General Pathology PDF

Vezzoli Victoria, Harvey 2023-24 GENERAL PATHOLOGY Pathology is the study of disease (or suffering). ▪ The Edwin Smith Papyrus is an ancient Egyptian medical text dating back to circa 1600 BC, which may have been a m...

Vezzoli Victoria, Harvey 2023-24 GENERAL PATHOLOGY Pathology is the study of disease (or suffering). ▪ The Edwin Smith Papyrus is an ancient Egyptian medical text dating back to circa 1600 BC, which may have been a manual of military surgery, describes 48 cases of injuries, fractures, wounds, dislocations and tumors; it’s a scroll 4.68 meters or 15.3 feet in length. The front side has 377 lines in 17 columns, while the back has 92 lines in five columns. Based on a recent “medically based translation” of the Smith papyrus, its enclosed treasures in diagnostic, prognostic and therapeutic reasoning are revisited. Although patient demographics, diagnostic techniques and therapeutic options considerably changed over time, the documented rationale on spinal injuries can still be regarded as the state-of-the-art reasoning for modern clinical practice. ▪ The Ebers Papyrus was written in Ancient Egypt in circa 1550 BCE, during the late Second Intermediate Period or early New Kingdom. It is believed to have been copied from earlier Egyptian texts. The Ebers Papyrus is a 110-page scroll, which is about 20 meters long. The scroll contains some 700 magical formulas and folk remedies. It contains many “magic spells” meant to turn away disease-causing demons and there is also evidence of a long tradition of knowledge based on experience and observation. The papyrus contains a section that focusses on the heart, defining it as the center of the blood supply, with vessels attached for every part of the body. Overall, the ancient Egyptians' understanding of disease was multifaceted, combining practical medical knowledge with spiritual beliefs. They did not have a concept of disease causation by microorganisms. Their observations of symptoms and treatments contributed to the early foundations of medical science. Socrates, Hippocrates, Plato, and Aristotle are four of the most prominent figures in ancient Greek philosophy and medicine, each making significant contributions to their fields and to Western thought. Their relationships are a mix of direct mentorship, influence, and parallel development within the intellectual traditions of ancient Greece. The ancient Greeks’ apprehension of health and illness was based on the theory of the four ‘fluids’ (blood, phlegm, yellow bile and black bile) that is in its turn based on the theory of the four elements: air, water, fire, earth, and their four corresponding qualities: heat, dry, humid and cold. Once Asclepius was recognized as the god of medicine, physicians who considered themselves followers of Asclepius had a clear obligation to treat the rich and the poor alike. The donations of the rich were used to subsidize care for the poor, in a way that the contemporary welfare state redistributes income through taxation and social insurance. Hippocrates became the father of medicine by defining important diseases and definitions without considering superstition and gods as their cause, thus separating medicine and religion. Galen became one of the main historical figures of pathology, pharmacology and anatomy, and wrote extensive medical theories. However, knowledge on human anatomy was missing; an unexpected contribution came from art. The Alexandrian school pioneered anatomical investigation for the purpose of understanding causes of death, often in animals. The first (recorded) autopsy done for the purpose of understanding cause of death in a human was performed in 1302 in Bologna. However, it was only at the end of the fifteenth century, after the Church and governments granted the authorization for the free exercise of anatomical dissection, that this became more common. Morgagni (1682 –1771) was an Italian anatomist, generally regarded as the father of modern anatomical pathology, who taught thousands of medical students from many countries during his 56 years as Professor of Anatomy at the University of Padua. Published in 1761, it describes the findings of over 600 partial and complete autopsies, organized anatomically and methodically correlated with the symptoms exhibited by the patients prior to their demise. Italian Marcello Malpighi (1628 – 1694) made outstanding contributions to the anatomical basis of respiration in amphibia, mammals, and insects and also greatly contributed to the fields of embryology and botany. Malpighi, while studying the structure of the lung, noticed its membranous alveoli and the hair-like connections between veins and arteries, which he named capillaries; his discovery established how the oxygen we breathe enters the blood stream and serves the body. Antonie van Leeuwenhoek (1632-1723), “The Father of Microbiology", often considered to be the first acknowledged microscopist and microbiologist. Van Leeuwenhoek is best known for his pioneering work in the field of microscopy and for his contributions towards the establishment of microbiology as a scientific discipline. The advent of microscopic anatomy marked a pivotal moment in the progress of pathology and medicine, transforming our understanding of diseases and their mechanisms. Before the use of microscopes, disease concepts were largely based on macroscopic observations and theoretical speculations. The ability to observe tissues, cells, and pathogens at a microscopic level introduced a new era in medical science, with profound implications for pathology. Robert Hooke studied and discovered the cell. Virchow (1821-1902) was a German physician, anthropologist, pathologist, prehistorian, biologist, writer, editor, and politician. He is known as "the father of modern pathology" and as the founder of social medicine, and to his colleagues, the "Pope of medicine". Virchow recognized that cells were the smallest viable units of the body and he formulated a new and lasting set of ideas about disease – cellular pathology. The cell is at the center of pathology, and it’s affected by many different factors: ▪ Nutritional imbalance: nutritional deficiency and nutritional excess ▪ Biological/infectious agents: viral or bacterial microorganisms ▪ Chemical agents: toxins and poisons, smoking, air pollution, CO or N2 ▪ Physical agents: mechanical trauma, high or low temperatures, radiation, change in pressure ▪ Hypoxia: lack of oxygen ▪ Genetic alterations: mutations, chromosomal abnormalities ▪ Immunologic reactions: autoimmune disease and allergens 1. CELLULAR HOMEOSTASIS (Maggi, Paladini) Cellular homeostasis is the maintenance of stable internal conditions within a cell, despite changes in the external environment. it depends on some factors: temperature, pH levels, nutrient levels, and waste removal. The goal is to ensure that the cell functions optimally. Each cell reacts to stimuli differently, and a cell injury can be reversible or irreversible. It’s important to note that a cell cannot adapt to a stimulus during its entire life. In the case of adaptation, the cell undergoes hypertrophy (reversible); in the case of inability to recover, cell undergoes necrosis (irreversible) The point at which the cell loses its homeostatic capability is defined as the point of cell necrosis; the concept of cell death evolved in time, and started with the definition of oncosis (“sudden death”) and then necrosis from Majno (1960); the concept of regulated apoptosis was raised by Kerr (1972). Other terms were introduced later: pyroptosis (Hersch, 1999), programmed necrosis (Chan, 2003), necroptosis (Degterev, 2005), ferroptosis (Stockwell, 2012), alcaloptosis (Tang, 2018). Factors that influence cell injury are: type of injury, duration of the stimulus, severity of the injury (how many cells are affected), cell type (e.g., neuron), cell status (some injurious stimuli like radiation are more affective on cells that are rapidly dividing), differentiation, adaptability of the cell (e.g., neuron is very specialized and can’t adapt well). The causes of cell injury, known as stressors, are: physical agents (trauma, radiation, heat), chemicals, infectious agents, immunological reactions, genetic alterations (e.g., Down’s syndrome), nutritional deficiencies (balanced diet gives the right amount of nutrients; if there is too much lipid, the cell will work overtime, thus leading to damage) and hypoxia (loss of oxygen supply to the cell). ▪ Morphological alterations of a cell are swelling and blebbing (formation of extroflexions), as well as mitochondrial and ER swelling. ▪ Biochemical alterations, which lead to loss of cellular function, are: - ATP depletion, the most dangerous for cell life. The biggest energy production is given by oxidative respiration (chain + chemiosmosis), which depends on pyruvate, obtained via glycolysis. Normal cells only use aerobic metabolism, which requires oxygen, as it is the last acceptor of electrons in the chain (in the mitochondrion), thus giving the higher amount of ATP. The mitochondrion is referred to as the "powerhouse of the cell" because it is the site of cellular respiration, where energy in the form of adenosine triphosphate (ATP) is produced through processes such as the citric acid cycle and oxidative phosphorylation; hence, mitochondrial damage leads to catastrophic effects, as it regulates energy and Ca2+ regulation. In the case of an energy shortage, anaerobic production is employed by pyruvate > L-Lactate, which drops the pH into a more acidic state. This leads to ATP depletion, and a consequence of lack of ATP is disruption of active transport function in the Na+/K+ ATPase; if in physiologic conditions, Na+ is higher outside and enters the cell (with water, since it’s an ion). The entrance of water leads to swelling, not only in the cell, but also in organelles such as the mitochondrion, which damages it. Another catastrophic consequence is loss of Ca2+ homeostasis (important for signal transduction). There are transporters in the membrane that maintain the concentration of calcium low in the cell, but that also maintain its concentration high in the organelles. There are two main transporters: Ca2+ ATPase (PMCA) and Na+/Ca2+ exchanger (NCX), both on the plasma membrane and both responsible for Ca2+ extrusion. If ATP is low, the transport is not regulated, the cell fills with water, leading to damage. - ROS accumulation; ROS are “friendly enemies” as they are always found within a redox window. Radicals are produced, especially oxygen radicals, by the cell’s productive functions, which are extremely reactive and are keen to donate electrons to neighboring molecules. These ROS are produced in the mitochondria and can be caused by injurious processes, such as inflammation; they can also be produced by radiation or chemicals. In order to inactivate these reactive molecules, a cell can remove them with NADPH or with specialized molecules that can inactivate them: specific enzymes such as superoxide mutase, which exists solely for this role, or molecules like glutathione, important for the redox regulation, or vitamins, which are scavengers of radicals in the cell. The most dangerous radical is superoxide, as it is highly unstable and possesses synaptic plasticity. Radicals can also be formed via physiological processes, such as the presence of iron, in which a free radical is formed; it’s a self-feeding reaction, so it continues until the substrates are removed. There can also be hydroxyl radical formation from peroxynitrite anion (RNS). Lipid peroxidation occurs: unsaturated lipids are very early oxidized and they obtain a radical which can be oxidized inside the membrane; when altering the structure of the lipid, the capability of the membrane to be flexible is also altered. In normal condition, termination occurs without harm (lipid peroxide is not harmful in itself), however, it doesn’t work in emergency. - Edema - Calcium release - Mitochondrial damage and RE/RER damage - Protein synthesis failure, which has no immediate effect. - Macromolecules shortage - Catabolites accumulation - Membrane damage, which can affect membrane permeability or membrane composition. - DNA and protein damage: side chains of aminoacids define the functions of the protein; hence, in a single base damage or a single-strand break, the protein will be modified and lose its function. Enzymes, which are protein, can also be altered and not function. 