Pathology and Diagnostics - Inflammation 1 - Clinical Pathology 1 PDF

Pathology and Diagnostics Garlanda – Clinical Pathology – lesson 01: Inflammation 1 19/10/2024 - Group #33 (Stefania Pesaresi - Valeria Viti) What will be told in this course can be found in this textbook: The foll...

Pathology and Diagnostics Garlanda – Clinical Pathology – lesson 01: Inflammation 1 19/10/2024 - Group #33 (Stefania Pesaresi - Valeria Viti) What will be told in this course can be found in this textbook: The following topics will be presented: - Laboratory tests of inflammatory responses - Laboratory evaluation of the cellular immune system - Laboratory tests for cancer MARKERS OF INFLAMMATION We briefly remember that the immune system has three lines of defense: First line of defense – skin and mucous membranes, the normal microbiota Second line – inflammation and the activity of innate immunity, which is the focus of some of the lessons Third line – adaptive immunity, present only in vertebrates, and we will only mention some of the modifications of leukocytes in the circulation mainly, associated with the specific condition, that may guide you in the diagnosis. Of course, the kinetics of the involvement of these three lines are very different: the innate immunity is the first response that we have, and we develop it in hours, whilst the adaptive immunity takes days. So, in a patient you may expect to find abnormal innate immunity parameters, or abnormal adaptive immunity parameters, depending on the kinetics of the condition that you are studying. The first topic is inflammation, because inflammation is present both in the initial part of innate immune response as well as in adaptive response. It is always present: in almost every condition you will face there is a component of inflammation. We know that inflammation is activated by conditions of infection, tissue damage, homeostatic imbalances, dysmetabolism (which means any metabolic condition that you may study related to metabolism of carbohydrates or lipids), dysbiosis (meaning an abnormal ratio between genera and species in our microbiome, either in the intestinal tract or in the skin or everywhere). 1 What is the meaning of inflammation? Essentially inflammation has phylogenetically the meaning of coming back to the homeostatic state, activating defense mechanisms and activating repair mechanisms. So, with all of this in mind, we will see that the markers that we are investigating in the patients are related to these steps or phases of the response. First of all, you know that the inflammation is activated upon recognition of tissue damage or microbial moieties of dysbiosis, by receptors that are expressed on the cells, which sense the presence of these imbalances in the environment. Cells that are involved in sensing these abnormalities are normally: Professional cells/Leukocytes: macrophages, neutrophils, dendritic cells, NKCs Non-professional cells: epithelial cells, stromal cells and endothelial cells They all contribute to activating an inflammatory response. In all cases these cells have different receptors, which may be located in the cell membrane, or in the cytoplasm, or in phagosomes. Others are then released in the extracellular environment, so they are soluble molecules released by the cells. They all play a peculiar role, and even if sometimes they share roles, some roles are specific for a certain type of receptors. So, in general, we classify them in: Receptors that activate an inflammatory response by activating the transcription factors, mainly the NFkB transcription factor, or the IRF, which are responsible for the regulation of the transcription of the genes associated with inflammation, like cytokines, interferons, but also acute phase proteins. Acute phase proteins are those that you will look for in your patients to determine whether an inflammatory response is present in the patient. All of these are activated downstream to the activation of transcription factors, under the activation of several receptors, like Toll-like receptors, several integrins and some sensors of viral molecules. So, any type of sensing will lead to this process and this kind of activation Group of molecules that are released, so soluble molecules that can be also used as markers for the inflammatory process. 2 In any condition of disruption of the homeostasis, microbes, dysbiosis, metabolism, we have the activation of a cytokine cascade, of which some are the leaders: TNF, IL-1 and IL-6 are really the leaders of this cascade. Some act locally, TNF and IL1 in particular, others, in particular IL-6, act at a systemic level: Locally: when these cytokines are produced, they will cause the recruitment of leukocytes because they induce the production of chemokines or colony stimulating factors or prostaglandins. So, all these factors regulated by the IL1 and TNF, will lead to the recruitment of leukocytes and promotion of their survival. And they will in turn activate a loop, a positive loop of activation of this innate immune response and they will eventually orient the adaptive immune response. Systemically: At the systemic level, IL6 will cause the production at the level of the liver of a huge number of proteins that we will discuss now, which are called the acute phase proteins, which are amplifying the systemic response. Further, with these positive regulators, also negative regulators are produced, examples are the glucocorticoids, downstream the hypothalamus and adrenal axis, TGF-beta, interleukin-10, and these are the main, the principal role to tune this cascade in order to avoid an excessive activation of inflammation. That means that you may look as a marker of inflammation, not only to the positive regulators but also to the negative regulator; so if you want to make a picture of the state of inflammation in the patient, you may look for the positive and negative regulators, and you will see that they may be activated in parallel. 