lipid hydroperoxides in cell membranes, while gutathione peroxidase destroys lipid hydroxy peroxides formed in the per oxidation of polyunsaturated fatty acids /, /.
Because of the nature and rapid reactivity ROS/RNS are difficult to measure directly with high accuracy and precision. A review of current methods for directly measuring these species and the secondary products they generate is presented in Ref. .
The following methods are commonly used:
- To determine the ROS: measurement of peroxides, malondialdehyde, and isoprostanes
- To evaluate the antioxidant defense: the total antioxidant capacity and enzymes such as super oxide dis mutase, glutathione peroxidase, and catalase or the concentration of antioxidant substances such as vitamin E, vitamin C, reduced glutathione (GSH), and uric acid
- Nitric oxide· can not be determined directly due to its short half-life. Indirect methods used include the determination of nitrite in the form of nitrate or nitrosylated tyrosine residues (3-nitrotyrosine) and of asymmetrical dimethylarginine (ADMA).
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11. Noiri E, Tsukahara A. Parameters for measurement of oxidative stress in diabetes mellitus: applicability of enzyme-linked immunosorbent assay for clinical evaluation. J Investigative Med 2005; 53: 167–75.
14. Griendling KK, Touyz RM, Zweier JL, Dikalov S, Chilian W, Chen YR, et al. Measurement of reactive oxygen species, reactive nitrogen species, and redox-dependent signaling in the cardiovascular system: a scientific statement from the American Heart Association. Circ Res 2016; 119: e39-e75.
- Temperature measurement
- White blood cell (WBC) count and differential
- Erythrocyte sedimentation rate (ESR)
- Serum protein electrophoresis and assessment of the α1-, α2- and γ-globulins
- Acute phase proteins, in particular CRP and serum amyloid A (SAA)
- Inflammatory cytokines (IL-1, IL-6, TNF-α); refer to .
The core body temperature is regulated by the temperature center of the anterior pituitary. Normal body temperature of an individual is an expression of the thermoregulatory set point. Temperatures above this value are referred to as fever and lower values as hypothermia. In routine diagnostics, fever is generally indicated by a rectal temperature of ≥ 38.0 °C (100.4 °F) in children or adults.
Normal body temperature is 37 °C (98.6 °F) and fluctuates by ± 0.6 °C (1.08 °F). The rectal temperature is around 0.6 °C (1.08 °F) higher than the oral temperature. Body temperature exhibits diurnal variation, being higher in the evening than in the early morning /, /.
Fever is characterized by an elevated core temperature due to an increase in the thermoregulatory set point. This set point does not normally exceed 41 °C (105.8 °F). Temperatures higher than this indicate extreme fever. In adults, such extreme elevation is typically caused by central nervous system disorders, drug-induced disturbances, or heatstroke. In children, temperatures of 40–41 °C (104.0–105.8 °F) are not unusual and can result from viral infections. Persistent temperature elevation above 41 °C (105.8 °F) can cause irreversible brain damage /, /.
- Temperatures ≤ 39 °C (102.2 °F) suggests non-bacterial disease such as acute myocardial infarction, acute pulmonary embolism, acute pancreatitis, gastrointestinal hemorrhage, hematoma, phlebitis, uncomplicated wound infection, cystitis, cholecystitis, viral hepatitis, tracheobronchitis, osteomyelitis, decubitus ulcers, deep vein thrombosis, neoplasia, antibiotic induced diarrhea, systemic lupus erythematosus, or acute respiratory distress syndrome (ARDS).
- Temperatures > 39 °C (102.2 °F) indicates an infectious origin such as pneumonia, sepsis, or pyelonephritis.
Fever of unknown origin
Fever of unknown origin is characterized by a temperature > 38.3 °C (100.9 °F) on several occasions for at least three weeks with failure to reach a diagnosis. It often occurs in intensive care units in patients with traumatic brain injury or neurological disorders, some of whom are mechanically ventilated, and those with some combination of urethral, central, and peripheral catheters. Findings include intermittent Staphylococcus positive blood cultures, pyuria with 10–20 thousand CFU/mL, or chronic watery stools with occasional proof of Clostridium difficile. CRP values fluctuate almost daily, making therapeutic decisions difficult, and patients have been repeatedly treated with antibiotics without success . A diagnostic algorithm for fever of unknown origin in children is shown in Ref. .
Hereditary periodic fever includes familial Mediterranean fever (FMF), hyper immunoglobulinemia D and periodic fever syndrome (HIDS), and tumor necrosis factor receptor-associated periodic fever syndrome (TRAPS). All three diseases are thought to be caused by disturbed cytokine balance. Common features are recurring fever with pleuritis, pericarditis and/or peritonitis, arthralgia, and exanthemas, with initial onset in childhood or early adolescence. Genetic investigations are used to verify the diagnosis: detection of mutations in the MEFV gene in FMF, in the MVK gene in HIDS, and in the TNFRSF1A gene in TRAPS.
Clinical features of fever
Elevation of the body temperature leads to chills, shivering, cold clammy skin, pallor, and generalized vasoconstriction.
Types of fever
- Remittent fever: the body temperature rises or declines by 1–2 °C (1.8–3.6 °F) over the course of a day; occurrence in patients, for example, with purulent processes and tuberculosis
- Intermittent or septic fever: the temperature rises from normal values or a subnormal temperature level by 2–3 °C (3.6–5.4 °F), often in combination with shaking chills and then falls again; occurrence in patients, for example, with pneumonia, cystitis, or malaria
- Continuous fever: the body temperature is continuously high and its daily variation does not exceed ± 1 °C (1.8 °F). This fever type was previously known as febris continua and is the classical fever type associated with microbial infection
- Biphasic fever pattern: febrile periods are interrupted by one or more days without fever; occurrence in patients, for example, with Hodgkin’s disease, borreliosis, or malaria.
Biochemistry and physiology
The temperature center in the hypothalamus regulates body temperature by controlling peripheral vasoconstriction and muscle metabolism. Fever results from the direct stimulation of the temperature regulation center by pyrogens, which are biochemical substances with various structures. Pyrogens include substances produced by microbes or exogenous substances (e.g., drugs). Endogenous pyrogens are produced following the activation of monocytes/macrophages and other inflammatory cells. The pro inflammatory cytokines synthesized by these cells (e.g., TNF-α, IL-1, IL-6) and chemokines such as prostaglandin E1, induce fever in the context of an acute phase response. The rationale for setting the body temperature higher is :
- To activate enzymatic reactions that run at normal activity at normal body temperature
- To promote phagocytosis, bacterial killing, and the immune response
- To increase energy provision.
The ESR is the relative rate of the red cell column in relation to the length of the total blood column in a graduated tube.
Screening test in a suspected inflammatory response as well as a method for monitoring such a reaction.
Westergren method: a citrated blood sample is aspirated into a glass or plastic pipette with millimeter graduation up to the 200 mm mark. The pipette remains in an upright position and the sedimentation of erythrocytes is read off in mm after one hour; in some tests, a result is also read off after two hours. The performance of the method follows an approved guideline .
Citrated blood (1.6 mL of blood + 0.4 mL of 3.8% sodium citrate solution): 2 mL
Values are expressed in mm for the first hour
During an inflammatory response, the plasma concentration of acute phase proteins, fibrinogen, and immunoglobulins rises. However, as an indicator of the acute phase response, the ESR responds slowly. Accordingly, a rise in the ESR occurs at the earliest 24 h after the initiation of an inflammatory response and, after completion of the acute phase response, the ESR declines with a half-life of 96–144 h.
The ESR is often considered to be a general indicator of disease. This is incorrect because:
- A normal ESR does not rule out non-inflammatory organ diseases, organ dysfunctions, or neoplasias. For instance, in a study of 1,000 asymptomatic soldiers in whom the ESR was measured monthly over the course of 15 years, 10 individuals developed a malignant tumor despite a normal ESR.
- An elevated ESR is only of supportive value in relation to data from case history as well as clinical and laboratory findings; in rare cases (less than 0.1%), it is the only clue to an important diagnosis .
A moderately elevated ESR should always be investigated if there is any association with a disease (i.e., in the medical history and/or clinically or laboratory-diagnostically relevant data or findings). Approximately 5% of all ESR elevations are essentially unexplainable. In one study involving more than 9,000 outpatients, 8% had an ESR elevation, whose underlying cause could not be determined in 43 patients. Follow-up ESR monitoring over the course of 10 years showed spontaneous normalization in 74% of these patients. Another study demonstrated that asymptomatic young individuals (18–33 years) with persistent, slight ESR elevation had an approximately 5-fold higher risk of disease than those with a normal ESR. The risk of disease was highest for myocardial infarction.
In comparison to the quantitative determination of an acute phase protein (e.g., CRP) the ESR is also raised by an increase in the concentration of immunoglobulins, immune complexes, and other proteins. It therefore covers a broader spectrum of diseases than CRP.
In the case of chronic inflammatory diseases such as SLE, polymyalgia rheumatica, and temporal arteritis, in which CRP is often normal or only slightly elevated, and for monitoring in patients with these diseases, the ESR is a better indicator of the inflammatory process. This is also supported by the fact that the ESR, due to its sluggishness, is subject to little influence by transient inflammatory diseases such as viral infections. In one study of patients with chronic inflammation and malignant tumors, however, the determination of CRP was shown to be slightly more advantageous. For the diagnosis of an inflammatory reaction, an ESR of 31 mm/h corresponded to a CRP concentration of 15 mg/L.