1.1 - NECROSIS Cell death and necrosis are two different things: cell death is a process that leads to the point of no return, which, for liver cells submitted to total ischemia, lies at 150 minutes, at which time scarcely any changes can be seen in histological sections. Necrosis is full-blown only after 12- 24 h. Hence, cells die long before any necrotic changes can be seen by light microscopy. To say cell death by necrosis implies that the cell dies when it becomes necrotic, which is patently untrue. Furthermore, necrosis has been used for approximately 2000 years to mean drastic tissue changes visible to the naked eye and therefore occurring well after cell death; it is important, both conceptually and didactically, to preserve this usage. Necrosis is signaled by irreversible changes in the nucleus (karyolysis, pyknosis, and karyorrhexis) and in the cytoplasm (condensation and intense eosinophilia, loss of structure, and fragmentation). Regarding histological aspects, after inducing necrosis (which is a time-dependent event), there’s a huge difference in gross anatomy of the liver after two days of necrosis: nuclei completely disappear after 48 hours and organelles cannot be recognized. N is necrosis, K is karyorrhexis, which is the complete loss a nucleus. Tissue necrosis can be: ▪ Coagulative, or “cooked”, such as the liver, because the necrotic tissue is maintained within the organ; ▪ Caseous, such as in the lung, due to its appearance, which is similar to cheese; ▪ Colliquative, such as in the brain, where it is more frequent although the reason is not clear. There’s a loss of tissue, because the necrotic tissue liquefies due to protein degradation: the unfolding proteins aggregate, thus losing them; it occurs via proteases which are in lysosomes. When the membrane is lost, the proteases exit and the cytosolic environment, having a lower and more acidic pH due to anaerobic glycolysis and availability of Ca2+, becomes the perfect activating environment for them. If the aggregation process is not fast enough, the liquification will prevail. ▪ Steatonecrosis, typical of the liver and pancreas; the state of it is indication of the cause of the necrosis, in particular after pancreatitis, because lipid cells will attack neighboring cells. ▪ Gangrene, which can be dry or wet; it’s similar to colliquative necrosis (wet) or coagulative necrosis (dry), but with the intervention of an infection. Inflammation is an important signal of emergency in the cell and it always accompanies necrosis. DAMPs (danger-associated molecular patterns) lead to pain, fever, erythrocyte sedimentation (SED) rate (which increases in inflammation), neutrophilic leukocytosis and serum enzymes. this is relevant for diagnosis of necrosis, as it is associated with marked rise in total bilirubin, transaminases (lower in physiological conditions, as they are set in hepatocytes; if hepatocytes are dying, they release their content in the serum), alkaline phosphatase, LDH along with PT-INR in acute hepatic injury cases. During suspicion of a heart attack, blood test must be done to mark whether myocytic substances are found in the blood, due to necrosis of cardiomyocytes; it’s also used to measure the quantity of enzymes to detect the spectrum of information regarding the diagnosis. In myocardial infarction, creatin kinase is the substance used, as it is specific for myocardium, or troponin. Is pancreatitis, three enzymes derived from pancreatic acinar cells - amylase, lipase, and the proenzyme trypsinogen - have been tested as biochemical markers of acute pancreatitis; serum amylase is the most commonly used of these in clinical practice. Calcification after necrosis can be mostly found, due to interactions of calcium in lipids, in muscles: ▪ Dystrophic calcifications: o Typical of necrotic tissues o Serum Calcium at normal levels ▪ Metastatic calcifications: o Present in living tissue o Hypercalcemia 1.2 - APOPTOSIS Apoptosis is defined as regulated cell death. Necrosis is a consequence of cell damage, while apoptosis is a regulative function of the cell, thus being organized and based on a clean removal of the cell. Necrosis is also not a one cell event, as it involves many cells, while apoptosis is. Apoptosis is particularly important in embryology (e.g., it removes extra skin between fingers). Physiological functions are: ▪ Embryogenesis, metamorphosis ▪ Hormonal stimuli ▪ Regulation of the immune system (homeostasis, regenerating tissues, autoimmunity) ▪ Elimination of cells that have exhausted their function Nematodes were used to describe apoptosis, as it their embryos had a very specific number and lineage of cell. In the last part of oncosis, the cell explodes, releasing all the content outside; in apoptosis, the content of the cell is never external, because the content is always maintained in vesicles (“clean death”). It is fact a single-cell event and it is hard to spot in histological slides. Caspases are cysteine-dependent, aspartate directed protease, with many isoforms (initiation caspases and effector caspases), thus having many interactions between cells. Active caspases are only found in apoptotic cells. The phases of apoptosis: 1. Cell volume reduction 2. Chromatin condensation 3. Extrusions 4. Apoptotic bodies 5. Phagocytosis There are two apoptotic pathways: ▪ INTRINSIC 1. Initiation: Various intracellular stresses, such as DNA damage, oxidative stress, or lack of growth factors, trigger the intrinsic pathway. These stresses activate pro- apoptotic proteins or inhibit anti-apoptotic proteins. 2. Mitochondrial Outer Membrane Permeabilization (MOMP): increased permeability of the mitochondrial outer membrane. This is regulated by the Bcl-2 family1 of proteins, which includes both pro-apoptotic (e.g., BAX, BAK) and anti-apoptotic (e.g., Bcl-2, Bcl-XL) members. In fact, this family is a mitochondrial permeability regulator, and transcription regulated by growth factors or stimuli. When pro-apoptotic proteins dominate, they form pores or channels in the mitochondrial outer membrane, leading to the release of pro-apoptotic factors from the mitochondrial intermembrane space into the cytoplasm. The regulation of apoptosis is mediated by Bcl-2: when anti-apoptosis proteins prevail, there will be no apoptosis; when pro-apoptosis proteins prevail, apoptosis will occur. They are specifically involved in the regulation of the intrinsic pathway. 3. Release of Cytochrome c: Cytochrome c is a key protein released from the mitochondria following MOMP. In the cytoplasm, cytochrome c binds to apoptotic protease activating factor 1 (Apaf-1) and ATP, forming a complex called the apoptosome. 4. Activation of Caspases: The apoptosome facilitates the activation of initiator caspases, particularly caspase-9. Activated caspase-9 then cleaves and activates executioner caspases, such as caspase-3, -6, and -7. 5. Execution of Apoptosis: Activated executioner caspases initiate a cascade of proteolytic events within the cell, leading to the dismantling of cellular structures, DNA fragmentation, and ultimately cell death. This process is highly regulated and ensures that the cell is dismantled in an orderly and controlled manner. Hence, when there’s a pro-apoptotic signal, BAX/BAK decrease (being inhibited by Bcl-2 and Bcl-xL), pores are formed in the outer mitochondrial membrane and Cytochrome C will be extruded from the mitochondria into the cytosol, where it can associate with Apaf-1, an apoptotic molecule that creates with cytochrome the apoptosome (7 repeats of C and Apaf-1). Pro-apoptotic molecules must oligomerize at the pores, where cytochrome C will be released. ▪ EXTRINSIC 1. Initiation: The extrinsic pathway is initiated by external signals, such as binding of specific ligands to death receptors on the cell surface. Examples of death receptors include Fas (CD95), tumor necrosis factor receptor 1 (TNFR1), and TNF-related apoptosis-inducing ligand receptor (TRAIL-R). 2. Ligand-Receptor Binding: Binding of the ligand (e.g., Fas ligand, TNF-α, TRAIL) to its respective death receptor induces receptor trimerization and conformational changes in the receptor complex. 3. Formation of Death-Inducing Signaling Complex (DISC): The conformational changes in the death receptor complex allow it to recruit adaptor proteins, such as Fas- associated death domain (FADD) and TNF receptor-associated death domain (TRADD), which in turn recruit pro-caspase-8 or pro-caspase-10 to form the DISC. 4. Activation of Caspases: Within the DISC, pro-caspase-8 or pro-caspase-10 molecules become activated through self-cleavage. Activated caspase-8 or caspase-10 then initiates the downstream signaling cascade by directly activating executioner caspases, such as caspase-3, -6, and -7, or by cleaving the BH3-interacting domain death agonist (BID) protein. 5. Execution of Apoptosis: The activated executioner caspases, as well as the cleaved BID (tBid), initiate a cascade of proteolytic events within the cell, leading to the dismantling of cellular structures, DNA fragmentation, and ultimately cell death. 1. BAK and/or BAX oligomers = membrane pores BH3 only = stress sensors, BAK/BAX oligomerization activators and BCL2 inhibitors IAPs: inhibitor of apoptosis.  Extrinsic pathway is also associated to Fas-mediated apoptosis: 1. Fas-Mediated Apoptosis: Cell death mediated by Fas is typical of cytotoxic T cells. Fas ligand binds to Fas receptors, leading to receptor oligomerization and the formation of the Fas-associated death domain (FADD), which has affinity for pro-caspase 8; hence, more are activated. FADD recruits pro- caspase 8, which becomes activated and initiates the extrinsic pathway by activating caspase-3, the main effector caspase responsible for apoptosis. 2. TNF-alpha-Mediated Apoptosis: TNF-alpha receptor activation leads to the assembly of the receptor-interacting protein (RIP) complex. If the RIP complex is assembled at the receptor level, caspase-8 will be activated, leading to apoptosis. 