3 Effects of IL1: One of the kings of cytokines is interleukin-1. This molecule is produced by different stimuli, including: microbes, the activation of the inflammasome, senescence, dysbiosis, alteration of the metabolism, tissue damage. Depending on the levels, we can have systemic inflammation or local inflammation. So, in a patient you may observe signs of systemic inflammation, which normally are due to IL1, and the target organs are the brain for instance, this is why you have fever and the induction of cortisol, because you have activation of the HPA axis, the liver, so this is why you have the acute phase proteins. Endothelial cells are also targeted, so this is why you have leukocyte recruitment in the tissues involved by information, and so on. You also have activation of leukocytes and functional polarization. So, we said that, upon sensing this alteration, interleukin-1 and interleukin-6 play this major role and activate the synthesis of proteins, the acute-phase proteins, either in the liver, IL6 in this case, or in other cells. So, you may find these acute phase proteins, which are shown here (image on the right), which may derive from the liver. Examples are the C reactive proteins, serum amyloid components, we will discuss them in a moment. Others that derive from cells in the tissues, such as the pentraxin-3. Others that may derive from both the liver or locally in the tissues: these include different molecules that you will use as markers to detect inflammation in the patient, like fibrinogen, fibronectin, serum amyloid component and complement molecules. And these molecules are in general not only biomarkers for inflammation, but also players of a pathological condition. So, several of these molecules are responsible for tissue repair or extracellular matrix remodeling, coagulation (which, in the simplest animals, is a form of defense) and humoral innate immunity, so recognition of the pathogen, activation of the complement and opsonization. So, they are both biomarkers and players of the process. These molecules are produced and released in the circulation with a kinetic which is specific for each molecule. So, this is (image on the right), for instance, what is occurring in terms of change in plasma concentration of all these molecules over time, days after the application of an inflammatory stimulus. 4 You see that we have some biomarkers that you daily use in your patients, such as C-reactive proteins, which has a relatively rapid induction (still in terms of days) and then it returns to the basal level when the inflammatory stimulus has been eliminated. Others have completely different kinetics, for example fibrinogen increases slowly over time and remains high over time. Others that we will discuss together now, have different values, so transferrin, for instance, is considered a negative acute phase protein because its production is reduced after inflammatory stimulus, and then it returns to the basal level. One of the molecules that we described, pentraxin-3, is not ready for clinical use, so you will not use it now in the clinic, but it is a molecule that Garlanda’s and Mantovani’s group has developed in Humanitas as a biomarker. It is a relative of the C-reactive protein, and its kinetics are much more rapid than that of C-reactive proteins. So, the peak of elevation of this molecule is in a few hours after an inflammatory insight, and then it returns to the basal level more rapidly than the C-reactive protein. For this reason, we define some positive and negative acute phase proteins depending on the concentration in the situation, if it increases or decreases. When you need to assess inflammation in a patient, that may be acute, chronic, severe, or mild, affecting different organs, you may use different tests: Erythrocyte sedimentation rate C-reactive protein Procalcitonin Other acute phase reactants Erythrocyte sedimentation rate (ESR) This is a very old test, so it is not surprising that you don't see it in a hospital like ours. The definition is: the rate at which red blood cells sediment in one hour. You just have to put your blood in a tube and wait to observe sedimentation. Sedimentation is measured in millimeters per hour. This is of course not expensive at all, any lab can do it, even in a developing country where maybe you don't have any other sophisticated instrument to test all the other markers that are at the moment on the market. The blood is collected with an anticoagulant which may be the sodium citrate or EDTA. You know that the tubes in which samples of blood are collected from a patient have a cap with a different color, and this color is associated with the different anticoagulant that is inside the tube. So, depending on your objective, on the test you have to perform, you have to pick the correct type of sample. Blood is