It must be kept in mind that the usefulness of the ESR is limited because it is a reliable indicator of inflammation only in diseases that cause moderate to strong dysproteinemia and have little to no effect on the volume and number of erythrocytes ().
In the absence of inflammation or anemia, ESR elevation in individuals over the age of 50 years suggests a monoclonal gammopathy. At monoclonal immunoglobulin concentrations of greater than 20 g/L, the ESR is often extremely high (above 120 mm/h). A normal ESR does not rule out the presence of multiple myeloma. This is especially true if the monoclonal immunoglobulin fraction is below 10 g/L or in light chain myeloma where polyclonal immunoglobulin synthesis is only moderately reduced.
The ESR testing should be started no later than 2 h after the blood collection. The 2-h value does not provide additional information. If the opportunity to read the 1-h value was missed, the blood cannot be remixed and used again for the ESR. The test should be performed at room temperature. At a temperature lower than 18 °C, the result is of no value because of alterations to the erythrocyte membrane. Temperatures higher than 20–24 °C lead to a rise in the ESR; at 27 °C, the ESR is approximately twice as high as at 20 °C .
The ESR can be determined within 3 minutes using quantitative centrifugation methods and photometric recording. The results obtained using these methods differ from those obtained using the Westergren method , particularly if monoclonal immunoglobulins are present in the blood .
Anti-inflammatory drugs such as acetylsalicylic acid, cortisone, indomethacin, and phenylbutazone, exert an inhibitory effect on the erythrocyte sedimentation.
The ESR is based on the sedimentation and aggregation of erythrocytes.
The erythrocyte density is 6–7% higher than that of plasma; the erythrocytes therefore sink to the bottom as a result of gravity. Simultaneously, plasma rises to the top and slows down the erythrocyte sedimentation. Since the erythrocyte surface is negatively charged (zeta potential), adjacent cells repel each other once the intercellular distance falls below a certain minimum and thus the erythrocytes stay afloat.
Plasma proteins attach to the erythrocyte surface. Depending on the presence of dysproteinemia in certain diseases, plasma proteins may reduce the zeta potential and thus cause the erythrocytes to approach each other more closely.
Plasma proteins such as fibrillary fibrinogen or pentameric IgM can adhere to two erythrocytes.
Overall, both effects of plasma proteins promote the formation of erythrocyte aggregates which, as large particles, undergo faster sedimentation than individual red cells. The contribution of the individual plasma proteins to the ESR are fibrinogen (55%), α2-macroglobulin (27%), immunoglobulins (11%), and albumin (7%).
Diseases that cause an increase in acute phase proteins (such as acute inflammation) and those that are associated with a polyclonal increase in immunoglobulins (chronic inflammatory diseases), but also monoclonal gammopathies, lead to a rise in the ESR. The rise in the ESR does not correspond to the extent of the inflammatory event when the fibrinogen has been consumed (e.g., as in the case of sepsis with hyperfibrinolysis and diffuse intravascular coagulation).
An increase or decrease in the number of polymorphonuclear granulocytes with or without an increased number of precursors such as bands, immature granulocytes, and myelocytes may be a sign of an inflammatory reaction (refer also to and ).
With the exception of acute leukemia and bone marrow toxicity, neutropenia is caused mainly by infection with Gram-negative bacteria, especially if sepsis or septic shock is present. The bone marrow reservoir can also be depleted transiently in the early stage of a severe infection. The neutropenia that results can be associated with the release of immature granulocyte precursors including promyelocytes (leukemoid reaction). This can also happen if patients suffering from infections have reduced bone marrow reserves (e.g., neonates, the elderly, alcoholics, and immunosuppressed individuals) .
Neutrophilia occurs within minutes of exposure to an inflammatory stimulus. The half-life of the subsequent increase in neutrophils is approximately 10 h. Neutrophilia may indicate an infection, but is a non-specific finding. Neutrophilia is also caused by :
- Inflammatory stimuli such as collagen disease, hypersensitivity reaction, tissue injury, necrosis, and neoplasia
- Acute hemorrhage, hemolysis, intoxication, and poisoning
- Metabolic diseases such as gout, uremia, ketoacidosis, and eclampsia
- Myeloproliferative diseases.
Acute purulent bacterial infection
Usually gives rise to a leukocytosis of greater than 15 × 109/L; more than 80% of the cells are granulocytes. In addition, the presence of a left shift is characteristic and may sometimes be the only sign. Granulocytes with toxic granulation are present in approximately three quarters of patients with sepsis but this is not specific for sepsis .
Tissue necrosis and sterile inflammation
Only slight to moderate elevation of the granulocyte count; a left shift is rare.
Normal leukocyte count or a slight rise, often monocytosis.
Acute allergic reaction, parasitosis
Normal leukocyte count or a slight rise, often eosinophilia
Normal, slightly increased or reduced leukocyte count, in most cases lymphocytosis.
The earliest sign of the acute phase response on protein electrophoresis is the increase in the α1-globulin fraction due to an increase in the concentration of α1-antitrypsin and α1-glycoprotein. This is followed by an elevation in the α2-globulin fraction due to an increase in the synthesis of haptoglobin and ceruloplasmin. Fibrinogen and CRP are located in the β-globulin fraction but are not associated with a fractional rise since serum is analyzed (fibrinogen is consumed) and CRP is not detectable because the concentration is too low. Significant changes in the α-globulins are not to be expected within the first 48–72 hours .
Chronic inflammation is associated with a rise in γ-globulins while chronic active inflammation is associated with a rise in α- and γ-globulins.
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CRP is the classic biomarker used to diagnose the acute phase response of an inflammatory process. An increase in plasma concentration of CRP results from the stimulation of pro inflammatory cytokines, such as interleukin-6. Pro inflammatory cytokines trigger acute systemic inflammation, also known as the acute phase response (refer also to ). The acute phase protein CRP is an integral component of the innate immune system. It is synthesized in the liver and reaches via the circulation to the site of tissue damage or is produced locally by activated monocytes/macrophages and fibroblasts.
The acute phase response causes an increase in the serum CRP level that corresponds to the extent of the inflammatory process. Inflammation is caused by infections, sterile tissue damage (surgical procedures), malignant tumors (especially metastatic), systemic malignant diseases (Hodgkin and non-Hodgkin lymphomas), and some autoimmune diseases.
A distinction is made between high-grade, moderate, mild, and low-grade inflammation. Low-grade inflammation is associated with obesity, diabetes mellitus, atherosclerosis, and cardiovascular disease . The CRP concentration is low in low-grade inflammation, which can be present for decades without clinical symptoms. For this reason, assays that could measure CRP in low-grade inflammation were also known in the past as high-sensitivity CRP (hs-CRP) assays. This designation will no longer be used here since most quantitative CRP assays can now detect low-grade inflammation.
To screen for acute phase response and monitor the course:
- In fever, leukocytosis, and suspicion of infection
- In the critical care setting and neonatology
- In the postoperative period (the first 6 days in particular)
- In premature rupture of membranes
- During cytotoxic therapy-related episodes of neutropenia and in bone marrow transplantation.
- Differentiation between viral and bacterial febrile illnesses (e.g., in meningitis or pneumonia)
- Monitoring response to antibiotic therapy
- Identification of intercurrent infections in patients with connective tissue disease.
Diagnosis of inflammatory processes:
- Confirmation of suspected acute organ disease (e.g., cholangitis, adnexitis, synovitis, and deep vein thrombosis)
- Confirmation of chronic inflammatory disease (rheumatic, gastrointestinal, respiratory)
- Differential diagnosis of arthralgia, myalgia, and atypical back pain.
To aid in the differential diagnosis of gastrointestinal complaints:
- To distinguish irritable bowel syndrome from organic disease (inflammatory bowel syndrome)
- To aid the differentiation of ulcerative colitis from Crohn’s disease.
To aid in the management of rheumatic diseases: evaluation of an optimal anti-inflammatory therapy (steroidal or non-steroidal) and to establish the minimal effective dose.
Management of patients with atherosclerosis:
- Risk stratification in patients with cardiovascular disease
- Prediction of future cardiovascular events in patients with myocardial infarction
- Prognostic indicator in unstable angina pectoris.
Principle: many different analytical procedures can be used to quantitatively determine CRP. In routine diagnostics, the most commonly used methods are particle-enhanced immunoassays, with nephelometric or turbidimetric measurement. These assays have a low detection limit and broad measuring range, to measure CRP concentrations in both low-grade and high-grade inflammation (refer also to ).
Serum, plasma: 1 mL
Elevated serum levels of CRP always signal the presence of a disease condition.
Elevated CRP concentrations are indicators for:
- Acute and chronic inflammation caused by microbial infections (bacterial infections are the most potent stimulators of CRP synthesis)
- Acute tissue damage due to major surgery, trauma, or malignant tumors. Even the tissue damage caused by heavy smoking and marathon running can cause a minor elevation of CRP and indicate pathology.
- Inflammation that accompanies autoimmune diseases such as rheumatoid arthritis and for immune-complex mediated diseases such as immune vasculitis.
Normal CRP levels do not exclude the presence of minor degrees of localized inflammation in which the acute phase response is minimal. Examples include chronic autoimmune diseases (inactive systemic lupus erythematosus, progressive systemic sclerosis, dermatomyositis, and ulcerative colitis).