3. DNA Degradation. The marker of apoptosis is mediated by proteins called CAD proteins (caspase- activated DNase), which cut DNA at certain levels in their 3D structure. CAD is inhibited by ICAD (inhibitor domain), but caspases can cleave ICAD, allowing CAD to degrade DNA. 4. Removal of Apoptotic Bodies: Apoptotic cells undergo changes such as exposure of phosphatidylserine on their surface, release of soluble factors, and expression of receptors that facilitate their recognition and engulfment by phagocytic cells like macrophages. Complement proteins such as C1-b can also aid in the clearance of apoptotic bodies. Phagocytosis involves: phosphatidylserine, soluble factors, receptors on macrophages and complement (C1-b). Apoptosis is involved in pathological processes, as it is triggered by: ▪ DNA damage ▪ Protein folding dysfunction ▪ Viral infections (HIV, CD4+ lymphocytes, and CTL) ▪ Transplant rejection (CTL lymphocytes) ▪ Pathological atrophy of parenchyma ▪ Cytotoxic treatments (for cancer or autoimmune disease, by selectively targeting memory cells) ▪ Immune/inflammatory response ▪ Mediated by TNFa or FAS and FASL Apoptosis is also a consequence of nutrient shortage; pro-apoptotic signals are activated because anti-apoptotic have no nutrients to be produced from. Loss of apoptosis2 is associated with: t(8;14): Burkitt’s lymphoma, t(14;18): Follicular lymphoma, t(11;14): Mantle cell lymphoma, t(11,18): Marginal zone lymphoma (MALT lymphoma), t(9;22): CML (Philadelphia chromosome), Bad prognosis ALL, t(8;21): AML M2, t(15;17): AML M3 (Promyelocytic AML), t(X;18): Synovial cell sarcoma, t(11;22): Ewing’s sarcoma, t(12;21): Pre-B ALL (good prognosis ALL), t(4;11) and t(1;19): Bad prognosis ALL. In humans, BCL-2 is overexpressed as a result of a t(14;18) chromosomal translocation in ~90% of follicular center B cell lymphomas; MCL1 or BCLX is amplified in diverse tumors; both BIM alleles are deleted in 17% of mantle cell B lymphomas; and BIM or PUMA expression is decreased in diverse malignancies owing to promoter hypermethylation or other causes. For example, the overexpression of Bcl-2 results in many tumors cells' resistance against therapeutic drugs and further experiments confirmed this phenomenon has no direct relation with the expression of multiple drug resistance gene following drugs accumulation. However, leukemia cells transduced with antisense oligonucleotide of Bcl-2 will regain the sensitivity towards chemotherapeutic drugs. Estrogen can up-regulate the expression of Bcl-2 in mammary carcinoma cells, so the use of Tamoxifen, which can act as an antagonist against estrogen, helps the cells reacquire the sensitivity to amycin by down- regulating the expression of Bcl-2. Other terms have been introduced over the past years to accompany and better specify apoptosis: ▪ Pyroptosis is closer to apoptosis because it’s clean, but it causes inflammation, unlike apoptosis, because of release of cytokines (IL-1 beta, which leads to fever). It is typically triggered by bacterial or viral infections, and it involves: inflammasome activation, caspase involvement, cytokine release, IL-1 activation by caspase 1. ▪ Necroptosis: cell resembling necrosis but triggered by specific signals, involved in inflammation and tissue damage; these initiator signals are: kinases (RIPK1 and , activating MLKL and membranes. Physiological necroptosis is responsible for cartilage formation, while pathological necroptosis is related acute pancreatitis, steatohepatitis and neurodegenerative diseases; it’s also mediated by Fas bound to RNA or viral DNA sensors. ▪ Ferroptosis: cell death due to iron overload and lipid damage, often seen in diseases like cancer and neurodegeneration. It’s regulated like apoptosis, but it damages the membrane, not mediated by specific proteins. Its initiator signals are: regulated signaling, mediated by oxidative damage (lack of scavenger systems or ROS neutralization) and damage to the membrane. it may have a physiological role which has not yet been discovered. Autophagy is a not physiological survival mechanism involved with degradation of damaged cells; it is in fact a cellular recycling process where damaged or unnecessary components are broken down and recycled to maintain cellular health and homeostasis, via lysosomal fusion. Alteration of the ability to do autophagy of cells correlates with neurodegenerative diseases and tumors. Reperfusion injury occurs when blood supply returns to tissue after a period of ischemia (lack of blood flow), after which tissues become deprived of oxygen and nutrients, leading to cellular damage and metabolic disturbances. When blood flow is restored (reperfusion), it can paradoxically exacerbate tissue injury due to several factors: 1. Oxidative stress: sudden reintroduction of oxygen during reperfusion leads to the production of reactive oxygen species (ROS), causing oxidative damage. 2. Inflammatory response: Reperfusion triggers an inflammatory cascade, involving the release of inflammatory mediators and recruitment of immune cells, which can exacerbate tissue damage. The neutrophil recruitment leads to: accumulation of ROS, production of chemokines and cytokines, complement activation, cell adhesion (P- selectins, CD18/11 and ICAM-1). 3. Calcium overload: Ischemia disrupts calcium homeostasis in cells. Upon reperfusion, there is an influx of calcium ions, leading to activation of calcium-dependent enzymes that contribute to cell injury. 4. Formation of free radicals: Reactive oxygen species (ROS) generated during reperfusion can react with other molecules, forming free radicals that further damage cellular structures. 5. Iron overload: While iron overload itself may not be a direct cause of reperfusion injury, it can exacerbate tissue damage during reperfusion through mechanisms such as promoting oxidative stress and lipid peroxidation. 2. "ALL" stands for Acute Lymphoblastic Leukemia, a type of cancer affecting the blood and bone marrow, characterized by the overproduction of immature lymphocytes (white blood cells). The term "bad prognosis" in ALL refers to a subtype of the disease associated with a less favorable outcome to treatment compared to other subtypes.  2. INFLAMMATION Any change in the environment can, in principle, harm our cells and tissues, in a process defined as inflammation. The causes of damage can be endogenous or exogenous, such as: infections and microbial toxins, tissue necrosis (ischemia, trauma, physical injury, chemical agents), foreign bodies (splinters, dirt, sutures, which cause traumatic tissue injury and carry microbes), endogenous substances (urate crystals, cholesterol crystals, lipids), immune reactions. DAMPs (Damage Associated Molecular Patterns) derive from injured cells that cast damage signals in the surrounding environment; DAMP is something that should not be there (e.g., leaked outside its natural compartment); PRRs (Pattern Recognition Receptors) are detectors (e.g., Toll-like receptors). Different types of cell death release different amounts of DAMPs in the extracellular space; DAMPs released after necroptosis and pyroptosis are in higher amount than those released after apoptosis. Typical DAMPs can be: poorly CpG methylated genomic DNA, mitochondrial DNA, genomic DNA + high mobility proteins, genomic DNA + endogenous antibiotics, RNA, ATP, Allarmins (e.g., IL-33), Heat shock proteins (HSP), Cytoplasmic calgranulins (s100), Monosodic Urate (MSU), Hyaluronic Acid Fragments. The counterpart of DAMPs are PAMPs (Pathogen Associated Molecular Patterns), which can be released by microorganisms (viruses, bacteria, fungi) and are specific molecules but not specific for one strain or another, as they are just molecules that shouldn’t be in the tissues, and PRPs. Examples of PAMPs are lipopolysaccharide LPS, N- acetylglucosamine (NAG), N-acetylmuramic acid (NAM, lipoteichoic acid, N-formil methionil peptides, Mannane, Glycolipids, DNA (NON methylated CpG), Double strand RNA, Single strand DNA, Zymosan, Glucans, Byglicans. PAMPs and DAMPs share the same receptors, the PRR, and sometimes also share the mechanism of response. Sometimes you can0t tell if a molecule is PAMP or DAMP (e.g., mitochondrial DNA-10.3389/fimmu.2018.00832). The combined action of PAMPs and DAMPs provides a strong reaction to set off inflammation. Sometimes, PAMPs alone are not enough, which is why the microbiome doesn’t cause response. Higher PAMPs and DAMPs can be induced by the necrotic tissue. The threats posed by a local injury such as a wound (tissue is destroyed; door is open to bacteria) are: ▪ Vessels are severed > bleeding > local mechanisms of homeostasis ▪ Loss of tissue > regeneration and local scavenger cells clear up the debris (given that skin is a labile tissue) ▪ Infection > local defenses are not adequate; macrophages are not enough in tissue to locally control the response, since it’s too expensive to maintain fully alert the defenses everywhere in the body. Hence, it’s preferrable to have some cells in the blood to check the body, so that cells can be recruited at the local level. Inflammation (from Latin “inflammare”, to set afire) is a response to injury of vascularized living tissues (because blood delivers defense material to the tissue). Its purpose is to deliver defensive materials (blood cells and fluid) to a site of injury; it’s not a state, but a process. PRRs are on and in many cell types; the tissue macrophages produce different mediators, which can do different things: a type 1 macrophage promotes inflammation (by recognizing damage), while type 2 macrophage switches off inflammation (because they can remove damage without the help of other components) M2 macrophages, however, can also promote tumor growth. M2 MACROPHAGES are NOT ENOUGH in the tissue to locally control the response to it. If there’s a damaged cell, with DAMPs and PAMPs acting on receptor: ▪ If in a tissue there’s a lot of M2 macrophages or a lot of DAMP and PAMP, there can be a silent response, because the local macrophages (M2) wipe out all the PAMPs and DAMPs, and no signs or symptoms occur. ▪ If PAMP/DAMP overwhelm the local M2 clearing capacity, M1 undergo activation, leading to signs and symptoms, which causes inflammation. The local silent response is privileged over inflammation. The removal of DAMPs and their source (dead cells, microbes) by M2 macrophages prevents the inflammation set off, by a bunch of mediators (such as IL-10, IL-1RA, IL-37, adenosine, resolvins). Switching off inflammation does not mean that there is not a response to the danger, but simply that you can locally control the response to it. Most of the times local response is enough to deal with local damage. In case this is not enough, inflammation will start, leading to the release of mediators that can change irreversibly our tissues. There’s always a balance between M1 and M2 macrophages, as it is mandatory to have both of them in the site of damage response. Inflammation is both a good alley for the body to detect problems, but it can also be bad, as most diseases have an inflammatory pathogenesis (chronic inflammation > death). When dealing with the damage response (inflammation), on one hand it must be enhanced, on the other, it must be stopped. One can’t avoid doing both activities at the same time, otherwise there will be fatal or very bad consequences. Acute inflammation is the response of a vascularized tissue to damage, and it relies on the local vascular tissue response: arterioles, capillaries, venules and in general it requires a microcirculation. Acute inflammation depends on: ▪ Production of soluble mediators ▪ Production of exudate, a liquid that leaks out from the vessel. ▪ Cell response This response should be the delivery of blood-borne materials because most of the defensive forces are in the blood: ▪ Leukocytes, which can be polymorphonuclear cells, monocytes or lymphocytes. ▪ Plasma components, such as opsonins, proteins that coat foreign materials and make them easier to phagocytize; opsonins include complement (20 proteins that can be assembled locally to build a bacteria-perforating machine) and antibodies (that bind to the surfaces of bacteria and other parasites). Cells and fluid must be extravasated out of the vessels without interrupting the blood flow itself; the defense reaction is triggered by the products of aggression, which are early mediators of inflammation. The turning point of inflammation is the endothelium