then put in a tube and then the time in which the red blood cells sediment is measured. The sedimentation rate is influenced by pro-sedimentation factors: there are factors associated with inflammation which promote the sedimentation. Examples are: fibrinogen, which we have 5 seen before, which is one of the acute phase proteins produced by the liver during inflammation, but also other proteins produced during inflammation, like alpha2 and beta gamma globulins. These pro-sedimentation factors are balanced by negative factors, which consist in the negative charge on the surface of the erythrocytes. During an inflammatory process, the pro-segmentation factors, in particular fibrinogen, will increase and these molecules normally neutralize the negative charge on RBCs. Therefore, the erythrocytes, instead of repulsing, tend to adhere one to the other, forming what we call ”rouleaux”, a french word indicating a group of cells attached one to the other. And since they are plastered, they settle faster than free red blood cells. Another important thing to remember is that the normal erythrocyte sedimentation rate differs between males and females and due to age. So, you will have to link the values you obtain with the age and the gender of the patient. Which are the conditions in which these sedimentation rate increases? Inflammation, first of all, since proteins mask the negative charge of RBCs Anemia, and this is due to an effect on red blood cells themselves Autoimmune disorders, that means again inflammation, Infections, that means inflammation Pregnancy, which probably is linked to the red blood cells. And then, there are other conditions which are less specific, but in a certain way associated with the balance between pro-sedimentation factors and negative charges. However, the sedimentation rate can also decrease, for instance, in condition of hyperviscosity of the blood, polycythemia (condition in which the bone marrow is producing an excessive number of cells, meaning that blood viscosity is increased), sickle cell anemia (abnormality of the shape of the red blood cells so that the sedimentation rate may be modified because of this), condition of neoplasia of leukocytes, so leukemia, for the same reasons: you change the ratio of the cells and so the density of the cellular part of the blood. Also, when you have low plasma protein because of a chronic condition affecting the liver or the kidney. So, as I said, you probably will see very rarely in a hospital like ours, but if you are going to have an experience in any developing country, you may experience this: they don't have any sophisticated instruments to test the other inflammatory markers, so they use this, since the cost is zero and it's very simple to do. 6 C-reactive protein C-reactive protein is actually one of the proteins that you will measure daily, even when it is not really necessary, because the cost is very low. But in any case, it's highly used because actually it is a useful molecule. This is the morphology of the shape of this protein (image on the right). It is a pentamer, so made of five units, identical. And it is the classical acute phase protein. It is produced by the liver in response to IL6 and it is a member of the pentraxin family. It was one of the first part-pattern recognition molecules identified in the history of immunology because this molecule recognised a component of the polysaccharide of the Streptococcus Pneumoniae, and by binding to it, it promoted the opsonization of this microbe and the clearance by phagocytes. Essentially this is an opsonin. Remember that an opsonin is a molecule that binds to pathogens but also to apoptotic cells, dead cells or damaged cells, promoting the clearance in an anti-inflammatory manner. So, whenever you have a tissue damaged, in terms of homeostasis, it's much better to have someone who is able to clear the debris, the dead cells, and avoid that an inflammatory response is activated because of the presence of damaged tissue. When you have an inflammatory signal, the liver starts producing this protein, so a few hours after the inflammatory stimulus and its half-life is relatively low (5h). That means that when you analyze the levels of CRP in the circulation, the levels are mostly due to the production by the liver because in any case it is rapidly eliminated. So, when you measure it, it is like a picture of what the liver is doing, not the clearance of the molecule, because the clearance is relatively rapid, so you don't care about what is eliminated, you care about what is produced. The normal levels of concentration of CRP in a healthy adult are about 0.8 mg/l to 3 mg/l. Note that milligrams per liter is a huge amount, seen that for example others are measured in micrograms per liter. So, when you have an inflammatory stimulus, CRP levels can rise to more than 500 mg/l. So really an enormous increase. So that means that you have a really wide window of concentration in which you can detect a pathological condition. These on the right are the patterns of response of CRP after an inflammatory stimulus: as we said, there is a peak in a few days and then it returns to the basal level in more or less one week. The erythrocyte sedimentation rate is much lower and indeed less useful. So, CRP is for sure more sensitive and more accurate than ESR and there are different ranges of normal and abnormal levels that