A single CRP measurement is only useful to indicate inflammation within 3 days of an acute event. In such an event, plasma CRP concentrations are elevated after 6 h, reach a peak at 48 h, and decline with a half-life of 48 hours .
Daily determinations provide fare more information in monitoring the progress than a one-time measurement. This is the case because:
- The intraindividual variation of CRP within the reference interval is high, with a coefficient of variation of 30–60%
- The reference interval is very wide. Approximately 25% of healthy individuals have a CRP value below 1 mg/L, which means that the CRP concentration for some people has to increase by at least a factor of 5 to exceed the upper reference interval value. Approximately 14% of healthy individuals have values ≥ 10 mg/L, which are suggestive of inflammation .
In many cases, the increase in CRP precedes the clinical symptoms. Therefore, the CRP level must always be interpreted in the light of the clinical picture.
The degree of elevation of CRP over the upper reference interval value reflects the product of the inflammatory activity and mass of the inflamed tissue and depends on the liver’s capacity for synthesis. Only in the light of the clinical picture is it possible to determine from the rise in CRP whether an inflammatory process is the primary component of the underlying disease or secondary to this disease. Examples of diseases in which inflammation is the primary component include adnexitis and rheumatoid arthritis, whereas inflammation is a secondary component in the case of a postoperative complication.
The degree of elevation of CRP reflects the mass of the inflamed tissue and, in acute inflammation and infection, the level of CRP correlates well with inflammatory activity. This is less often the case in chronic infections, although in a few important diseases, such as rheumatoid arthritis, Crohn’s disease, and poly myalgia rheumatica, the correlation is used for therapeutic monitoring.
Disease activity is classified as follows based on the CRP concentration:
- Low-grade inflammation (> 3–10 mg/L): the reasons include obesity, type 2 diabetes mellitus, and atherosclerotic disease
- Mild inflammation (> 10–40 mg/L): causes include local abscesses, minor operative and accidental trauma, myocardial infarction, deep vein thrombosis, quiescent rheumatic disease, metastatic malignant tumors, and isolated viral infections
- Moderate inflammation (> 40–100 mg/L): diseases include severe inflammatory processes such as purulent cystitis, bronchitis, purulent dental infections, urinary tract infections, and genital infections. These infections require treatment (e.g., with antibiotics).
- High-grade inflammation (> 100 mg/L): diseases include acute generalized bacterial and fungal infections (sepsis) as well as serious tissue damage following multiple trauma or major surgery ().
In cases of suspected infection, it is essential to evaluate the clinical picture of the patient in relation to the CRP level, since non-inflammatory events like deep vein thrombosis and pulmonary embolus may also induce an acute phase response.
Bacterial endotoxin is the most potent stimulus of the acute phase response. The highest levels of CRP are seen in sepsis caused by Gram-negative bacteria and S. aureus, sometimes reaching more than 500 mg/L. Sepsis caused by fungi is also associated with levels of 100–200 mg/L. Parasitic infections usually cause a more modest acute phase response, typically not exceeding 50 mg/L and rarely more than 100 mg/L. Sequential CRP determination can therefore be used to detect intercurrent infection in high-risk surgical patients and following cytostatic therapy in order to detect intercurrent infections.
Viral and bacterial infections can often be differentiated on the basis of the extent of CRP elevation. For example, in meningitis and respiratory tract infection, CRP concentrations > 100 mg/L indicate a bacterial origin. The median CRP concentration in viral infections is 15 mg/L; values > 40 mg/L are rare.
CRP measurement is valuable since it can indicate anatomically closed infections that are not accessible to conventional microbiological diagnosis. The rapid response time of the rise and fall in CRP allows diagnosis before bacteriological tests provide a result.
The behavior of CRP in conditions involving mild to high-grade inflammation is shown in . In a study chronic low level CRP concentrations (> 3 to 10 mg/L) were associated with increased risk of bacterial infections, and in particular Gram-negative infections .
When using CRP for therapeutic monitoring, it is important to note that changes in plasma level lag behind changes in inflammatory activity by some 12–24 h. This is relatively unimportant, though, since changes in the clinical symptoms are usually much slower. For example, following antibiotic therapy for pyelonephritis or bacterial cystitis, improvement in the clinical picture is not expected for at least 24–48 hours and following anti-inflammatory therapy for rheumatoid arthritis, improvement is not expected for at least 4–6 weeks. In most cases of inflammatory disease, therefore, CRP provides a much earlier assessment of the therapeutic response than the change be made from the clinical picture. A persistently raised CRP concentration generally indicates that therapy is ineffective.
Serial measurements of CRP can be used to monitor therapy in the following situations:
- Optimization of antibiotic therapy in acute infections
- Optimization of antibiotic therapy in high-risk patients in the absence of a microbiological diagnosis
- Dis continuation of antibiotic therapy when CRP has returned to normal
- Selection of appropriate anti-inflammatory therapy in various rheumatic diseases that are difficult to assess clinically
- Titration of the dose of anti-inflammatory drugs
- Prediction of the development of complications (e.g., the development of giant cell arteritis in a patient with poly myalgia rheumatica).
- Simple infection: CRP declines precipitously or exponentially following antibiotic administration. The intensity of the decline is determined by the half-life of CRP. This pattern is observed in focal infection or in bacteremia.
- Suppurative infection: there is a time delay between the administration of antibiotics and the decline in CRP. This pattern is observed in purulent effusions, suppurative bronchitis, and empyema in the third space and when antibiotic doses are inadequate. The source of a persistent infection must always be sought.
- Complicated infection: the CRP concentration fails to decrease or it continues to increase despite antibiotic therapy. This can be caused by the wrong choice of antibiotic, surgical complications, or serious non-infectious disease.
- Recurrent infection: CRP shows a bimodal pattern with an initial decline followed by a second rise. This suggests either reinfection with the same causative microorganism at the same site or a new infection.
In Europe and North America, two-thirds of the population over 45 years of age have CRP values below 3 mg/L, less than 5% have values above 10 mg/L, and the rest have values between 3 and 10 mg/L . According to a statement issued by the Centers for Disease Control and Prevention and the American Heart Association, CRP determination in addition to the classical risk factors is beneficial in the risk stratification of cardiovascular disease. The following CRP threshold values for cardiovascular risk were specified :
- Below 1.0 mg/L = low risk
- 1.0–3.0 mg/L = moderate (normal) risk
- Above 3.0 mg/L = high risk.
This recommendation is based on the following:
- Inflammation plays an important role in the development and course of atherosclerosis
- Population studies have shown an independent relationship between the basal CRP value and future coronary events
- The serum CRP is useful for detecting patients with metabolic syndrome or type 2 diabetes
- Low-grade inflammation is associated with cardiovascular disease, the metabolic syndrome, type 2 diabetes, and vascular endothelial dysfunction .
The association between CRP in low-grade inflammation and the body mass index is shown in:
- In apparently healthy individuals, the intraindividual day-to-day variation in CRP is 46%, compared to just 9% for cholesterol. This means CRP measurements of samples collected on different days could differ by up to 84% and the individual could be assigned to more than one risk category. This background variation is attenuated and balanced out in large studies but is important at individual level. For example, 27.5% of women over 20 years of age in the USA have a CRP value of 3–10 mg/L .
- Non-inflammatory stimuli influence lower CRP concentrations such as genetic factors , physical activity, high protein intake, alcohol consumption, depressive syndromes, chronic fatigue syndrome, and obesity in particular () .
Method of determination
High concentrations of rheumatoid factor can mimic high CRP concentration because the rheumatoid factor can bind to CRP to form a CRP/anti-CRP complex.
- The 5th percentile ranged from 0.12 mg/L in men aged 25–34 years to 0.43 mg/L in women aged 65–74 years
- The 95th percentile ranged from 4.9 mg/L in men aged 25–34 years to 15.5 mg/L in women aged 55–74 years.
Reference interval for adult women in the USA: 24.5% have CRP concentrations below 1 mg/L, 29.7% have concentrations of 1–3 mg/L, 31.8% have concentrations of 3–10 mg/L, and 14% have levels above 10 mg/L .
Reference interval for children and young adults : mean value 1.6 mg/L; median 0.4 mg/L. The CRP concentration increases with age. Women aged 16–19 years have higher concentrations than men of the same age. Individuals of Mexican origin have higher concentrations than black or white Americans.
Prevalence of CRP values above 3 mg/L
The CRP concentration is influenced by age. Values increase from an average of 0.21 mg/L for individuals aged 5–13 years to 0.86 mg/L for men and 0.75 mg/L for women in the age group 50–75 years.
Body mass index (BMI)
Individuals who are considerably overweight have higher CRP values than those with normal BMI.
CRP concentrations are two times higher in women receiving treatment with estrogens and progestogens than among women not receiving treatment. The effect of oral contraceptives on CRP and serum lipids is shown in .
Increases the CRP concentration by 16–52% in men aged 29–75 years.
CRP as an indicator of cardiovascular risk
CRP should be measured in two blood samples, taken two weeks apart. If a value greater than 10 mg/L is identified, the test should be repeated and, if the value is confirmed, a search should be initiated for any source of mild to moderate inflammation .