activation, because after the recognition of damage, endothelium is activated to release exudate and remove the damage itself. M1 macrophages cast a lot of signals to the endothelium, especially the endothelium of venules, thus undergoing activation. In fact, inflammation can’t take place without endothelial activation. Endothelial activation allows fluids and leukocytes (white blood cells) to move out of the vessel and leak out onto the tissue. Sometimes, it happens that there is the creation of an excess of exudate which can constitute the beginning of a disease. If inflammation is not proportional to the damaging agent, or it doesn’t happen in the right place, time and for the appropriate time-lapse, it can destroy the healthy tissue and become a disease itself. Regarding the movement of fluids outside the vessels during inflammation: fluids (acellular exudate) and leukocytes (cellular exudate). Within the first few minutes from damage, acellular exudate (proteins) occurs; then, a cellular exudate is introduced: the first one is enriched in polymorphonuclear cells, the second with macrophages. A clear-cut time limit for duration cannot be identified: the only known aspect is that the process of inflammation must have a point of start and stop after a certain time- lapse, otherwise, if the point of stop cannot be defined, inflammation can become dangerous and not properly work anymore. Inflammation lasts as long as required to eliminate the cause and to repair the damage. With time, it usually changes in character: ▪ Vascular dilation tends to subside ▪ Redness tends to abate ▪ Predominance of mononuclear cells (but there is a second recruitment of monocytes) Mediators can be involved in the local and systemic response; some of them can taper the response to prevent tissue’s damages, leading to the onset of the response. Inflammation is the first response of the immune system; it is hard to differentiate inflammation from innate immunity, but the two are involved with each other (inflammation is a response to innate immunity). Inflammation is a stereotypical response, hence there are similar phases and stages regardless of the stimulus. Acute inflammation is aimed at: ▪ Identifying the area of damage ▪ Ridding of the causes of damage ▪ Restraining the effects of damage ▪ Laying the ground work to restore the normal anatomical and functional framework (restitutio ad integrum) in a relatively short time (minutes, hours, days). The degree of the response is tuned by: ▪ Duration of the injury and extent of the tissue damage ▪ Characteristics of the patient: health status, age (e.g., in children the response differs and is a reduced response), gender (e.g., in men there’s a more intense response), age*gender (e.g., in old men, there’s an exaggerated response). At the beginning, the response is local and non-inflammatory; sometimes, the damage requires a systemic involvement with a local and limited involvement. Systemic effects are normally beneficial, as a rule; however, they may turn out to be deleterious (e.g., fever or sepsis). Fever is normally a good sign during inflammation, but an excess of fever can be dangerous. Inflammation can also pave the way for the onset of specific immune response: the acute inflammation which leads to that specific immunity crucial for recovery. Thus, acute inflammation, in this way, is just the first step of a longer response which requires the onset of specific immunity (e.g., measles recruit Th1). 2.1 - HISTORY OF THE STUDY OF INFLAMMATION The study of inflammation as a disease started in 2700 B.C., when an Egyptian papyrus described inflammation as a disease, in which redness is due to an excess of red blood; hence, the therapy was blood-letting. In 30 B.C.-38 A.D., Cornelium Celsus, a Roman physician, described “rubor et tumor cum calor et dolor”, while in 130-200 A.D. Galen, another Roman physician, described the “function lesa” (then by Sydenham and Virchow). Other important dates that defined inflammation as a useful reaction are: 1794 - Hunter: Inflammation in itself is not to be considered as a disease, but as a salutary operation, consequent either to some violence or some disease. 1859 - Virchow: pus contained pus corpuscles, the emigrated leucocytes. 1824 - Dutrochet: emigration of leukocytes from blood vessels 1846 - Waller: origin of the pus globules from blood corpuscle perforating the capillaries 1867 - Cohnheim: diapedesis 1882 - Metchinkoff (Nobel prize 1908): inflammation brings phagocytes to the injury 1890 - Behring and Kitasato: inflammation bring antibodies to the injury, defined then by Ehrlich (Nobel prize 1908) 1927 - Sir Thomas Lewis: role of chemical mediators, mainly histamine, and nervous transmission (axon reflex) 1930 - Tillett and Francis: C-reactive protein 1996 - Hoffman: Toll-like receptors (Nobel prize 2011 with Bruce Beutler and Ralph Steinman were pioneers in the field of innate immunity) After them, other studies described as a useful reaction related to muscles. 2.2 - CARDINAL SIGNS OF INFLAMMATION The cardinal signs of inflammation are: ▪ Dolor – pain, due to release of inflammatory mediators; however, pain is not a sign, but a symptom. It’s considered a sign only if observed during medical examinations or procedures. A symptom is subjective evidence of disease or physical disturbance observed by the patient (e.g., the patient can have pain but the clinician can’t detect it). There are many types of pain: Nagging pain > (dolore costante) Fleeting pain > (dolore migrante) Prick (acute pain) > (una fitta, un attacco) Stabbing pain > (dolore ripetuto) Sore > (dolore); soreness > (indolenzimento) Twinge (acute pain) > (una fitta, un attacco) Shooting pain > (ripetuto e in movimento) Tender sore > (dolore alla pressione) Stitch (acute pain) > (una fitta, un attacco) Stinging pain > (dolore pungente) Sharp pain > (dolore acuto) Pang (acute pain) > (una fitta, un attacco) Piercing pain > (dolore da crampo) Dull pain > (dolore sordo) Throe (acute pain) > (una fitta, un attacco) Excruciating pain > (dolore insopportabile) Burning pain > (dolore urente) Pins and needles > (formicolio) Agonizing pain > (dolore insopportabile) Radiating pain > (dolore irradiato Itching > (prurito) The mechanisms of pain depend on specialized nerve-fiber endings, which are stimulated by mediators (e.g., bradykinin). Prostaglandins sensitize the nerve endings to the effect of bradykinin; it’s possible, but there’s no good evidence of correlation between tissue swelling and pain. Non-inflammatory edema (transudate) is painless, as it doesn’t stimulate the nervous system. ▪ Calor – heat, due to increased blood flow and consequent heat dissipation. The increased perfusion of the tissue allows higher temperature, but the temperature of the tissue can never be higher than that of blood, hence skin is normally cool and inflamed inner organs cannot become hotter because they are already as hot as they can be. Hence, heat is related to the skin. There is a theory according to which warmer temperatures move leukocytes faster. ▪ Tumor – swelling due to increased vascular permeability causing fluid leakage into surrounding tissue (cells and fluid). Exudate is made of leukocytes and plasma, while pus is exudate rich in death cells (not the cells of the tissue, but neutrophils that, after the phagocytosis, ended their lives). The mechanism of swelling is due overwhelmingly excess fluid; plasma escapes from the blood vessels, which have somehow become leaky; leaks in the microcirculation are present and filtration pressure has increased due to arteriolar dilatation. Reabsorption of fluid into the bloodstream is impaired because plasma proteins have escaped into the extracellular spaces where they tend to neutralize the osmotic pressure of the plasma in the vessels. The vascular leakage occurs because the endothelial layer is interrupted, but the basement membrane remains and holds the vessels together: - Direct injury: leakage starts immediately and continued until the damage vessels are repaired and closed. - Histamine-type mediators: histamine (early), platelet serotonin (shortly thereafter), PAF and leukotrienes (leukocyte membrane). - Cytokines: IL-1, TNF, IFN-gamma after 2h (6-12 h). - Neutrophils: maintain vascular leakage as long as they are present - Vasodilatation by prostaglandins: increase blood flow and, therefore, tend to exaggerate vascular leakage due to any mechanism. - Leaky sprouts of regenerating vessels (granulation tissue) ▪ Rubor – redness from increased blood flow and dilated blood vessels. By taking a ruler and putting on of its corners firmly along the skin of the forearm, a red line appears within seconds (3-15 s), a result of vasodilation; a red flare appears after 15-30 s, spreading all around the line to a distance of several centimeters, which is another vasodilation effect (just other capillaries); the red line becomes a wheal after 1-3 min (transient swelling of the skin, such as is produced by the sting of a nettle), due to vascular leakage. At first, the wheal is read; as it swells further, it tends to pale. The mechanism of redness is described by the triple response of Lewis: 1. Trauma by the ruler breaks up or degranulates the mast cells in the dermis > histamine > local vasodilatation. 2. Histamine cannot diffuse several cm in a matter of seconds > axon reflex. 3. Vascular leakage ▪ Functio lesa – loss of function. It can be subjective evidence, hence it is closer to a symptom; however, if the function loss is observed, it becomes a sign. Rubor is a positive sign, because it means that there is more blood supply for the defenses; dolor is incidental, pointing to an area of trouble; calor is limited to inflammation of the skin; swelling is positive because it’s related to leaks; loss of function is related to chronic inflammation, but it’s positive because it defines a conclusion of the inflammation. 