you have to try to fix in your lab. 7 There are factors that affect the erythrocytes sedimentation rates, for example pregnancy and anemia. For CRP it is not the case: C-reactive protein is much less affected by conditions that would in contrast affect the ESR. Which are the conditions in which CRP increases? Any inflammatory diseases, including rheumatic disease, autoimmune conditions, infections, trauma, necrosis, cancer even (you can ask for CRP to investigate the inflammatory component of the cancer) or allergic reactions. CRP levels: Considered that the normal levels are 0.8-3 mg/liter, you can have abnormal ranges, which depend on the type of condition the patient has: metabolic inflammation, for instance, atherosclerosis or type 2 diabetes, range between 2-10 mg/l, just a bit above the normal value. mild inflammation, for instance, a mild viral infection, and you may range between 10 and 40 mg/l active inflammation, for example a bacterial infection, let's imagine pneumonia, or urinary tract infection, levels may reach 200 mg/l severe bacterial infection or a burn, for instance, levels may be above 200 mg/l. In this case, it may be a very severe condition that may evolve into sepsis. So, once the problem has been eliminated, CRP level may return towards the basal levels relatively rapidly because the half-life is short, so production is not persisting anymore and elimination is rapid because the half-life is short. We test this molecule by a technique which is called nephelometry at the moment, but it can be tested by other immune-mediated approaches, such as ELISA and immunoturbidimetry. For nephelometry, you collect your sample, and if the C-reactive protein is there, antibodies will recognise it. So, antigen-antibody complexes form, and these can be measured, since they form aggregates that will scatter the light that passes through the sample. So, the light that is passing through the immune complexes will depend on the amount of the immune complexes that have been 8 formed in the sample, meaning that the light will be proportional to the quantity of the antigen you have in your sample. You will observe when you will be in the clinics that in several cardiovascular diseases, C-reactive protein is used as a very good marker to detect cardiovascular disorders. But the levels of patients in the range of metabolic inflammation (such as in atherosclerosis), are relatively close to the normality, as we said before. So, in these types of conditions, you will use not the normal C-reactive assay, but a high-sensitivity CRP test (has-CRP). This is made with a laser nephelometry, which does not really matter, but the point is that the resolution and the sensitivity is much higher. This way, you may detect even 0.04 mg/l. So, in the range of normality, there are in any case individuals who have higher than absolutely normal levels and this is associated with increased susceptibility to develop cardiovascular disorders, since it reflects a very mild but chronic inflammatory process. It has been shown that the high-sensitivity CRP test should be used when you have in front of you a patient in which you suspect or you want to study the presence of atherosclerotic cardiovascular disease. So, based on the levels of normality (we said from 1 to 3 mg/l), we can identify: patients with low risk to develop cardiovascular disorders - less than 1 mg/l, average risk - 1 to 3 mg/l, high risk - above 3 mg/l There has been a very important trial, the JUPITER trial, reported in a paper in the New England Journal of Medicine, in which it was shown that even if normally to detect the presence of risk to develop cardiovascular disorders, we measure the levels of LDL cholesterol, these levels may be normal in a patient that develops a cardiovascular disease. It showed that half of all vascular events occur in patients in which the LDL levels are normal. So, this study, Jupiter, decided to investigate the risk to develop cardiovascular disorders based on the high sensitivity CRP, and then based on this, use the Rosuvastatin to prevent these vascular events. So essentially the study suggests that even if the cholesterol levels are normal, you may use statins to reduce cardiovascular risks based on the CRP levels. If you use this high sensitive CRP, you may detect patients at risk to develop a myocardial refraction, stroke, peripheral arterial disease, sudden cardiac death, even if they have a normal level of LDL cholesterol. Student’s question: inaudible Answer: yes if you will work in the cardiology world you will use this type of test because your patients have cardiovascular disorders, it is not used for sepsis or pneumonia unless they are mitigated of course, since you use this type of test to measure the subtle abnormalities of chronic inflammation which is cause of cardiovascular disorders. 