The molecular weight of CRP is 118 kDa. CRP is a non glycosylated protein and the gene that encodes it is located on chromosome 1q21-q23. Two Ca2+ ions are bound to the protein chains and are an integral component of the binding site for ligands. The pneumococcal c-polysaccharide is a typical ligand. Phospho choline residues of the pneumococcal c-polysaccharide are the main determinants of Ca2+ ion-mediated binding to CRP. In the presence of Ca2+ ions, CRP also binds histone, chromatin, fibronectin, laminin, oxidized LDL, and other phospho choline-containing polysaccharides .
CRP is synthesized in the hepatocytes under the control of IL-6 but its synthesis and secretion are also influenced by IL-1 and TNF-α. CRP is synthesized in the liver following induction by IL-6, and at the maximum point of an acute phase response, as much as 20% of the liver protein synthetic capacity may be directed toward its synthesis. The normal synthetic rate is 1–10 mg/day, increasing to more than 1 g/day in acute inflammation. In patients with surgical trauma, the doubling time is 8–10 h in the plasma. In the absence of an IL-6 stimulus, the synthetic rate normalizes within 2–4 h but the half-life in the blood of patients following surgery is 24–48 h; this does not reflect the half-life of CRP in blood, but rather the disappearance of a non-infection dependent inflammation. The biological half-life of CRP is around 19 h but it may be cleared more rapidly once it is bound to ligands.
The functions of CRP include the detection, clearing and elimination of apoptotic tissue cells and their products, such as DNA, which may be toxic or allergenic. CRP also acts as a non-adaptive defense mechanism by opsonizing invading microorganisms for phagocytosis. CRP only binds to cells when the normal structure of the lipid bilayer has been disrupted, leading to the exposure of internal phospholipids.
Once bound to one of these endogenous or exogenous ligands, CRP is able to activate a number of biological systems that result in removal of the ligand by the following processes:
- Complement is activated by the classical pathway with the consecutive clearance of CRP ligand complexes by macrophages in tissues, the blood, and the spleen. C3b deposited on the surface of these complexes is recognized by macrophages. In addition, anaphylatoxin and chemokine production results in inflammation and macrophage activation.
- CRP binds to both high and low-affinity receptors on phagocytes such as CD32. CRP is thus exposed on the cell surface, allowing direct cellular contact with the ligands, the activation of complement and the initiation of phagocytosis.
- Spleen macrophages clear CRP-coated ligands and material from the circulation
- CRP binds to IgG FcR on T, B, and null cells and activates natural killer cells (NK cells)
- CRP bound to the cell membrane of neutrophils at the inflammatory focus is cleaved by proteases to generate small tuftsin-like fragments, which activate macrophages and inhibit neutrophil function
- CRP cross-links soluble and particulate ligands whereby these precipitate and become localized in the tissues
- CRP in the vascular wall has a dominant role in the development of atherosclerosis. It up regulates the expression of adhesion molecules, activates complement, and induces the expression of tissue factor by macrophages () .
Basal plasma levels of CRP depend on the genetic polymorphism of the CRP gene. The allele frequency of haplotype-associated single nucleotide polymorphisms (SNPs) varies significantly in ethnic groups. In the white population, for example, rs3093068, rs1130846, and rs1417938 are associated with higher CRP concentrations while rs1205 and rs1800947 are associated with lower CRP levels . However, such variants are not significantly associated with an increased risk of diabetes in postmenopausal women .
Pentameric CRP is found in the plasma whereas monomeric CRP is expressed in tissues and vascular walls. It is assumed that both forms have different biological activity at endothelial cells. CRP exerts its effects primarily via the Fc receptor CD32 (FCRII) and up regulates the LOX-1 receptor, which facilitates the uptake of oxidized LDL (oxLDL) by endothelial cells. In contrast, monomeric CRP inhibits the uptake of oxLDL in endothelial cells independently of LOX-1, CD32, or CD16 .
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The CALC-1 gene encodes procalcitonin and calcitonin gene-related peptide. In healthy individuals, PCT is produced by the C cells of the thyroid and by neuroendocrine cells in the lung and pancreas. PCT undergoes successive cleavage to form three molecules:
- Calcitonin with 32 amino acids
- Katacalcin with 21 amino acids
- An amino terminal end with 57 amino acids.
All parenchymal organs and differentiated cells in the body produce PCT in response to stimulation by bacterial toxins and mediators of inflammation. The plasma PCT concentration rises within a matter of hours in systemic bacterial infection but local bacterial infection, viral infection and bacterial colonization do not induce a rise in PCT.
- In suspicion of sepsis
- To guide the use of antibiotics in systemic bacterial infection
- To determine whether or not antibiotics are indicated in patients with upper respiratory tract infection
- Prognostic marker in infection
Principle: the PCT containing specimen is incubated in a test tube coated with monoclonal antibodies directed against PCT. After removal of the unbound material the solid-phase bound PCT-antibody immuncomplexes are labeled using a second monoclonal antibody coupled to an acridinium ester. After this step the addition of NaOH and H2O2 produces a light signal with an intensity corresponding to the concentration of PCT in the sample.
Assays of different functional sensitivity (lowest concentration with CV% below 20) are available:
- Assays with functional activity of 0.3–0.5 ug/L are used for diagnosis and monitoring of sepsis
- Ultra sensitive assays with functional activity of 0.06 ug/L are used for the differentiation of upper respiratory tract infection and pneumonia.
Principle: the test is a semi quantitative one-step solid phase immunoassay. A polyclonal sheep anti-calcitonin antibody is immobilized on a solid phase and a monoclonal gold-conjugated mouse-anti-catacalcin antibody is used as a tracer in the soluble phase. The serum of the sample solubilizes the tracer when applied to the provided test area and proceeds into the test area. The PCT-antibody immuncomplex becomes visible when it is immobilized and binds the tracer. A red colored line develops at concentrations of about 0.5 µg/L. The non-bound tracer permeates into the area of the control line, developing a dark red second line, indicating the positive function of the assay. The following PCT levels by comparison with the reference scale are differentiated < 0.5 μg/L, 0.5 to < 2 μg/L, > 2 to ≤ 10 μg/L, and > 10 μg/L.
Plasma (EDTA, citrate, heparin) or serum: 1 mL
Any attack against the body, whether inflammatory, traumatic, or chemical, stimulates the production of pro inflammatory cytokines. The PCT level is below 0.5 μg/L.
In severe systemic inflammation, especially that caused by bacteria, the body launches a vigorous immune response that triggers many changes such as the synthesis of proteins that can be used as biomarkers of inflammation and infection. The PCT level may increase to ≥ 0.5 μg/L.
PCT levels show a large scatter between patients with a seemingly similar clinical condition, hence single absolute values are difficult to interpret. However, there is overwhelming evidence that in most cases high PCT concentrations indicate bacterial infection. The shortcomings of PCT absolute values might be compensated when the kinetics of PCT is taken into account to indicate infection .
Unlike CRP and IL-6, PCT concentrations do not increase ≥ 0.5 μg/L in sterile inflammatory processes such as connective tissue disease and rheumatic disease, postoperative inflammation, and many febrile conditions unless bacterial superinfection occurs.
In systemic bacterial infection, plasma PCT rises rapidly and continues to rise if inflammation is uncontrolled, antibiotic therapy is not used, or the correct antibiotics are not applied. The sensitivity and specificity of PCT to indicate bacterial infection is 75–85%.
PCT is not elevated in viral infections (e.g., viral meningitis) and is only moderately elevated in systemic fungal infections. PCT cannot be used to reliably distinguish sepsis within the systemic inflammatory response syndrome ().
Transient elevations in PCT can occur due to weakness of the intestinal barrier caused by hypotension, surgical intervention, or intestinal paralysis. In all of these cases, PCT declines again within 24 h.
Following contact with bacterial endotoxin, PCT is detectable within a few 4–6 h, reaches its peak within 24 h and then starts its decline in the case of adequate treatment with levels reducing by approximately 50% daily. In contrast, serum CRP is elevated only after 12 h and reaches a peak at 24–48 h. While increased PCT concentrations return below the threshold value within 2–3 days, CRP remains elevated for 3–7 days. The temporal course of the rise in PCT compared to other inflammatory markers is shown in .
In inflammatory disorders of unknown etiology, PCT indicates a possible bacterial etiology. In non bacterial disease (postoperative inflammation, viral infection, automimmune disease, transplant rejection), the PCT concentration is low (< 0.5 μg/L or rarely 0.5–2 μg/L) in cases of comparable clinical severity.
During the course of non bacterial inflammatory diseases, PCT concentrations may rise due to secondary bacterial infection, sepsis, or organ dysfunction. It is also important to bear in mind that PCT elevation can occur without infection in multiple trauma, burns, extensive surgery, or prolonged circulatory shock.
PCT is used as a biomarker for monitoring the course and treatment of inflammations with high-grade CRP elevation. In sepsis the PCT level correlates with disease severity and mortality. If the initial concentration is elevated, PCT should be determined daily for monitoring purposes. Consistent PCT elevations indicate an unfavorable prognosis. The pattern of the PCT concentration during the course of the disease provides information about the severity of inflammation. PCT is used as a biomarker to guide therapy and as a prognostic indicator.
Because of its short half-life, PCT is a useful parameter for monitoring the course of sepsis. It is therefore used to monitor infection in critically ill patients.
PCT has a high negative predictive value. Low levels rule out systemic bacterial infection with a high degree of certainty.
Since PCT does not rise in response to local inflammation and trivial infections, it is a better diagnostic tool than CRP, temperature measurement, or the WBC count.