3. ACUTE INFLAMMATION Inflammation is a reaction; an aseptic injury will trigger inflammation and is one of the effector arms of the immune response. Infection is a contamination with microorganisms. For example, COVID-19 is an inflammation caused by an infection by the virus; the main effects of the disease (such as long COVID) actually depend on the inflammation, and not on the action of the virus. An inflammation, in medical terminology, is defined with the suffix “-it is” and it is specific for the inflammation of an organ or tissue (appendicitis, meningitis, pleuritis). Acute is an inflammation that lasts for hours or days, while chronic is an inflammation that takes weeks, months or years; both acute and chronic inflammations can come sharply to a climax, although it is more common in acute situations. An injury can be local or systemic. Factors that affect the inflammation response are: ▪ Intensity and duration of the stimulus ▪ Quality of the stimulus (mechanical, chemical, bacterial, physical, immunological) ▪ Entities regressive phenomena ▪ Type of cells and tissue (different responses) A dysfunctional inflammation is excessive (in most of cases) or not sufficient (less frequently). When the intensity of inflammation is disproportionate, the condition turns out to be pathological. Some people deal with SARS-COVID-19 infection unproperly, setting off an exaggerate inflammatory response which overwhelms the capability of the body to cope with that response, thus, causing a disease. Pneumonia caused by COVID 19 is a serious complication, but the inflammatory damage deriving from it is usually worse than the capability of the virus itself of destroying pneumocytes and lung structures: the problem is not the virus itself, but the excess of cytokines release. A chronic inflammation occurs when the duration of the response is disproportionate. There is a “grey” zone between acute and chronic inflammation, as there is not a clear limit that determines the end of one and the beginning of the other; there are no signs that tell the exact time when acute inflammation becomes chronic. Acute inflammation sooner or later has to stop and if it doesn’t, it progressively becomes chronic inflammation, which brings to disease. With chronic inflammation, one cannot time when the acute phase started, and the two states are not recognizable. Some examples are: - Super infection (e.g. burns, abscesses) - Loss of substantial amounts of tissue (gashes, surgical severing, ischemic damage) - Long-lasting action of the damage - Completely different features from healthy or acute tissues. The scar is a fibrous tissue in place of the previous one which can be made of skin or lung parenchyma, for example. (I and II-degree burns). Causes can include: infection, loss of tissue, ischemia, damage; cancer is fueled by the inflammation Atherosclerosis: an inflammation beneath the endothelial layer of arteries, especially involving the innermost layer (tunica intima) of arterial vessels ACUTE INFLAMMATORY RESPONSE CHRONIC INFLAMMATORY RESPONSE - Early and rapid response (minutes-hours) - Follows acute inflammation or arise de novo - Short duration (minutes, hours, days) - Long duration (weeks, months, years) - Bacteria, fungi, dead cells, edema - Agents difficult to eradicate (bacteria, viruses, fungi, self-antigens, environmental antigens) - Edema: exudation of fluid and plasma proteins) - Proliferation of several cell types - Migration of leukocytes (neutrophils) - Lymphocytes and macrophages - Tissue destruction and fibrosis (deposition of extracellular matrix) - Inflammation and healing Acute inflammation presents densely packed Chronic inflammation presents lymphocytes polymorphonuclear leukocytes (double-headed arrow), plasma cells (arrows) (PMNs) with multilobed nuclei and a few macrophages (arrowheads). (arrows). Acute inflammation resolution leads to the fact that tissues before and after the damage are not recognizable anymore, as if the damage has never taken place; this occurs via elimination of the initial stimulus and apoptosis of inflammatory cells. There are a lot of mediators that lead the inflammatory mechanism to resolution: in the past, mediators of resolution were poorly defined, only during the past 5 years it has become possible to identify them. Resolution is determined by the removal of damaging agents and the clean-up of tissues’ DAMPs and PAMPs. The major actors of this removal are M2 macrophages which possess high phagocytic/efferocytic activity. Phagocytosis is related to the capability of engulfing molecules through changes in the cytoplasm while efferocytosis is related to the removal of ‘what is in excess’, of apoptotic cells, to prevent a damaged site from becoming a site of inflammation. M2 macrophages’ activity doesn’t take place once in a time in our body but every day, every second; M2 macrophages can also produce plenty of anti-inflammatory/proresolutive. Molecules like IL (interleukins)-10, IL-1RA, IL-37, adenosine, resolving are also involved. In summary, M2 work in order to remove DAMPs and PAMPs; M1, on the contrary, overwhelm M2 capability of removal and keep DAMPs and PAMPs levels high; M1 action leads to lack of resolution: it happens when the amount of DAMPs/PAMPs overwhelms the wiping out capability of M2. The phases of inflammatory response to tissue injury are: ▪ RECOGNITION by receptors of resident cells. ▪ VASCULAR PHASE - Changes increasing nearby blood vessel permeability to plasma, causing rapid flooding of injured tissues with fluid, coagulation factors, cytokines, chemokines, platelets and inflammatory cells, particularly neutrophils. Exudate is a fluid that oozes out of blood vessels (mainly capillaries and postcapillary venules) and it is deposited in nearby tissues. This fluid is rich in proteins, as well as cellular debris, and its presence implies the existence of an inflammatory process. Acellular exudate is the first type of exudates to appear on the site of inflammation; cellular exudate is composed of two important groups of cells: ▪ Polymorphic nuclear cells (PMNs) which are also the first ones to appear after acellular exudates ▪ Macrophages which appear after PMNs exudate. There are 2 kinds of macrophages as well, both of which work together during the process of inflammation: - M1: pro-inflammatory macrophages (in red) - M2: anti-inflammatory macrophages (in blue) Transudate3 is the non-inflammatory exudate. Edema is the accumulation of exudate inside a tissue; effusion is the exudate present in large quantities in body cavities: pericardial effusion (pericarditis), peritoneal effusion (peritonitis) and pleural effusion (pleuritis). Most of the proteins that make up the exudate are synthesized by the liver. Inactive precursors in the vessels are activated by: ▪ Mechanisms of proteolysis, by proteases enzymes, outside the vessels ▪ Mechanisms of proteolysis through the contact system (proteins get into contact with dead or activated/damaged endothelium they undergo activation) These proteins can be divided into several classes: a. Acute phase proteins are triggered by inflammatory cytokines (i.e., IL1, IL6, TNFα). They bind to apoptotic and necrotic cell and to microbial structures, where they amplify the inflammatory response and trigger the coagulation cascade, enhancing chemotaxis and phagocytosis. They pack up RBCs, leading to enhanced erythrosedimentation rate (ESR): high ESR (inflammation in the body), low ESR (no inflammation in the body). CRP (C reactive protein); SAA, Serum amyloid A; MBP-Ca2+ dependent Mannose binding protein; other pentraxins. CRP increases hundreds of times, as proteins go to the tissue and, outside the tissue, promoting opsonization, phagocytosis and complement activation. Pentraxin family (as serum amyloid A) is also important, as the concentration of these proteins is mediated by IL6, IL1 and TNFα, i.e. IL6 increases CRP and Fibrinogen concentration; IL1 and TNFα increase SAA concentration. b. Contact system proteins c. Complement system d. Coagulation system Acute inflammation presents specific morphological patterns: ▪ Serous inflammation is marked by the exudation of cell-poor fluid into spaces created by cell injury or into body cavities (effusion) lined by the peritoneum, pleura, or pericardium. The fluid in serous inflammation does not contain microbes or large numbers of leukocytes. ▪ Fibrinous inflammation is a fibrinous exudate, which develops when the vascular leaks are large or there is a local procoagulant stimulus. Characteristic of inflammation in the lining of body cavities, such as the meninges, pericardium and pleura. ▪ Purulent (suppurative) inflammation and abscess: exudate consisting of neutrophils, the liquefied debris of necrotic cells, and edema fluid. ▪ Ulcers is a local defect, or excavation, of the surface of an organ or tissue that is produced by the sloughing of inflamed necrotic tissue 3.1 - RECOGNITION PHASE Recognition is done via receptors of resident cells, which can be macrophages (M1, M2), dendritic cells and mast cells. All cells involved in recognition are cells that belong to the immune system, mainly the innate one. After the recognition, the vascular phase occurs: blood vessel permeability to plasma, rapid flooding of injured tissues and fluid, coagulation factors, cytokines, chemokines, platelets and inflammatory cells, particularly neutrophils. Exudate is a fluid that oozes out of blood vessels (mainly capillaries and post-capillary venules) and it’s deposited in nearby tissues. This fluid is rich in proteins, as well as cellular debris, and its presence implies the existence of an inflammatory process. The proteins of exudate are produced in the liver mainly; a second source is bone marrow. Hepatocytes are reprogrammed by IL1, TNF and IL6, which are cytokines that stimulate the liver; the reprogramming post- recognition occurs via: ▪ Major acute phase proteins: serum amyloid A (SAA), C reactive protein4 (CRP) ▪ Complement system, C2, C3, C4, C5, C9, C1 inhibitor, Protein binding C4. ▪ Coagulation system: fibrinogen, Von Willebrand factor ▪ Protease inhibitors, metal binder proteins The goals of acute phase proteins are: to bind apoptotic and necrotic cells; to bind microbial structures; to amplify the inflammatory response; to trigger coagulation cascade; to enhance chemotaxis and phagocytosis. CRP increases hundreds of times; proteins go to the tissue and d, outside the tissue, promoting opsonization, phagocytosis and classic complement activation (e.g., C3b). it is part of the pentraxin family (as serum amyloid A) and the concentration of these proteins is mediated by IL6, IL1 and TNFα i.e. (IL6 increases CRP and Fibrinogen concentration; IL1 and TNFα increase SAA concentration). 3. Transudative edema has different denominations for different body cavities: hydrocephalus (cerebral ventricles), hydrothorax (pleural cavity), hydropericardium (pericardial cavity), ascites (peritoneal cavity), hydrocele (testicle), anasarca (generalized). 4. Diagnostic biomarker for inflammation. 3.1.1 - COAGULATION SYSTEM The coagulation cascade is a series of amplifying enzymatic reactions that lead to the deposition of an insoluble fibrin clot. Proteins are involved in the conversion of liquid blood into a solid jelly clot (this is expected to happen extra-vascularly otherwise we have disease i.e., thrombosis). Coagulation factors are passed from one partner to the next. Each reaction step involves an enzyme (an activated coagulation factor), a substrate (an inactive proenzyme form of a coagulation factor), and a cofactor (a reaction accelerator). A number of proteases and cofactors are produced by the liver in an inactive form. Factor XII is activated in presence of negative charged surfaces (e.g collagen, basement membrane, proteolytic enzymes, lipopolysaccharides, foreign materials, elastin, bacterial walls, urate crystals or/and trypsin). Once activated (through cleavage) it triggers the activation of: ▪ Plasminogen to plasmin which dissolves the clot → fibrinolysis. Fibrinopeptides are chemotactic factors able to attract the cells closer increasing endothelium permeability and therefore leading to increased inflammation ▪ Complement, by cleavage of some complement components that generate biologically active products ▪ Kinins: circulating proteases that are inactive inside the vessel but they are activated by factor XII and other proteases outside the vessel. ▪ Coagulation system (Thrombin factor or factor II-interplay) The anti-coagulation system leads to activation inside the vessel leads to clotting inside the vessel. Vitamin K-dependent coagulation factors FX, FIX, FVII, FII (thrombin) are coagulation factors and they work with platelets to form a blood clot. Their production is vitamin K dependent and it takes place in the liver. Anticoagulant drugs can be administered in order to block the synthesis of these factors, e.g., dicumarole, a competitive vitamin K inhibitor of reductase epoxide (VKOR). Other substances that can be used are: antithrombin III, plasminogen (protease that destroys the fibrin clot) and endogenous heparin. Kinins are formed in plasma and tissues by the action of serine protease kallikerins on specific plasma glycoproteins kininogens (high molecular weight), stimulating local tissue cells and inflammatory cells to generate additional mediators such as prostanoids, cytokines (TNFα and other interleukins), NO, tachykinin. Bradykinins: contraction and relaxation of smooth muscles, plasma extravasation, cell migration, inflammatory cell activation and inflammatory mediated pain responses, amplification of inflammatory effects by stimulating local tissue cells and inflammatory cells to generate additional mediators like prostanoids, cytokines (TNFα and interleukins), NO and tachykinins. 