9 A second study, the CANTOS trial, was based on the same principle, and it wanted to show that if you treat patients who have high CRP levels, with anti-IL-1, you prevent cardiovascular disorders. So, you can perform anti-inflammatory therapy in patients with atherosclerotic disease, diagnosed atherosclerotic disease, or previous events of acute myocardial infarction, for instance. So, once you have defined your population at risk to develop a severe cardiovascular event, then you may start this type of therapy to inhibit inflammation. You will see that depending on the type of patients you have to treat, you will ask for one or the other test, and really, the scenario is completely different. If you are in the world of infectious diseases, where pneumonia, urinary tract infections, viral infections are your daily life, you will not use high-sensitivity CRP, because it is useless. You use high sensitivity when you want to measure a very mild form of inflammation, but chronic, which is absolutely deleterious. Pentraxins and the long pentraxin PTX3 CRP is a member of the pentraxin family, and it is called a short pentraxin. PTX3 is another pentraxin, a long pentraxin. In the 90s, when professor Garlanda was part of Alberto Mantovani's group, the group identified the pentraxin 3, which is called the long pentraxin because the X domain is quite long (see picture on the right) (all these molecules have an highly conserved domain in evolution, and then the X domain which varies). We have seen before that CRP is produced by the liver. In contrast, PTX3 is produced by different cell types of the immune system: leukocytes (most of them are neutrophils, macrophages, dendritic cells), but also stromal cells, fibroblasts, adipocytes, endothelial cells. And these all produce pentraxin-3 in response to an inflammatory stimulus, microbes, or tissue damage. This molecule has different functions, some overlapping with CRP, so it opsonizes microbes and eliminate them. PTX3 is also working as an acute phase response biomarker and indeed, we are exploiting this as a biomarker. IL6 induces CRP in the hepatocytes, all the other cytokines and microbial moieties induce pentraxin-3 in all the other cell types. Once they are released, they both have a diagnostic potential. So, when we discovered this molecule, since the molecule is produced by different cell types, in particular endothelial cells, we hypothesized that this marker could reflect disorders of the vascular bed. Indeed, this is what the professor’s team has done in the last 20 years, and we have shown that pentraxin-3, once released, may be used as a marker of prognosis of several conditions. 10 One example is for COVID: the group analyzed PTX3 in the circulation thinking that it could be a marker of the severity of the condition. We observed that it was produced by macrophages and endothelial cells, and it was associated with the severity of the condition. The picture on the right shows an immunohistochemical analysis, in which in brown are the cells that produce this molecule. In the picture we find a thrombus, and we see that endothelial cells that surround the thrombus are particularly dark and brown. That means they produce PDX3. Then, the group collected the samples from the patients inside the hospital and we analyzed pentraxin-3 levels and observed that the patients who died earlier presented higher levels compared to patients who were discharged from the hospital. This is a Kaplan-Meier curve, a graph which reports the survival of a patient. So, these sort of steps in the graph are the number of dying individuals over time. The blue line indicates the patients with the lower levels of pentraxin-3; in red the intermediate level and in green those who had the highest levels. With this it was shown that pentraxin-3 was a predictor of mortality and since the samples were collected at the entrance in hospital, the molecule levels already reflected the severity of the condition, even of a patient who will eventually die 28 days after hospitalization. This study was then confirmed by other groups independently: when you discover a potential marker, it's always useful that someone else confirms your data. And indeed, these are three independent studies where all of them found that the pentraxin-3 levels in Covid patients was associated with the severity of the condition and mortality. For example, an interesting study made in the UK in London, by doing a proteomic analysis, so collect the plasma and analyze all the proteins present in the circulation found out that among the non-survivors, pentraxin-3 was one of the highest molecules in terms of levels compared to the non-survivors. During this last year, we found out that PTX3 is a good biomarker reflecting cardiovascular diseases, inflammatory conditions, sepsis, infections, and even the genetic polymorphism of the molecule are associated with the condition. 11 PTX3 and myocardial infarction This is a classical kinetics of behavior of C-reactive protein in the situation of patients with acute myocardial infarction. If a patient arrives at the ER with pain in the thorax or the stomach, immediately this type of marker is analyzed, because immediately acute myocardial infarction is suspected based on the symptoms of the patient. During the first 24 hours, nothing will occur in these patients in terms of CRP levels. Whereas for pentraxin-3, you have an immediate rise and increase in the concentration, then return to the basal level. And indeed, the two molecules have completely different kinetics. PTX3 is very rapid, produced directly by the heart undergoing infarction, whereas CRP is produced by the liver in the acute phase response. Indeed, the two molecules do not correlate one to the other because the kinetics are completely different. And then another study confirmed this, showing that PTX3 was the best predictor of mortality, much better than the classical biomarkers used in acute