PCT levels in healthy children are < 0.5 μg/L, with a slight increase (0.5–2 μg/L) during viral infections, non-infectious inflammation, stress situations, and focal bacterial infections. In systemic bacterial infections, PCT increases > 2 μg/L and can reach concentrations as high as 50–100 μg/L .
In approximately 20% of children who attend the emergency department with fever, clinical examination fails to reveal a source for the fever . Although the vast majority of these children have viral infections, 10–20%, in particular those under the age of 3 years, may have unrecognized serious bacterial infections such as pyelonephritis, pneumonia, osteomyelitis, or bacterial meningitis. The WBC count and CRP measurement have comparable diagnostic specificity and a sensitivity of only 70–86% . The additional determination of PCT increases the positive likelihood ratio. In severe bacterial infection, the positive likelihood ratio is 10.6 by a combination of WBC > 15 × 109/L, CRP > 50 mg/L, and PCT > 2 μg/L .
Bacterial infections are the main cause of neonatal morbidity and mortality, especially in premature infants. Neonatal infections are difficult to diagnose through medical examination alone since the clinical symptoms (hypoglycemia, respiratory distress syndrome) are either non specific or are not present at all if infection occurred shortly before delivery or is still in the early phase. Blood and CSF cultures are often negative and need an assay time up to 24 h. In such cases, it is often difficult to decide whether or not to initiate antibiotic therapy and to decide when antibiotics should be discontinued. Although PCT is an indicator of sepsis in adults and children, its use in suspicion of neonatal sepsis is not recommended. The reasons are:
- PCT exhibits physiological increase in the first days of life ()
- In full-term neonates, PCT levels peak at up to 20 μg/L in the first 48 h and then return to normal after 48–72 h .
- In pre term infants, values are lower but may take 72–96 h to return to the reference interval .
Because of the afore mentioned reasons the IL-6 concentration should be determined if neonatal sepsis is suspected.
Method of determination
In a study the evaluation of Diazyme PCT on Roche Cobas c702 (PCT-D), and Brahms PCT on Roche Cobas e602 (PCT-BR) analyzers was compared with Brahms PCT-sensitive Kryptor (PCT-BK). Almost 40% of the samples at concentration < 0.5 μg/L seem to show sample-related interferences using PCT-D. PCT-BR and PCT-BK showed a good correlation.
In healthy individuals, plasma concentrations of PCT are in the range of 0.005–0.05 μg/L. However, clinical studies have indicated a threshold value of < 0.5 μg/L to rule out sepsis.
In whole blood at room temperature, PCT decreases by 9% after 6 h and 13% after 24 h. At 4 °C, concentrations do not decline significantly within the first 6 h and have declined by 7% after 24 h. Repeated freezing (–20 °C) and thawing of samples does not lead to a loss in PCT concentration . In cerebrospinal fluid (CSF) stored at 20 °C and 4 °C a degradation over the first 72 h of 5% and 8%, respectively was measured .
PCT is not glycosylated and, in plasma, the N-terminals of two amino acids are cleaved off by the enzyme dipeptidylpeptidase 4 (DPP4, CD26), which is present in renal, epithelial, and endothelial cells. Other fragments of PCT can also be detected in the plasma. PCT exists in two forms (PCT-I and PCT-II), which can be distinguished based on their eight C-terminal amino acids. PCT-I is the predominant form found in the plasma in sepsis. Commercial assays can detect both forms.
While endotoxin is a powerful stimulus for PCT synthesis, pro-inflammatory cytokines such as TNF-α and IL-6 can also induce the synthesis of PCT, albeit to a lesser extent. PCT synthesis is induced in various body cells in response to infection and inflammation but the greatest proportion is synthesized by the liver . Its half-life in the plasma is 25–35 h, which may be prolonged by up to 30–40% in severe renal insufficiency without resulting in an accumulation of PCT .
PCT is cleared by hemofiltration using PMSF 1200 membranes with a sieving coefficient of around 0.2 . At normal filtration solution flow rates (< 1–2 L per hour), effects on the plasma level are minor but when high-flux systems and other membranes are used, a reduction in the plasma concentration can be observed.
The biological functions of PCT are not fully known. The protein may not have any measurable effects in healthy individuals but is associated with increased mortality in sepsis. Microbial infection generally causes increased expression of the CALC-1 gene, with the subsequent release of calcitonin-1 precursors in all body tissues. In bacterial infections, the serum PCT concentration can rise from values below the measurement range (0.005 μg/L) to as much as 1,000 μg/L. Such a rise correlates with the disease severity and mortality.
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6. Turner D, Hammerman C, Rudensky B, Schlesinger Y, Goia C, Schimmel MS. Procalcitonin in preterm infants during the first few days of life: introducing an age related nomogram. Arch Dis Child Fetal Neonatal Ed 2006; 91: F283–F286.
10. Guibourdenche J, Bedu A, Petzold L, Marchand M, Mariani-Kurdjian P, Hurtaud-Roux MF, et al. Biochemical markers of neonatal sepsis: value of procalcitonin in the emergency setting. Ann Clin Biochem 2002; 39: 130–5.
11. Chiesa C, Pellegrini G, Pandero A, Osborn JF, Signore F, Assumma M, Pacifico L. C-reactive protein, interleukin-6, and procalcitonin in the immediate postnatal period: influence of illness severity, risk status, antenatal and perinatal complications, and infection. Clin Chem 2003; 49: 60–8.
12. Ceriotti F, Marino I, Motta A, Carobene A. Analytical evaluation of the performances of Diazyme and Brahms procalcitonin applied to Roche Cobas in comparison with Brahms PCT.sensitive kryptor. Clin Chem Lab Med 2018; 56: 162–9.
13. Meisner M, Tschaikowsky K, Schnabel S, Schmidt J, Katalinic A, Schüttler J. Procalcitonin – influence of temperature, storage, anticoagulation and arterial or venous asservation of blood samples on procalcitonin concentrations. Eur J Clin Chem Clin Biochem 1997; 35: 597–601.
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22. Uzzan B, Cohen R, Nicolas P, Cucherat M, Perret GY. Procalcitonin as a diagnostic test for sepsis in critically ill adults and after surgery or trauma: a systematic review and meta-analysis. Crit Care Med 2006; 34: 1996–2003.
24. Haasper C, Kalmbach M, Dikos GD, Meller R, Müller C, Krettek C, et al. Prognostic value of procalcitonin (PCT) and/or interleukin-6 (IL-6) plasma levels after multiple trauma for the development of multi organ dysfunction syndrome (MODS) or sepsis. Technol Health Care 2010; 18: 89–100.
25. Jensen JU, Heslet L, Jensen TH, Espersen K, Steffensen P, Tvede M. Procalcitonin increase in early identification of critically ill patients at high risk of mortality. Crit Care Med 2006; 34: 2596– 2602.
27. Bouadma L, Luyt CE, Tubach F, Cracco C, Alvarez A, Schwebel C, et al. Use of procalcitonin to reduce patients’ exposure to antibiotics in intensive care units (PRORATA trial): a multicentre randomised controlled trial. Lancet 2010; 375: 463–74.
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31. Mantadakis E, Plessa E, Vouloumanou EK, Karageogopoulos DE, Chatzmichael A, Falagas ME. Serum precalcitonin for prediction of renal parenchymal involvement in children with urinary tract infections: a meta-analysis of prospective clinical studies. J Pediatr 2009; 155: 875–81.
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SAA is an acute phase protein that is bound to plasma high-density lipoprotein (HDL). Like CRP, SAA is synthesized by the liver in response to stimulation by IL-6 as part of an acute phase response. SAA is also the precursor protein of reactive amyloid deposits in the organs. SAA is used to diagnose inflammation due to viral or bacterial infection in particular but also low-grade inflammation that is not caused by microbial infection.
- Inflammatory marker in viral infections
- Biomarker of transplant rejection
- Investigation of patients with amyloidosis
- Prediction of cardiovascular disease.
Serum, plasma: 1 mL
The serum SAA concentration is a very sensitive, but non-specific marker both in diagnosis, prognosis and monitoring of inflammatory and infectious diseases. Under pathological conditions i.e., inflammatory or infectious disease or neoplasia SAA concentrations of more than 10 mg/L and up to 1,000 mg/l are found in serum. SAA rises approximately 8 h after the start of the inflammatory response but exceeds the upper reference interval value earlier than CRP. Whereas in the case of CRP, the median value of healthy individuals differs from the upper reference interval value by approximately a factor of 10, this factor is only 5 in the case of SAA. In milder infections (e.g., in many viral infections) SAA is therefore more commonly elevated than CRP . In infections, the rise in SAA is higher than that of CRP ().
Since SAA is a more sensitive indicator of low-grade inflammation than CRP, it has an advantage over CRP in the diagnosis and differential diagnosis of infections. A positive correlation has been observed between SAA and CRP in bacterial infections when the CRP exceeds 100 mg/L, with the concentration of SAA being approximately 10–15 times higher than that of CRP . SAA is more effective at discriminating between normal and disease states than CRP, especially in mild APRs ()
Method of determination
Calibration is carried out using an SAA reference preparation created in 1997.
SAA is the generic name of a family of proteins with 103–104 amino acids that share high levels of sequence homology, but are encoded by different genes. In humans there are 4 SAA genes (SAA1, SAA2, SAA3 and SAA4). Of these genes SAA1 and SAA2 code for acute-phase SAA proteins. SAA4 encodes a constitutively expressed SAA protein, and SAA3 is a pseudo gene .