3.1.2 - COMPLEMENT SYSTEM The complement system is a group of proteins found in plasma and in cell surfaces, needed for defense against microbes. It includes 30 different proteins including plasma enzymes, regulatory proteins and cells able to cause the lysis of other proteins. They are mainly made in the liver and activated as a cascade of activation by proteolytic cleavage. The exudate contains many proteins can activate the complement system. Activation of complement system may be performed in three ways 1) Alternative pathway – C3 recognize bacterial surface structures > activation of C3a, C3b > activation of C4a, C4b > activation of C5 > activation of C5a, C5b. ▪ C3 spontaneously hydrolyzes ▪ Factors B and D act on C3i to generate C3b ▪ C3b recruits Factor B ▪ C3b Factor B is cleaved by Factor D ▪ C3bBb: alternative C3 convertase ▪ Properdin (P) binds C3 convertase stabilizing the complex: C3bBbP ▪ Another C3b molecule yields C3bBbPC3b that is C5 convertase 2) Classical pathway – C1 recognize antibodies expressed on surface antigen > activation of C3a, C3b > activation of C4a, C4b > activation of C5 > activation of C5a, C5b. ▪ C1 circulates in the plasma (C1q, two C1r, two C1s subunits) ▪ C1q subunit binds Fc region of two antibodies ▪ Binding activates C1r subunits and they activate C1s subunits. ▪ C1s cleaves serum C4 in C4a and C4b ▪ C4bC2a: C3 convertase ▪ C3 convertase cleaves C3 in serum ▪ C4bC2aC3b: C5 convertase 3) Lectin pathway – acute phase proteins activated > activation of C3a, C3b > activation of C4a, C4b > activation of C5 > activation of C5a, C5b. ▪ MBL (mannose-binding lectin) binds to carbohydrate moieties in pathogen proteins ▪ MBL-associated serine protease (MASP) associates with MBL and cleaves C4 ▪ C4b binds motifs in proteins on the pathogen surface ▪ C2 binds C4b and is cleaved by MASP complex ▪ C4bC2a: C3 convertase The terminal steps include: ▪ C5 convertase, fixed on a pathogen surface, cleaves serum C5 ▪ C5b remains bound to the convertase ▪ C6 stabilizes the complex ▪ C7 binding exposes hydrophobic regions that facilitate complex penetration into the pathogen membrane ▪ C8 stabilizes the complex in the membrane and the formation of a pore is initiated ▪ Four C9 subunits complete membrane attack complex (MAC) ▪ Pathogen lysis due to osmotic balance (ions and water go through the pathogen) In the complement system, C5, C6, C7, C8 are mainly responsible for bacterial killing; C3a, C4a, C5a are responsible for the increasing in permeability and in chemotaxis; C3b is responsible for phagocytosis and opsonization; C5a and C3a activate leucocytes; C5a and MAC stimulate endothelial cells. The system amplifies the inflammatory response and self-amplify its own response, it stimulates endothelial cells and leukocytes. When the components of the complement are activated, they promote inflammation. Some mediators of the complements are called anaphylatoxins because if injected in animals they are able to induce anaphylactic shock. These proteins can lead to massive leakage of fluid inside the vessels and induce the death of the animal. Complement system can also promote opsonization and, when cells are layered by complement particles, it can be easily recognized and eaten by macrophages. They also stimulate chemotaxis. The intravascular activation of complement proteins: a. Can lead to shock. b. Is involved in organ transplantation side effects. c. Is involved in COVID-19 side effects. d. Might be related to the development of cognitive diseases. 3.1.3 - PRESSURES In physiologic conditions, microcirculation involves the continuous movement of fluid from the intravascular compartment to the extravascular compartment. Microcirculation is given by the arterioles and precapillary sphincters, capillary bed, venules and nodes. Normal vascular microcirculation is governed by four different pressures: ▪ Hydrostatic pressure: from blood flow and plasma volume which forces the fluid out of the vessels ▪ Oncotic pressure: due to plasma proteins which draws fluid into the vessels ▪ Osmotic pressure: reflecting the amount of Na+ and H2O in vascular and tissue spaces ▪ Lymph flow: continuously drained out of the tissues in the lymphatic vessels and nodes The fluid interchange between the vascular and the extravascular compartments reflects a balance of forces that draw fluid into vascular spaces and out into tissues. The venular and capillary endothelium are kept at rest (very low permeability) by M2 macrophages which produce many anti-inflammatory mediators (e.g. IL-10, IL-1RA, Resolvins, ADP). This leads to fluid leakage, which can occur through transudate, which is an increase of hydrostatic pressure (e.g. increase in venous resistance) or reduction of colloid osmotic pressure (e.g. hypoproteinemia), or though exudate, which is an increase of hydrostatic pressure (vasodilation and postcapillary district stasis) and reduction of oncotic pressure: solute spill (after the increase of vascular permeability). 3.2 - VASCULAR PHASE The vascular response to this is represented by: ▪ Changes in arteriolar and precapillary sphincters: HYPEREMIA > RUBOR and CALOR a) Brief vaso-constriction of arterioles, which is aimed at preventing bleeding and is elicited by neurogenic and chemical mediators (endothelin and noradrenaline). It usually resolves within seconds to minutes. b) Long-term vasodilation of precapillary arterioles ▪ Changes in endothelial permeability (mainly in postcapillary veins then in capillaries): EDEMA > TUMOR a) Increased permeability of post-capillary venules allows fluid and plasma components to accrue in the tissue: endothelial cell contraction and then cytoskeletal reshape b) To avoid the incidental occurrence of the phenomenon, endothelial cells are connected to each other by tight junctions and separated from the tissue by a limiting basement membrane. The disruption of this barrier function is a hallmark of acute inflammation. ▪ Venous stasis: slowing of the blood cycle ❖ LEUKOCYTE ADHESION DEFICIENCY (LAD) is a rare hereditary syndrome defined as the “selective deficiency of leukocyte adhesion receptors”, such as β2 subunit of leukocyte integrins, alterations of endothelial selectins (dysfunction of glycosylation process) and defects in platelet aggregation. Patients with LAD are highly susceptible to devastating surface bacterial infections due to the impaired ability of leukocytes to adhere to the blood vessel walls and migrate to sites of infection. This leads to a compromised immune response and increased vulnerability to infections. Patients present inflammation signs (tumor, rubor, calor) and an exudate without neutrophils. In this condition: a. Vascular response precedes the entry of leukocytes b. Leukocyte entry is partially independent to vascular response c. Vascular changes are not sufficient to determine a passive outflow of leukocytes from the circulatory stream d. Leukocyte extravasation is an active process that requires an interaction between leukocyte and vascular endothelium 5. Vascular changes depend on resident cells and resident cell-derived mediators ❖ MAST CELLS are granules (vesicles) filled with ready-to-use preformed mediators. C3a and C5a receptors bind their ligands, leading to the degranulation; TLR activity promotes the expression of pro-inflammatory genes. Degranulation consists of granules fusing with the plasma membrane releasing the contents in the extracellular microenvironment. Granule mediators are: histamine, serotonin, proteases, proteoglycans and cytokines (TNF, IL4) 1. Vasodilatation: release of muscle sphincters of precapillary arterioles. Histamine H2 receptors expressed on smooth peri-arteriolar muscle cells. 2. Increase of permeability by endothelial cell contraction mediated by Histamine H1 receptor. Weakening of intercellular joints and loss of functional integrity. ❖ MACROPHAGE play a crucial role in the innate immune response by recognizing pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs). Upon recognition of PAMPs, macrophages initiate a series of immune responses, including the expression of pro-inflammatory genes. The expression of pro- inflammatory genes by macrophages leads to the production of cytokines such as IL-1 (Interleukin-1), IFN-I (Type I Interferons) within several hours of activation, as well as TNF (Tumor Necrosis Factor), which is responsible for: ▪ Endothelial cell contraction: TNF causes endothelial cells to contract, which can contribute to vascular constriction and reduced blood flow. ▪ Increased vascular permeability, allowing immune cells and proteins to migrate more easily to the site of inflammation. ▪ Increased expression of NF-κB dependent genes: TNF activates the NF-κB signaling pathway, leading to increased expression of adhesion molecules such as ICAM-1, VCAM, and E-selectin on endothelial cells. These molecules facilitate the adhesion and recruitment of leukocytes and platelets to the site of inflammation. ▪ Pro-adhesive and pro-coagulation properties: By promoting the expression of adhesion molecules and activating endothelial cells, TNF enhances the adhesion of leukocytes and platelets to the endothelium, as well as promotes coagulation processes, essential for containing infections and repairing damaged tissues. During the phospholipase A2 cascade, cPLA2 migrates close to lipidic membranes, mainly of intracellular organelles (mediated by pro-inflammatory stimuli). Arachidonic acid (AA) by COX1 and COX2 > prostacyclins (PG12: vasodilatation) and prostaglandins (PGE: increased vascular permeability); Lyso-phosphatidylcholine (LPC) by LPC acetyltransferase > Platelet-Activating Factor (PAF): increase vascular permeability. ❖ NERVOUS FIBERS are relevant due to the pain signals being sent to the brain through nociceptive fibers. The brain will process the information and will send efferent signals; these will cause vasodilation by use of substance P (Takikinin), calcitonin-gene-related peptides (CGRP), Neurokinin A and B. Diabetic patients have an impairment of peripheral sensory fibers and an impairment of the damage response. Increase infections in the skin which lead to gangrene and then amputation (the diabetic foot). ❖ PROSTAGLANDINS include eicosanoids, due to having diverse hormone-like effects, which are enzymatically derived from the fatty acid arachidonic acid; every prostaglandin contains 20 carbons including 5-carbon ring. COX-1 is responsible for baseline prostaglandins synthesis; COX-2 produces prostaglandins through inflammatory stimulation. The structural differences between prostaglandins account for their different biological activities. Opposite effects in different tissues, in some cases: determined by the type of receptor to which the prostaglandin binds. They act as autocrine or paracrine factors with their target cells present in the immediate vicinity of the site of their secretion. Main traits of prostaglandins are: - Differ from endocrine hormones, not produced at a specific site but in many places throughout the human body. - Powerful vasodilators and inhibit the aggregation of blood platelets. - Prostaglandins are produced by almost all nucleated cells. - Prostaglandins are produced following