myocardial infarction, including CRP, creatine kinase, troponin T, or the protein P. This (graph on the right) is another study carried out on patients with heart failure, a chronic condition, and again patients with higher levels of PTX3 died at a higher frequency over time at cardiac events, compared to those of normal levels. Another study was carried out on sepsis. At least a few years ago, people with a severe infection, critically ill patients, were classified based on specific features of the condition in different levels of severity. Starting from systemic inflammatory response syndrome (SIRS), progressing to sepsis, severe sepsis and then septic shock. Now the classification is different, but the concept is that starting from less severe condition to the most severe condition, PTX3 levels were progressively higher. 12 And PTX3 correlated with the other classical biomarkers used in this type of condition and correlated with the survival of the patients. In another study, performed by Professor Jaillon, PTX3 was measured in urine. So, you don't have only the plasma to measure an inflammatory biomarker, but you can use any biological fluid. Urine samples were collected from patients with a severe form of a severe UTI, meaning acute pyelonephritis. These patients displayed higher levels compared to patients with a minor form of the condition or patients that already started antibiotics. And the last one is a study that has just been published by the Professor’s group, in which they investigated the potentiality of secondary infections in patients with COVID-19. When you have a viral infection in which there is damage to the epithelial layer, so our first line of defense, then you have other microbes that may exploit this condition and cause an infection and a disease. In particular, in patients hospitalized because of the COVID-19, one of the problems was the development of secondary infections, which are normally bacterial or fungal. You need to follow these patients because you have these inflammatory markers which are already high because of the viral infection and you have to understand whether you can discriminate another infection developing in the patient to decide whether to add an antibiotic or not. So, in this study, they observed that PTX3 levels at the time of diagnosis of an in-hospital acquired infection had much prognostic and predictive levels and specificity compared to CRP. This (graph on the right) is a ROC curve which defines the sensitivity and specificity of an assay: moving higher and to the left, sensitivity is higher to detect a real problem and specificity is higher, meaning that you do not make a mistake in the diagnosis. So in this case we found that PTX3 was acting better than CRP. Question: inaudible Answer: If you measure the PTX3 levels in a patient who is hospitalized, let's imagine your patient is in the hospital because of COVID, so for sure he has a fever. But one morning, you find it very strange with symptoms that suggest another infection (maybe the fever is higher and the clinical conditions are worse than the previous day). So, you may suspect a secondary infection, which is quite common (15% of patients with COVID develop a secondary infection) and you should immediately start an antibiotic, otherwise your patient dies because of this secondary infection. So, you collect a sample, let's imagine, from the respiratory tract, and you send it to the laboratory of analysis to detect this eventual secondary infection. But if in the meantime you 13 measure PTX3 levels you observe that the patients in addition to having fever, will have signs recalling a secondary infection and also increased PTX3 levels. These levels will be better reflecting the complication compared to the levels of CRP, since CRP will rise and go down too fast, so you do not detect a secondary bacterial infection because of this kinetic of induction, you need something more rapid. Procalcitonin (PCT) Procalcitonin is a peptide which is the precursor of calcitonin, a hormone involved in calcium homeostasis. It is normally produced by the parafollicular cells (C cells) of the thyroid and by the neuroendocrine cells of the lung and the intestine and normally processed by endopeptidases in order for the active form to be released, calcitonin, which has a role in regulating calcium homeostasis. However, in inflammatory conditions it has been shown that other cell types, in addition to the parafollicular cells, may produce this protein, which is then used as a biomarker of inflammation. On the right (in blue) is shown the normal functioning of the molecule, on the left (in green) the functioning during inflammatory conditions, usually a bacterial infection. The normal levels are very low, normally below detection levels (0.01 μg/L). When an inflammatory stimulus develops, especially of bacterial origin, you have the production of this molecule as an acute phase reactant, that would be measured in the circulation. Reminder: bacterial infections induce a universal increase in the expression of this gene in any type of cell. The gene is not induced in any form of viral infection or sterile inflammation (meaning not due to a microbe). The levels of this molecule may increase from 0.01 to 100 μg/L, in case of a bacteria infection, and its half life is relatively longer compared to CRP (25-30h) These are the definitions of sepsis valid