The N-terminal region of SAA is predicted to harbor an HDL binding site, whereas the C-terminal segment constrains the overall SAA structure such that its removal facilitates aggregation of the cleavage product AA, forming highly ordered β-sheets as in amyloid fibrils .
SAA exists in the serum as two isoforms, SAA1 and SAA2, whose primary structures are 93% identical. SAA1 is the predominant isoform in the acute phase response. The SAA1/SAA2 ratio is about 3 to 1 and remains stable during the acute phase response . Whereas the promoter genes for both SAA and CRP have a high sensitivity for IL-6, the SAA promoter genes are more sensitive than those of CRP to stimulation by IL-1β.
Like CRP, SAA is synthesized mainly by hepatocytes. After SAA is secreted, it binds to HDL, LDL, and VLDL but especially to HDL3. During the acute phase response, the SAA plasma concentration increases 100–1,000-fold as a result of increased incorporation of SAA into the HDL particles ().
SAA is an acute-phase protein and possesses pro inflammatory cytokine-like activity and is chemotactic for phagocytes. The results of a study indicate that the N- and C-terminal sequences contain structural determinants for the pro inflammatory and chemotactic activities of SAA, and their removal switches SAA to an antiinflammatory role.
SAA is catabolized in hepatocytes after it is taken up into these cells along with the HDL particle. During an acute phase response, this catabolism is reduced. This indicates that the SAA increase during an acute phase response is due to increased synthesis and reduced degradation of SAA .
SAA is the main constituent of the fibrillary component of amyloid A deposits in the tissues. The homozygosity at the SAA1 allele is a strong predictor for type AA amyloidosis.
4. Lannergard A, Friman G, Ewald U, Lind L, Larsson A. Serum amyloid A (SAA) protein and high-sensitivity C-reactive protein (hsCRP) in healthy newborn infants and healthy young through elderly adults. Acta Paediatr 2005; 94. 1198–1202.
11. De Buck M, Gouwy M, Wang JM, van Snick J, Openakker G, Struyf S, van Damme J. Structure and expression of different serum amyloid A (SAA) variants and their concentration-dependent functions during host results. Current Medical Chemistry 2016; 23: 1725–55.
14. Mangiarotti P, Moulin F, Palmer P, Ravilly S, Raymond J, Gendrel D. Interferon-alpha in viral and bacterial gastroenteritis: a comparison with C-reactive protein and interleukin-6. Acta Paediatr 1999; 88: 592–4.
15. Lindback S, Hellgren U, Julander I, Hansson LO. The value of C-reactive protein as a marker of bacterial infection in patients with septicemia/endocarditis and influenza. Scand J Infect Dis 1989; 21: 543–9.
17. de Beer FC, Fagan EA, Hughes GRV, Mallya RK, Lanham JG, Pepys MB. Serum amyloid A protein concentration in inflammatory diseases and its relationship to the incidence of reactive systemic amyloidosis. Lancet 1982; ii: 231–3.
21. Johnson DB, Kip KE, Marroquin OC, Ridker PM, Kelsey SF, Shaw LJ, et al. Serum amyloid A as a predictor of coronary artery disease and cardiovascular outcome in women. Circulation 2004; 109: 726–32.
Gertrud M. Hänsch
Polymorphonuclear neutrophils (PMNs) play a key role in the clearance of pathogens. They are capable of affecting many aspects of both innate non-specific, non-adaptive cellular immune response and adaptive immunity. They are one of the front-line defenders involved in bacterial ingestion and subsequent killing but are also involved in defending the body against parasites, viruses, and tumors .
Abnormal granulocyte function generally manifests as frequent, recurrent, or treatment-resistant bacterial infection, in some cases with banal or generally nonpathogenic microorganisms (). It makes sense to investigate granulocyte function if other causes of immunodeficiency such as antibody deficiency or complement deficiencies have been ruled out.
- Increased susceptibility to infections
- Treatment-resistant infections
- Recurrent infections with banal or generally harmless microorganisms
- Severe periodontitis
- Impaired wound healing.
The following can be determined:
- Granulocyte count, morphology, and receptor expression
- Functions such as chemotaxis, phagocytosis, and oxygen radical production
- Genetic defects and genetic polymorphisms.
These investigations can be used to confirm or rule out neutropenia, PMN deficiencies, and important functional deficiencies. If the results indicate pathology, the investigations should be repeated after a few weeks and family members should also be tested if possible. If a disorder of PMN function is confirmed, further investigations are performed to determine the type of defect involved. A diagnostic algorithm is shown in .
If the neutrophil count in the blood falls below the lower reference interval value, neutropenia is present. This can be caused by a range of factors, such as infections (Parvovirus), drugs (chemotherapy, antibiotics), or abnormal neutrophil maturation (refer to ). Molecular defects have been described in the case of congenital neutropenias. Mutations in the ELA2 gene occur in cyclic neutropenia, mutations in the Hax1 gene occur in Kostmann’s disease, and mutations in the WAS gene occur in X-linked neutropenia. Although the relationships between the gene defects and neutropenia have not yet been established, demonstration of the respective mutation results in a reliable diagnosis .
Altered granulocyte morphology can be demonstrated using stains in a conventional blood smear. A deficiency of secondary specific granules is associated with increased susceptibility to infections. It is caused by a lack of transcription factor C/EBPε and results in reduced chemotaxis and reduced production of oxygen radicals .
A variety of in vitro assays are available for assessing PMN function, some using whole blood and some using isolated cells. Various procedures are used to isolate PMNs, such as density gradient sedimentation or centrifugation or hypotonic lysis of erythrocytes. Although contamination with monocytes and lymphocytes can occur, this does not affect fast functional assays.
Principle of Boyden chamber technique /, /: in this technique, isolated granulocytes are allowed to migrate through a filter with the appropriate pore size toward a specific chemoattractant. This takes place in a chamber (Boyden chamber), which is separated by a filter (pore size 3 μm) into two compartments. The chemoattractant is placed in the lower compartment and the granulocytes (1 × 109/L) are contained in the upper compartment. Yeast-activated plasma is used as a chemoattractant and the chemoattractant peptide f-Met-Leu-Phe (formyl-methionine-leucine-phenylalanine) or interleukin 8 is used as a source of complement 5a.
The chambers are incubated for 2–3 h at 7 °C to migrate through the filter. The filter is then removed, and the cells are fixed, stained, and examined under a microscope. When granulocyte chemotaxis is assessed, a comparison must always be made with granulocytes donated by healthy controls.
By microscopic analysis, the following criteria are used to assess chemotaxis:
- Number of cells on the surface and at different levels within the filter are counted. The number of cell at each level is plotted against the distance from the top. The area under the resulting curve is calculated and used as a chemotactic index.
- Greatest granulocyte migration distance. In this method, known as the leading-front method, the greatest distance at which 5 granulocytes are still visible in the view field is determined.
- Number of granulocytes that have reached the bottom of the filter.
Analytical evaluation: the testing of patient samples allow the following functions to be assessed:
- Spontaneous motility of the PMN
- Chemotactic activity of the PMN
- Ability to generate chemotactic activity in the serum.
Medical assessment: a low level of spontaneous migration and/or chemotaxis suggests an adhesion defect. Flow cytometry is used to detect CD11/CD18 adhesion proteins. If expression of these proteins is normal, a possible deficiency can be investigated further by determining CD15, actin polymerization, complement receptors, and membrane signaling. Refer to .
Principle des phagocytosis and killing assays: radioactively labeled, serum-resistant bacteria, (e.g., E. coli) are opsonized by incubation with serum and presented to isolated PMN for phagocytosis. After 20 min., the granulocytes are exposed to ultrasound to stimulate the release of phagocytosed bacteria. The phagocytosis rate is determined from the number of bacteria presented for phagocytosis and the number of bacteria actually phagocytosed. Intracellular killing is determined by plating the released bacteria and counting the colonies 20 hours later. The advantage of this method is that phagocytosis and killing can be determined simultaneously in the same assay. The disadvantage is that radioactive labeling is expensive, even with low levels of radioactivity.
Phagocytosis can also be detected using fluorescein (FITC)-labeled bacteria, using microscopy or flow cytometry. Heparinized blood can be used for this method, keeping experimental costs low, and the isolation procedure eliminates artefacts. Fixed, FITC-labeled bacteria are available commercially, which improves the standardization of the method. A disadvantage is that bacterial killing cannot be measured at the same time.
As in all functional assays, patient PMNs and PMNs donated by a healthy control are tested in parallel. If bacteria from patient isolates are used in this assay, their resistance to the body’s defense mechanisms can also be tested. This is relevant because bacterial variants have been described that, although they can be phagocytosed, cannot be killed.
Analytical evaluation: the ratio of phagocytosed bacteria to bacteria that are phagocytosed but not killed is determined.
Reference interval: 80–90%.
Principle: this method allows the phagocytic capability of PMN and their ability to produce oxygen radicals to be investigated in parallel . Isolated PMN are incubated with a suspension of Candida albicans, serum, and the dye NBT. If oxygen radicals are produced, the dye is reduced and a blue precipitate forms in the PMN. Light microscopy is used to detect any intracellular Candida or blue coloration. A blank test is also run, using a buffer solution instead of patient serum.
The number of PMN with intracellular Candida particles (phagocytosis) and the number of PMN with blue coloration (phagocytosis and oxygen radical formation) are counted.