the sequential oxygenation of arachidonic acid by cyclooxygenases COX-1 or COX-2 and terminal prostaglandins synthase. ❖ LEUKOTRIENES are eicosanoid inflammatory mediators produced in leukocytes by the oxidation of arachidonic acid (AA) and the essential fatty acid eicosa-pentaenoic acid (EPA) by the enzyme arachidonate 5-lipoxygenase. Leukotrienes use lipid signaling to convey information to either the cell producing them (autocrine signaling) or neighboring cells (paracrine signaling) in order to regulate immune responses, thus triggering contraction of smooth muscle lining the bronchioles; their overproduction is a major cause of inflammation in asthma and allergic rhinitis. The lipoxygenase pathway is active in leukocytes and other component of the immune system such as mast cells, eosinophils, neutrophils, monocytes and basophils. Leukotrienes act principally on a subfamily of G-protein coupled receptors; they may also act upon peroxisome proliferator-activated receptors. Chemotactic effect on migrating neutrophils (LTB4) and as such help to bring the necessary cells to the tissue. Powerful effect in bronchoconstriction and increased vascular permeability. ❖ NITRIC OXIDE (NO) is produced in M1-macrophages by nitric oxide synthase (NOS). NOS oxidases can be present three main NOS isoforms: constitutively expressed neuronal nitrogen synthase (nNOS), endothelial nitrogen synthase (eNOS), which are endothelium derived relaxing factor, vascular smooth muscle relaxation, and inducible nitrogen synthase (iNOS). Inflammatory cytokines increase the expression of inducible nitrogen synthase, generating intracellular and extracellular NO which has many roles in vascular physiology and pathophysiology. In physiologic concentration is found alone and coupled with O2 as intracellular messenger. NO prevents platelet adherence and aggregation at sites of vascular injury, reduces leukocytes recruitment and scavenges oxygen radicals. ❖ PLATELET-ACTIVATING FACTOR (PAF) is a phospholipid-derived lipidic mediator, whose production is controlled by the activity of PAF acetylchol-hydrolase and secreted by a variety of cells including mast cells, endothelial cells and macrophages. It is a mediator of many leukocyte functions, platelet aggregation and degranulation, inflammation, anaphylaxis, changes to vascular permeability (1000x more powerful than histamine’s effect-short lived), oxidative burst, chemotaxis of leukocytes and increased arachidonic acid metabolism in phagocytes. It is largely produced during inflammatory response by inflammatory cells in response to specific stimuli. PAF initiates an inflammatory response in allergic reactions. The inflammatory response is enhanced by the use of vasodilators, including prostaglandins PGE1 and PGE2 and inhibited by vasoconstriction. It is an important mediator of bronchoconstriction. It causes platelets to aggregate and blood vessels to dilate therefore is important in the process of hemostasis.  Nitric oxide Vasodilation Histamine Vasodilation, Permeability, Bronchoconstriction (contraction of smooth muscle); Itch (pain equivalent) in allergic reaction with leukotrienes Platelets-activating factor Vasodilation, Permeability Prostaglandins Vasodilation, Permeability, Pain. Leukotrienes Vasodilation, Permeability, Bronchoconstriction (asthma and rhinitis). The vascular response to injury or inflammation involves several coordinated processes and mediators that aim to contain the damage, facilitate immune cell recruitment, and promote tissue repair. ▪ Changes in the flow blood and permeability of vessels 1. Vasodilation: induced by chemical mediators (histamine) 2. Increased permeability of the microvasculature, via: - Formation of gaps between postcapillary venule endothelial cells by mediators (histamine, bradykinin, leukotrienes) that act on vascular smooth muscle inducing inter-endothelial gaps. Short lived (15-30 minutes) - Retraction of endothelial cells by TNF and IL-1: cytoskeleton modification (4-6 hours) - Endothelial injury by physical agents, toxins, neutrophils 3. Stasis: vascular congestion due to concentration of red cells, increased blood viscosity ▪ Escape of fluid, proteins and blood cells from the vascular system into the interstitial tissue or body cavities (Edema-peaking 3-4 hours after injury). Exudate has a high protein concentration (specific gravity >1.020 Kg/L); transudate has a low protein concentration (specific gravity Plasma Clot formation, fibrin degradation pathway. Complement, Kinins Complement fragments Liver > Plasma Chemotaxis Kinins Liver > Plasma Vasodilation, pain 3.4 - PRODUCTIVE PHASE The productive phase of inflammation involves the resolution of the inflammatory response and the initiation of tissue repair and remodeling: 1. Migration of macrophages, as they phagocytize remaining pathogens, debris, and apoptotic cells to clear the site of inflammation. Macrophages also produce growth factors and cytokines that stimulate tissue repair and activate fibroblasts to produce extracellular matrix components essential for tissue remodeling. 2. Migration of lymphocytes (particularly T and B cells), which migrate to the site of inflammation in the productive phase to participate in the adaptive immune response. T cells help regulate the immune response and promote tissue repair, B cells differentiate into plasma cells and produce antibodies to neutralize pathogens and facilitate their clearance. 3. Fibrosis refers to the deposition of extracellular matrix proteins, primarily collagen, to repair and replace damaged tissue. Fibroblasts, activated by growth factors released by macrophages and other cells, proliferate and synthesize collagen and other matrix proteins to form scar tissue and restore tissue integrity. While fibrosis is essential for wound healing and tissue repair, excessive or prolonged fibrotic response can lead to tissue fibrosis and impaired organ function. 3.5 - RESOLUTION Resolution is an active process restoring the tissue to its original form (homeostasis); the main actors of resolution are M2 macrophages. There are several mediators and it’s carried out by different cells. If the differentiation is towards M1, the inflammation will be maintained, if not, there will be resolution. The main steps are: ▪ Phagocytic activity (efferocytosis) by M2 macrophages, which have phagocytic/efferocytic activity, aimed at getting rid of DAMPS and PAMPS. Annexin 1, expressed on dead neutrophils and on debris cells, signals to the M2 to “eat” the cell. CD47, the opposite of annexin, prevents efferocytosis and it’s expressed during inflammation to cross the endothelial barrier. M2-mediated efferocytosis allows higher secretion of anti-inflammatory mediators (such as adenosine resolvins or antagonists of IL-1, e.g., IL-10, IL-1RA, IL-37). The involvement of M2 macrophages in inflammation is related to cancer, because some mediators create a microenvironment that encourages the growth of cancer; IL-10 is also a mechanism of resistance to immune checkpoint inhibitors, hence the presence of inflammation can influence the development of cancer. ▪ Endothelium deactivation by M2 - M2 can deactivate the endothelium in order to return to a resting state; in this way, leukocytes can no longer pass the endothelium and move to the tissue. Thus, it’s no longer a problem of permeability, but also in the adhesion of leukocytes in the endothelium: it loses its stickiness baring leukocytes from adhering onto it and squeeze through (M2 modulates the expression of the receptors). The exudate will no longer be in the tissue, both the cellular and acellular. ▪ Stem cell activation - Stem cell can restore the anatomical and functional status of the tissue, leading to the restitutio ad integrum. In order to maintain the functional status, the first phase of stem cell activation presents a limitation of the stromal activation, in order to reduce the amount of fibrotic tissue and prevent scarring. If inflammation lasts for too long, stroma will be overactive and the process will lead to scarring. Stem cells can differentiate into any cell, thus being particularly valuable for cancer tissues. In non-labile tissues, scars are present after damage and inflammation. The full repair doesn’t exist, but there is a maintaining of the architecture and functionality of the structure. Many mediators derive from arachidonic acids, thus being fatty acids-derived, produced via phospholipase-A2 and COX2: there can be prostaglandins (vasodilation, permeability, pain, fever) and leukotrienes. COX1 has a physiological activity and is responsible for the production of prostaglandins and gastric mucous in the glomerular fluid and uterine tone. From arachidonic acids, mediators are made via: ▪ COX1: is responsible for baseline prostaglandins > gastric mucus, glomerular flow, uterine tone ▪ COX2: produces prostaglandins through stimulation > PGE, PGD > vasodilation, permeability and pain ▪ 5-LOX: production of LTA, LTB > vasodilation, permeability and pain Corticosteroids are broad-spectrum anti-inflammatory agents that reduce the transcription of genes encoding many proteins involved in inflammation, including COX-2, phospholipase A2, proinflammatory cytokines (e.g., IL-1 and TNF), and iNOS. The use of corticosteroids can lead to delayed wound healing. NSAIDS are meant for inhibition COX1 and COX2, thus inhibiting pro-inflammatory prostaglandins (pain and fever). As a consequence of COX1 inhibition, there can be gastric ulcerations. Inhibition of these enzymes and the reduction of prostaglandins also defines that lipoxins (anti-inflammatory mediators) synthesis is enhanced. 5-LOX or leukotrienes are pharmacologic agents that inhibit leukotriene production or block leukotrienes receptors (LT receptor antagonist); they are useful in the treatment of asthma. 5-LOX is not inhibited by aspirin and ibuprofen, as well as other NSAIDs. Lipoxins are a pro-resolutive mediators produced by 12-LOX (lipoxygenase); they have an anti-inflammatory activity. Lipoxins can be A or B depending on their action: they hamper inflammation, PMNs chemotaxis, PMNs free radicals, PMNs TNF-α response and pain. For the resolution of inflammation, there are platelets in the injured tissues, which interact with neutrophiles (inflammation), leading to conversion of leukotrienes-4 to lipoxin A and B; hence, platelets can be used as pro-regenerative mediators. Platelets Rich Plasma Products (PRPP) are found in a number of (chronic) inflammatory conditions: non-healing ulcers, chronic tendinopathy, ligamentous and acute muscle injuries, cartilage tendons lesions. M1 macrophages, eosinophils and airway epithelial cells generate 15S-HETE, which can be transformed via neutrophil-associated 5LOX into Epi-lipoxins, which resemble lipoxins in form and action. In presence of little inflammation, proinflammatory lipids are converted into anti-inflammatory ones, leading toward quick resolution. Aspirin irreversibly acetylate the COX enzyme, inactivating it; when aspirin (only) is administered in inflammation, 15R-HETE is generated by COX-2. Activated neutrophils convert 15R-HETE to 15-epi-LXs (aspirin-triggered lipoxins, ATL-EPI). In endothelial cells, 15 epi-LXA4 and 15 epi-LXB4 trigger prostacyclin (PGI2) and nitric oxide (NO) vasodilators. Low doses of aspirin prevent cardiovascular disease relapse and some cancers (colon, prostate). However, because they block COX2, they halt production of pro-resolutive (Epi-Lipoxins) and anti-platelet (PGI2) molecules, leading to high risk of thrombosis (> PGI2 inhibits platelet aggregation and acts as vasodilator). EPA and DHA can compete with arachidonate