until less than 8 years ago. SIRS → Severe Inflammatory Response Syndrome Sepsis Septic shock 14 The definition of SIRS is based on different parameters, like temperature, heart rate, respiratory rate, the number of leukocytes in the circulation and the presence of immature forms, reflecting the high activity of the bone marrow producing leukocytes. These types of alterations are what defines sepsis, although it is not important for the purpose of this exam to know the values and ranges listed below. The intermediate form is sepsis, in which the condition is already life threatening because some organs are dysfunctional. The most severe form is septic shock where the patient is really close to death because of organ failure. PCT was developed as a parameter to diagnose early sepsis. Since it is a very severe condition causing death in a few hours, it requires immediate intervention. This is a ROC curve similar to the previous one. When the curve is towards the left it means that the molecule has a high sensitivity and specificity for the condition you are investigating. PCT showed a good performance in terms of diagnosing early sepsis. Its kinetics are very close to the other inflammatory mediators that govern the cascade, while CRP is produced later on. You can also see IL-10, an anti-inflammatory cytokine, which is produced together with the pro-inflammatory molecules, for balance. As you can see here, the concentration of this molecule is associated with the survival of the patients. This curve indicates the percentage of survival of patients depending on the level of the biomarker present. You may establish a threshold above which or below which you can divide your population, discriminating a low or high level of the molecule, studying the event you want to study, in this case the mortality. In this case high levels are associated with higher mortality. Survival is rapidly decreasing in these patients. 15 For this reason, PCT was shown to better discriminate sepsis from SIRS compared to CRP. These markers are unfortunately never enough for the diagnosis of such complex conditions, in parallel you need to take into account all the other parameters of the patient. The professor proceeded to read the table above containing the different levels of PCT measured and what they mean. These are values that should be known because they dictate the behavior in case of severe conditions avoiding death. PCT has limitations, because sometimes, as already mentioned, sepsis can be caused by viruses and levels in this case are not modified. We can have abnormal variations in PCT in sterile conditions in the case of: neonates < 48 hours of life (physiological elevation) the first days after a major trauma, major surgical intervention, severe burns, treatment with drugs stimulating the release of pro-inflammatory cytokines patients with invasive fungal infections, acute attacks of Plasmodium falciparum malaria patients with prolonged or severe cardiogenic shock, prolonged severe organ perfusion anomalies, small cell lung cancer, medullary C-cell carcinoma of the thyroid In these cases antibiotics would not be useful. Furthermore, when the levels are low, you cannot exclude the presence of an infection. When we studied patients that developed a secondary infection after Covid-19 and studied in parallel the levels of PTX3, CRP and PCT, we found that PCT was not really informative compared to the other 2. It is rapidly induced after an infection, normally a few hours are enough compared to CRP which is slower; it is stable, so you can collect your sample and for some hours the molecule remains stable when refrigerated and therefore measurable. At -20 °C, it is stable for 6 months. There are several companies who have developed kits to study and test this molecule, for example bioMérieux. It is unfortunately extremely expensive, it is paid by our health system but 16 still, some medical doctors are not convinced about its efficiency. These companies are now trying to persuade them, but the health system is not willing to invest more money if people are not sure. The FDA cleared one of the tests (VIDAS) also to guide the use of antibiotics, since their abuse is the major cause of the increasing resistance to them. The FDA therefore established clear guidelines, listed in the image on the right: The FDA cleared the use for lower respiratory infections, such as acute bronchitis, pneumonia, and acute forms of chronic obstructive pulmonary disease (COPD) since these conditions may be caused by viral and bacterial. In this case PCT would represent a clear marker to discriminate between the 2, not using antibiotics in the case of a viral infection. Acute phase response and iron metabolism When I showed you this graph at the beginning of the lecture, I also mentioned molecules produced by the liver during the acute phase response, which are molecules known to bind iron. Iron is an important growth factor for microbes, they use it, so the molecules that bind iron have a dual role: they are responsible for eliminating the available iron from microbes, competing with them; they are responsible for the use of iron for our molecules, for example for the generation of hemoglobin; The liver is producing several molecules which are related to iron metabolism: some hormones (hepcidin, ferritin, haptoglobin, and hemopexin) are upregulated during the acute phase reaction. Transferrin is a negative acute phase protein, downregulated during the acute phase response. All these molecules have a specific role, for example promoting transport (hepcidin and ferroportin), or binding to heme (haptoglobin and hemopexin). You will use some of these molecules because it is important for you to know the metabolism of iron in the patient, and others because they reflect the inflammatory process. 