Phagocytic PMN (%)
O2 radical producing PMN (%)
A number of methods are available to quickly and easily test the ability of PMN to produce oxygen radicals in response to stimulation. Depending on the laboratory equipment available, fluorescence or luminescence assays can be used.
Cytochrome c assay
Principle: isolated granulocytes are suspended in a cytochrome c solution and stimulated by PMA (phorbol 12-myristate 13-acetate), independently of receptor activation, to release oxygen radicals. The radicals produced reduce the cytochrome c and the resulting change in absorption is measured spectrophotometrically. The quantity of oxygen radicals produced is calculated from the change in absorption and the absorption coefficient for cytochrome c .
Analytical evaluation: the ability of the patient’s PMN to produce oxygen radicals is compared with those of a control individual tested in parallel. The amount (in nmol) of reduced cytochrome c produced per 1 million cells per unit of time is measured.
Reference interval: 5–15 nmol/106 PMN
Medical assessment: reduced or absent oxygen radical production is suggestive of defects in the enzyme cascade that leads to the reduction of molecular oxygen. Some of these enzymes or coenzymes can be determined and used to investigate granulocyte function.
Fresh whole blood (heparin, 50 IU/mL) that has been collected simultaneously from the patient and a healthy control and stored for the shortest time possible.
Disorders of PMN function increase susceptibility to infections. lists the microorganisms detected in patients with abnormal PMN function. PMN deficiencies are extremely rare (less than 1 : 200,000) and mainly affect infants and young children; a number of hereditary disorders exist. In these cases, family studies can help to support the diagnosis. PMN deficiencies can be primary or secondary to other diseases or associated with particular underlying diseases, even though the association is not always clear at molecular level /, /. Refer to .
Disorders of PMN function may not manifest until adulthood, especially in cases where residual functions are preserved.
When evaluating the results of investigations, it is especially important to take the influence of medication into account. Antibiotics in particular can affect chemotaxis, phagocytosis, adhesion, and oxygen radical production /, /. Refer to .
Leukocyte adhesion deficiency is divided into two subtypes: LAD I and LAD II.
LAD I is results from an autosomal recessive inherited deficiency of the β2-chain (CD18), which also results in reduced CD11 expression. In clinically severe cases, no CD11/CD18 is detectable on the granulocyte receptor, whereas in less severe cases, 3–10% of the normal quantity of CD11/CD18 is expressed. The cell adhesion deficiency is a distinct clinical entity .
LAD II is caused by abnormal fucosylation that affects various ligands, including those for E-selectin. As a result, the initial binding of granulocytes to the endothelium fails to take place. This in turn leads to a reduced integrin reaction, reduced granulocyte adhesion, and finally, to reduced granulocyte migration out of the blood vessel. Children with LAD II also experience other severe symptoms in addition to increased susceptibility to infections . The fucosylation defect can be detected by determining CD15 (Sialyl Lewis x) using cytofluorometry.
Other disorders of chemotaxis and migration have been described for which the molecular causes are not yet evaluated. A variant of LAD I has been identified (referred to by some authors as LAD III) that is due to a reduction in the binding capacity of β2integrins . Lazy leukocyte syndrome is another isolated deficiency that has only been described in individual case reports and is characterized by impaired PMN migration .
- Chemotactic activity is reduced in children with Down’s syndrome, juvenile periodontitis, and pyoderma gangrenosum. This is thought to be due to defects in signal transmission.
- Neutrophil-specific granule deficiency (SGD) is characterized by reduced chemotaxis. SGD is caused by a functional deficiency of a transcription factor (CCAA/enhancer binding protein epsilon), which is thought to lead to defective differentiation associated with an absence of granule protein synthesis. As well as a chemotaxis deficiency, granulocytes also exhibit other functional deficiencies (e.g., reduced bacterial killing and receptor expression) /, /.
Microbial infections lead to the production of chemoattractants, which promote the infiltration of PMN into the tissues. In the tissues, the PMN fulfill their most important function: the phagocytosis and intracellular killing of foreign organisms. A prerequisite for phagocytosis is the ability to recognize and bind to microorganisms. Pathogen-associated molecular patterns (PAMPs) are among the most important recognition structures present on bacteria. They are associated with groups of bacteria and include lipopolysaccharides, lipoteichoic acid, flagellar proteins etc. These PAMPs are recognized by pattern recognition receptors such as those from the Toll-like receptor family and by LPS receptor CD14 /, /.
Bacterial recognition is increased significantly by opsonization, which involves the binding of endogenous proteins to bacteria so that they can be identified by special receptors on granulocytes. Opsonization with fibronectin, for example, promotes the recognition of S. aureus or Streptococcus spp. .
Opsonization is improved by coating bacteria with specific IgG antibodies or the complement activation products C3b and iC3b. C3b is recognized by complement receptor CR1 and iC3b is recognized by CR3 (CD11b/CD18). Particles to which both C3b/iC3b and IgG are bound are phagocytosed optimally.
Specific receptors for IgG are found on granulocytes. These are called Fc receptors (FcγR) because they bind the constant fragment (Fc region) of the antibody.
Although there is some overlap between Fc receptor functions, the receptors differ in their binding capacity and avidity and may bind monomeric IgG, aggregates of IgG, or particular IgG subclasses preferentially. Allotypes of CD32 and CD16 have also been described. The CD32 allotypes, for example, differ by a point mutation at position 131, which may be occupied by either arginine or histidine (FcγRIIa-R131 and FcγRIIa-H131).
Variations in the binding capacity of IgG2 have also been described, with FcγRIIa-H131 having high binding capacity and FcγRIIa-R131 binding only weakly. Because encapsulated bacteria (Streptococcus spp.group B, H. influenzae type b, N. meningitides type B, and K1-positive E. coli) stimulate the production of IgG2 mainly, the FcγII allotype may play a role in the phagocytosis of these bacteria. In fact, studies have shown that patients with FcγIIa-R131 are at higher risk of developing meningitis .
Two isoforms of CD16 are also known: the FcγRIIIa isoform exists as an integral transmembrane protein on natural killer (NK) cells and macrophages and the FcγRIIIb isoform is bound to granulocytes via glycosylphosphatidylinositol (GPI). FcγRIII binds IgG aggregates, in particular IgG1 and IgG3, with a high degree of avidity. A CD16 polymorphism based on an amino acid substitution (NA1/NA2 polymorphism) has also been associated with different degrees of phagocytic efficacy but its effect on the immune system is not yet clear, especially since CD16 deficiencies are relatively common but do not seem to lead to an increased risk of infection /, , , , /.
Bacterial binding to PMN induces intracellular signal transduction cascades, the quality and quantity of which depend on the recognition of bacterial structures (PAMPs) and opsonization. PMN activation leads to the formation of pseudopodia, which surround the bacterium and enclose it in a vesicle formed by the cell membrane (phagosome) within the granulocyte. Phagocytosis increases the oxygen requirements of the cell as well as glucose metabolism, which is associated with the reduction of molecular oxygen (). Phagocytosis can only take place if cells are sufficiently motile and the cytoskeleton is intact. This is why disorders of phagocytosis are often associated with disorders of chemotaxis.
The ultimate purpose of chemotaxis and phagocytosis is intracellular bacterial killing. Oxygen-dependent and oxygen-independent mechanisms can be distinguished. Ingested bacteria are enclosed in a membrane vesicle (phagosome), which then fuses with azurophilic granules (lysosomes) to form a phagolysosome. This leads to the release of lysosomal enzymes. Meanwhile, oxygen radicals are produced by the stepwise reduction of molecular oxygen. This reaction is known as an oxidative burst or respiratory burst and is crucial to the intracellular killing of pathogens (). The oxygen radicals can also reach the area around the cells, where they are deactivated by serum proteins such as ceruloplasmin. Excessive production of oxygen radicals can lead to tissue damage and contribute to local inflammation.
Defects in the enzyme cascade leading to the production of oxygen radicals are rare (incidence 1/200,000) and manifest as recurrent bacterial infections, usually associated with cellular infiltrates and granuloma formation. This clinical syndrome is known as chronic granulomatous disease (CGD). Defects can affect the cytochrome b subunits p22phox or gp91phox or factors p47phox or p67phox of NADPH oxidase, which are located in the cytosol. Cytochrome defects are more common .
Neutrophil proteins such as myeloperidase (MPO) and proteinase-3 play a key role in the development of autoimmune disease. The proteins act as auto antigens against which a pathogenic immune response has been generated . MPO is an auto antigen in immune vasculitis and associated with MPO-ANCA (see ).
Myeloperoxidase (MPO) deficiencies, although relatively common (with an incidence of approximately 1 : 2000), are rarely clinically evident, which suggests that they are compensated effectively by other mechanisms.
Knowledge about bactericidal substances secreted by PMNs has grown in recent years. This applies to:
- The production of NO·, which has bactericidal as well as vasoactive effects
- Antibacterial proteins and peptides such as bacterial/permeability-increasing protein (BPI), defensins, and cathelins. The potency and mode of action of these natural antibiotics are comparable to those of classic antibiotics. Their role in defending against bacteria is difficult to assess.
- Deficiencies of defensin and cathelin-LL37 in patients with congenital neutropenia (Kostmann syndrome) are responsible for the increased risk of infection that persists even after the granulocyte count has been increased using granulocyte colony-stimulating factor (G-CSF).