during synthesis of resolvins, promoting resolution after inflammation. Exogenous administration of EPA and DHA can act as pro-resolution drugs. ▪ Docosahexaenoic acid (DHA) from α-linolenic acid (peanuts, nuts, pumpkin, flax, hemp seeds, black currant, sacha inchi, soy) or breast milk, fish oil, or algae oil ▪ Eicosapentaenoic acid (EPA) from breast milk, oily fish/fish oil cod liver, herring, mackerel, salmon, sardines, algae. D/E series resolvins are pro-resolution mediators which block superoxide production, block PMN migration and diapedesis; they also switch NF-κB off, switch endothelium off, trigger efferocytosis (M2), block TNFα and trigger IL-10. They also prevent platelets adhesion/aggregation. Synthesis of resolvins can be triggered during inflammation by the contact between neutrophils and endothelium: ▪ Through action of 5-LOX synthesis of D1-D6 resolvins and D1 protectin can be performed from 17R-DHA ▪ Trough action of COX2 can generate resolvins (Resolvin E, Epi-resolving D, Epi-Protectin D1) from EPA and DHA ▪ Through action of acetylated COX synthesis of D1-D6 resolvins and D1 protectin can be performed from 18-HPETE (from arachidonate) Systemic activity of DHA/EPA acts on peroxisome proliferator activated receptor (PPAR) gamma in the liver and in macrophages of adipose tissue lowering of cholesterol and glycemia; this is why inflammation is negatively associated with good health. Neuroprotectins protect neural tissue from inflammation (brain protection) and control pain by TRP (transient receptor potential) channels on C fibers. Maresins are pro-resolution macrophage-derived cells, needed to block PMN migration, switch endothelium off, promote efferocytosis (M2) and secretion of IL10. ATP is transformed by ATPases into adenosine, which is an anti-inflammatory molecule. The enzyme is highly concentrated on the membrane of M2 macrophages, endothelial cells and T regulatory cells. This is one of the basic principles of acupuncture: a small damage due to a thin needle sets off a wealth of DAMPS including molecules (i.e., ATP) that turn to be anti-inflammatory (anti-pain, pro-resolutive, pro-regenerative). However, adenosine excess leads to conversion of adenosine by xanthine oxidase into uric acid, which triggers inflammasome. AICAR, a purine intermediate compound that accumulates due to methotrexate action, acts as an AMP mimetic and therefore the AMP status of immune cells may influence their ability to respond to MTX. Peptides IL-10, IL-1RA, IL-37 promote M2 formation, antagonize cytokines, trigger resolvins, promote efferocytosis and trigger/protect stem cells. Lipocortins (lipocortin 1/annexin 1) are peptides whose production by PMNs is induced by cortisol and in general by glucocorticoids to reduce inflammation. They switch off Phospholipase A2, deactivate the endothelium, promote PMNs apoptosis and M2-mediated efferocytosis, trigger IL-10, inhibit the release and effect of TNF-α. But how does the RESOLUTION OF PAIN occur? Anti-inflammatory lipids (neuro-protectins and D/E series resolvins) modulate TRPV1 7 channel activity on nociceptive C fibers. ▪ Endocannabinoids have high affinity for TRPV1 channels and are produced from our body under stressful conditions enhanced by paracetamol). Their mechanisms are: - Reduce mast-cell degranulation and subsequent release of histamine; Reduce PMNs activity; - Perform analgesia at CNS level by acting on specific receptors and open TRPV1 performing peripheral analgesia; - Deplete substance P. Acetylcholine is a neuro-mediator which triggers M2 action. Crosstalk between nerve fibers and macrophages is also mediated by Acetylcholine. M2 macrophages possess A7 acetylcholine receptors, whose activity can be increased by yoga and meditation ▪ Exo-cannabinoids are exogenous molecules with similar structure and function, and they can be found, among others, in hemp and marijuana. One of these compounds is called Anandamide, from the Sanskrit word for paradise. The name indicates its tremendous psychotropic effect. 3.6 - CELLULAR ADAPTATION Cellular adaptation is a state that lies intermediate between the normal, unstressed cell and the injured, overstressed cell. Adaptations are reversible changes in the size, number, phenotype, metabolic activity, or functions of cells in response to changes in their environment. Cellular adaptation: ▪ Permits survival and maintenance of cell function ▪ By altering gene expression, enables a cell to change size or form ▪ Is a normal response to an appropriate stimulus ▪ Abnormal cellular changes may also occur (pathologic adaptation) Cellular adaptation can occur in different ways: ❖ HYPERTROPHY, an increase in cell size with subsequent increase in organ size: no new cells, just larger cells. It’s typical of non-dividing cells (e.g., muscle cells). It can be pathologic, or physiologic, due to increased functional demand or hormonal stimulation (e.g., skeletal muscles after increased workload, uterus during pregnancy due to estrogen, breast in pregnancy and lactation due to human chorionic gonadotropin, progesterone, prolactin, puberty, like androgens, GH in male); weight-lifting with large weight loads leads to hypertrophy of type II fibers and increases muscle mass: these fibers favor anaerobic glycolysis. In addition to Akt mediated mechanisms, increased n. of mitochondria is seen. Demand-driven adaptation of skeletal muscle occurs via endurance training (repeated, prolonged exercise with small loads), which: 1. Raises the adenosine monophosphate-to-adenosine triphosphate (AMP:ATP) ratio, stimulating AMP kinase activity, increases cytosolic calcium concentration 2. Peroxisome activation triggers TFAM (transcription factor-activating mitochondrial transcription), which leads to replication and transcription of mitochondrial DNA. 3. Increase of muscle content of slow myosin H chains, increased numbers of mitochondria and improved endurance without muscle cell hypertrophy. Pathological causes of hypertrophy include: ▪ Excessive growth factor or neuroendocrine stimulation: high level of TSH ▪ Excessive hormonal stimulation: anabolic steroids ▪ Compensatory mechanism: enlargement of the heart in response to pressure overload, usually resulting from either hypertension or valvular disease. Hypertrophy can affect different organs: - Myocardial hypertrophy: pathways involved in muscle hypertrophy are the phosphoinositide 3-kinase (PI3K)/AKT pathway (physiologic hypertrophy) and signaling downstream of G-protein-coupled receptors (induced by many growth factors and vasoactive agents, and thought to be more important in pathologic hypertrophy). Physiologic hypertrophy and cavity enlargement are the heart's normal adaptation to rigorous athletic training. - Smooth muscle hypertrophy: as a consequence of the obstruction of the outflow of the content of a hollow muscular organ (e.g., stomach in pyloric stenosis, intestine in Chron’s disease, bladder in prostatic hyperplasia). ❖ ATROPHY, a decreased size or function of cells or organs. Atrophy of an organ differs from cellular atrophy: reduction in an organ's size may be caused either by reversible cell shrinkage or by irreversible loss of cells (cell death). Physiological causes include loss of endocrine stimulation and decreased workload, e.g., normal development (embryonic structures, thymic atrophy) and uterus following childbirth. Pathological causes include: DISEASE CONDITIONS IN WHICH ATROPHY OCCURS Aging Most organs that do not continuously turn over (e.g., muscles sarcopenia) Chronic disease Cancer; seen in congestive heart failure, chronic obstructive pulmonary disease, cirrhosis of liver and AIDS (untreated); diabetes; autoimmune disease. Ischemia Hypoxia, decreased nutrient availability, renal artery stenosis Malnutrition Generalized atrophy Decreased functional demand Limb immobilization, as in a fracture Interruption of trophic signals Denervation atrophy following nerve injury; menopause effect on the endometrium and other organs Increased pressure Decubitus ulcer, passive congestion of the liver Atrophy mechanisms are: ▪ Decreased protein synthesis ▪ Increased protein degradation due to the activity of lysosomes with hydrolytic enzymes and the ubiquitin-proteasome pathway ▪ Decrease in use of free fatty acids as an energy source Atrophy occurs for example, after limb immobilization in a cast (muscle cell lose mass and strength > activation of myostatin/Akt pathway) or during Alzheimer disease (marked atrophy of the frontal lobe characterized by thinned gyri and widened sulci). In muscle atrophy, the protein kinase Akt is central to atrophy: 1. Muscle disuse increases extracellular myostatin, a protein in the transforming growth factor-β (TGF-β) family. 2. Myostatin binding activates its receptor, which inhibits Akt. 3. A transcription factor, FOXO, which is normally curbed by Akt, is thereby released from that suppression. 4. FOXO activation increases production of ubiquitin ligases (E3), which mediate the degradation of muscle proteins by proteasomes. ❖ HYPERPLASIA, an increase in the number of cells in an organ (hypercellularity) which may then increase organ size. It can have physiological stimulation, such as hormonal stimulation (female breast at puberty and pregnancy), increased functional demand (lymphoid tissues during immune response), compensatory hyperplasia (liver regeneration after partial resection; erythroid precursors in secondary polycythemia). The stimuli and mechanisms vary greatly from one tissue and cell type to another; it’s typical of cycling cells. Pathologic hyperplasia can occur due to: excess hormone (endometrial hyperplasia due to estrogens), benign prostatic hyperplasia, chronic injury (e.g., calluses from ill-fitting shoes; inflammation), psoriasis (epidermis in skin). Hyperplasia is not a neoplastic process, but it may be soil for malignancy; “atypical hyperplasia” in the endometrium carries an increased risk for development of endometrial adenocarcinoma. HYPOPLASIA is an incomplete development of an organ so that it fails to reach adult size. It is unsure whether hypoplasia is a cellular adaptation, as the cellular process opposing to hyperplasia in adult tissues is programmed cell death (apoptosis). Other terminology include: hypoplasia (congenitally underdeveloped organ or tissue), aplasia (undeveloped organ), agenesis (complete absence of an organ and its outline) and involution (reduction of physiological volume of some organs). DYSPLASIA is not a cellular adaptation; it’s an atypical proliferative change due to chronic inflammation and is a typical pre-malignant change (DNA mutations). ❖ METAPLASIA, a reversible change in which one differentiated cell type (epithelial or mesenchymal) is replaced by another differentiated cell type that’s more resistant. It’s an adaptive response to persistent injury or stress (defense mechanism) and represent an altered differentiation pathway of tissue stem cells. In smokers, there can be a metaplasia of the bronchial epithelium, which is normally columnar and becomes a stratified squamous epithelium, because it’s more resistant to the components of tobacco, but less protective against other agents. In endocervix metaplasia (squamous metaplasia), the normal columnar epithelium is replaced by squamous; in Barrett’s esophagus, the columnar epithelium (glandular mucosa) replaces the squamous epithelium (dysplasia and adenocarcinoma may occur). ❖ SENESCENCE is an intrinsic mechanism that count cell divisions: primary mammalian cells have a finite life span in tissue culture (Hayflick limit). Cellular "senescence" may reflect aspects of organismal aging and i