17 Ferritin is a clear example of this. The levels of ferritin are associated with the levels of iron in the cells, so it is an indirect marker of the total amount of iron present in our body in normal conditions, and on the other hand it is an acute phase protein. You will use ferritin to diagnose iron deficiency and anemia (iron levels are below the needed normal amount), but also to diagnose an overload of iron in the body (like hemochromatosis or hemosiderosis). Ferritin is also used as a marker in the acute phase response. For instance, in Covid-19 it was always analyzed combined with other biomarkers to analyze the severity of the infection. There are 2 forms of anemia: due to iron deficiency: ferritin will be lower chronic inflammation-associated: in this case the liver will produce higher than normal levels of ferritin You don't need to know these values by heart In chronic liver disease the situation is even more complex: you might have iron overload because there is an abnormal production (reduced) of molecules that transport the iron inside and outside the cell, or you might have iron deficiency because of portal hypertension associated with chronic liver disease. When you have chronic liver disease, do not use ferritin as a marker without knowing all these complexities and factors that may impact on the level of ferritin or iron. Some of the acute phase proteins are also involved in tissue repair and coagulation. The first example is fibrinogen,which is upregulated and highly used during an inflammatory process. During Covid-19, it was one of the molecules that were investigated, along with its byproducts. Fibrinogen is normally degraded in byproducts that can be measured in the circulation, and d-dimer is one of them, widely used in clinical practice. Fibrinogen, in inflammation, bind platelets together allowing the formation of an insoluble clot. Covid-19 was associated with an increased thrombotic disorder, and patients often died because of thrombi in the periphery or in their lungs. This is why molecules like fibrinogen and d-dimer were highly used to demonstrate that the infection was a coagulation and inflammatory disorder. Tissue repair and coagulation evolve together as processes, so the insoluble fibrin that forms as a result of fibrinogen cleavage represents a provisional matrix, essential for tissue repair. When you have tissue damage, you have fibrinogen exiting blood vessels because of increased permeability, which is then transformed in its insoluble form, fibrin. This net of insoluble 18 molecules form a provisional matrix that is essential for the immediate repair. This matrix must be remodeled through a process of fibrinolysis and then it will mature into a normal extracellular matrix. Fibrin is indeed very important. In addition to this, in the acute phase response, you have the release of fibronectin or inhibitors of proteolytic enzymes aimed at reducing tissue damage (α1-antitrypsin, α2-macroglobulin, and α1-acid glycoprotein). These molecules are part of the acute phase response and are associated with the principle of inflammation, tissue repair and return to homeostasis. In evolution, fibrinogen and fibronectin in invertebrates were used as predecessors of antibodies, and they acted as opsonins. The acute phase reaction can be interpreted as what remains of a very ancient mechanism of defense and it is linked to elimination of microbes, as well as the aim to return to tissue homeostasis. On the right are the markers that you may read on your own, associated with Covid-19. Most of the acute phase proteins mentioned in this lecture are shown to increase during the infection, and used as markers of severity of the disease. Lactate Dehydrogenase (LDH) Lactate Dehydrogenase is another marker that you will use in case of suspected inflammation or tissue damage, reflecting cell death. LDH is part of the metabolism of glucose and converts pyruvate to lactate, responsible for the anaerobic pathway of glycolysis, so when oxygen is not present in the environment. LDH is made of 2 possible subunits that may combine forming tetramers, at the end having 5 isoforms. Each of them is preferentially used from one tissue or the other. Depending on the total amount of LDH, you can measure the amount of tissue damage in a patient, distinguishing the specific organ undergoing damage in case you measure the specific isozyme. It is a marker of tissue turnover that increases when there is tissue damage. You may have this in different conditions, like muscle injury, heart attack and infection. The normal range of LDH is between 140 to 280 U/L. However, levels are impacted by strenuous exercise, several drugs, age (infants and young children usually have much higher levels). These are the 5 isoenzymes of LDH. They have a different mobility in gel, so based on this you can define which one is highly produced. When you ask for the test, you will have the total amount and single isoenzyme amounts. 19