PMN are active for a limited time only in the blood or following isolation. Complex activities such as chemotactic migration decline particularly quickly as a result of in vitro aging (2–6 h). When reduced PMN function is identified, therefore, it is important to check whether the blood or isolated cells have been stored for too long time. Isolation conditions, temperature, media, and buffers also influence PMN function and must therefore be kept constant and monitored.
Contamination by lipopolysaccharides (LPS) in particular must be avoided, because even very low concentrations of these substances can inhibit PMN function and the expression of surface receptors.
About 5% of PMN are found circulating in the peripheral blood with a half-life of 6–8 h or attached to the vascular endothelium as a marginal pool. The rest remain in the bone marrow, from where they can be rapidly recruited as required (e.g., to sites of infection) resulting in leukocytosis.
- Alteration in the shape and function of PMN to enable them to migrate actively instead of being transported passively in the blood
- The ability to recognize the focus of infection.
Local alterations in the vascular endothelium are necessary to allow PMN to migrate through blood vessel walls. Mediators that are released at the site of inflammation (e.g., histamine) stimulate the expression of adhesion molecules (selectins) by endothelial cells. PMN are able to recognize selectins and bind to them via a surface glycoprotein, whose carbohydrate component, mainly fucose, plays a decisive role in the binding. This results in the (unstable) adhesion of PMN to the endothelial cells and the activation of PMN adhesion proteins known as β2-integrins.
Integrins are heterodimeric plasma membrane receptors, each consisting of one α and one β chain bound non covalently. Both chains are required for normal receptor expression and binding to the corresponding ligands. The main integrins involved in leukocyte adhesion to the vascular endothelium are the β2-integrins, which have the α chain CD18 in common: CD11a/CD18 (LFA-1), CD11b/CD18 (MAC-1), and CD11c/CD18 (gp150, 95). CD11b/CD18 is a complement receptor (CR3) that binds activated complement C3 (iC3b).
The contact between the PMN and the endothelium causes the PMN to proliferate and then migrate actively between the endothelial cells and through the tissues to the focus of inflammation. This directed movement is referred to as chemotaxis, to distinguish it from spontaneous migration (random migration of non stimulated leukocytes) and chemokinesis (random movement of leukocytes in response to chemicals).
Chemotaxis is triggered by soluble mediators produced at the site of infection known as chemotactic stimuli. The main chemotactic stimuli are anaphylatoxin C5a, formylated peptide chemoattractants produced by bacteria [in experiments, the corresponding synthetic peptide N-Formylmethionyl-leucyl-phenylalanine (f-Met-Leu-Phe) is used], leukotriene B4 (LTB4), platelet-activating factor (PAF), interleukin 8, and transforming growth factor β. Others include neuropeptides and fragments of matrix proteins /, , /.
Granulocyte receptors transmit signals and trigger increased adhesion, deformation, and migration. The chemoattractants released at the site of infection diffuse locally and bind to granulocyte receptors. At the point of maximum chemoattractant concentration, the chemotactic receptors become polarized, which determines the direction of active migration. Because the highest chemoattractant concentration is usually found at the infection site, the cells migrate to this location (leukocyte infiltration) ().
All currently known chemokine receptors are associated with G proteins, which transmit chemotactic signals to the cell. Phosphatidylinositol-3 kinase (PI3K) and p38 MAP kinase are important signaling molecules that ultimately control cytoskeletal dynamics and, therefore, cell motility /, /.
Die Myeloperoxidase (MPO) is a cationic, heme-containing, glycosylated enzyme which is found in the azurophilic granules of PMN. The PMN contain the MPO/HOCl system for intracellular killing of bacteria and fungi (). MPO is involved in neutrophil functions in innate and adaptive immunity .Refer to ).
Activated PMN release NETs, structures composed of de condensed chromatin, histones, and various antimicrobial substances (e.g., myeloperoxidase, elastase) in the circulation, thus aiding in the overall elimination and spread of pathogens . NETs can trap extracellular microbes and can kill some, but not all species.
7. van Furth R, van Zwet TL, Leigh PCJ. In vitro determination of phagocytosis and intracellular killing by polymorphonuclear or mononuclear phagocytes. In: Weir DM, ed. Handbook of experimental immunology. London: Blackwell, 1978.
11. Brenneis H, Schmidt A, Blaas-Mautner P, Wörner I, Ludwig R, Hänsch GM. Chemotaxis of polymorphonuclear neutrophils (PMN) in patients suffering from recurrent infection. European J Clin Invest 1993; 19: 693–8.
16. Etzioni A, Frydman M, Pollack S, Avidor I, Phillips ML, Paulson JC, Gershoni-Baruch R. Recurrent severe infections caused by a novel leukocyte adhesion deficiency. N Engl J Med 1992; 327: 1789–90.
26. Bredius RGM, Fijen CAP, De Haas M, Kuijper EJ, Weening RS, van de Winkel JG, Out TA. Role of neutrophil FcRγIIa (CD32) and FcγRIIIb (CD16) polymorphic forms in phagocytosis of human IgG1- and IgG3-opsonized bacteria and erythrocytes. Immunol 1994; 83: 624–30.
27. Sanders LAM, Feldman RG, Voorhorst-Ogink MM, de Haas M, Rijkers GT, Capel PJA, et al. Human immunoglobulin G (IgG) FcγReceptor IIa (CD32) polymorphism and IgG2-mediated bacterial phagocytosis by neutrophils. Infect Immun 1995; 63: 73–81.
30. Wagner C, Hänsch GM. Genetic deficiency of CD16, the low-affinity receptor for immunoglobulin G, has no impact on the functional capacity of polymorphonuclear neutrophils. Eur J Clin Invest 2004; 34: 149–55.
PMN, polymorphic mononuclear granulocytes; HETE, hydoxyeicosatetraenoic acid; HPETE, hydoxyeicosatetraenoic acid
RANTES, regulated on activation, normal T-cell expressed and secreted
Clinical and laboratory findings
FIO2 ,fraction of inspired oxygen; PaO2 ,partial pressure of oxygen; MAP, mean arterial pressure; *with respiratory support. The SOFA score is not meant to indicate the success or failure of interventions or to influence medical management.
Clinical and laboratory findings
Clinical and laboratory findings
ESR, erythrocyte sedimntation rate; SPE, serum protein electrophoresis; TM, temperature measurement
Clinical and laboratory findings
Clinical and laboratory findings
Score points and
Values are median, 2.5 and 97.5 percentiles
Values expressed in mg/L. Threshold values were determined in small groups of 27–80 individuals.
Median values and ranges (in parentheses) for 52 patients.
* Minimal effective SAA concentration required to exert biological activity
? = questionable; contradictory data
Primary granulocyte disorders
Disorders of granulocyte function in other diseases
Transient secondary granulocyte disorders
Reduced granulocyte function
* Sometimes combined with disorders of phagocytosis
Figure 19.1-1 The activation of NFκB takes place through phosphorylation and subsequent proteolytic degradation of the inhibitor protein IκB by IκB kinases. Free NFκB, a heterodimer composed of a 50 kDa and a 65 kDa unit, migrates to the nucleus, where it binds to specific sites on the promoter regions of genes that are responsible for the synthesis of inflammatory proteins such as cytokines, enzymes, and adhesion molecules. p, protein; mRNA, messenger RNA. Modified from Ref. .
Figure 19.1-2 Activation of the pro inflammatory cascade by lipopolysaccharide (LPS). This interacts with the LPS-binding protein (LBP) and binds to the CD14 receptor on macrophages. Via the signal pathway of NF-κB, transcription of cytokine genes takes place, followed by translation and secretion of pro inflammatory cytokines and chemokines within 15 min. In addition, anti-inflammatory cytokines that down-regulate inflammatory activity such as IL-10 and TGF-β are activated. The inflammatory reaction is modulated by positive and negative feedback (+ve; -ve). Abnormal regulation of cytokine production leads to a disproportionate inflammatory response. Modified from Ref. .
Figure 19.1-3 Induction and regulation of heat shock protein (Hsp) expression. Modified from Ref. . Physical or chemical stress induces the synthesis of proteins that are not folded or that are folded incorrectly. Monomeric heat shock factor in the cytoplasm forms trimers, which are phosphorylated and trans located to the nucleus. In the nucleus, they bind to Hsp promoter regions and induce the synthesis of Hsp. Hsp is typically released upon cell destruction but can also be released by certain cells such as smooth muscle cells and islet cells in response to oxidative stress alone, without cell destruction.
Figure 19.1-4 Hydrolysis of phospholipids by phospholipases. As a result of the action by phospholipase A2, arachidonic acid and a corresponding lysophospholipid are produced. The residue X may represent: inositol (PI, phosphatidylinositol), choline (PC, phosphatidylcholine), ethanolamine (PE, phosphoethanolamine), or serine (PS, phosphatidylserine).
Figure 19.1-6 Cytoplasmic membrane with receptors, G-proteins (α, β, γ) and bound phospholipase A2 (PLA2). The figure depicts the metabolic pathways into HPETE/HETE, leukotrienes (LT), and lipoxins (LX) via lipoxygenases, into prostaglandins (PG), thromboxane A2 (TXA2), and prostacyclin (PGI2) via cyclooxygenase (CO), and into PAF via lyso-PAF and acetyltransferase. 20:4-AA, arachidonic acid; O-CO-R, fatty acid in an ester bond; alkyl, fatty acid in an ether bond (with kind permission from U. Tibes).