Inflammation Outcomes: Healing, Sepsis

1. Inflammation Outcomes: Healing, Sepsis

3. Summary of acute inflammation • Stimulated by physical injury, infection, foreign body • Resident macrophages and/or damaged endothelium, mast cells—IL-1, TNF, endothelin, histamine • Vascular response: vasodilation, endothelial contraction, exudation of plasma • Neutrophils: marginate (selectin-glycoprotein), adhere (integrin-CAM), extravasate (CD31), migrate (IL-8, chemotactic stimuli) • Phagocytosis: recognition, engulfment, killing • Phagocytosis receptors bind mannose, oxidized lipids, lipopolysaccharides, lipoteichoic acids, opsonins • Killing is O2-dependent (respiratory burst, NADPH oxidase generated H2O2; myeloperoxidase generated HOCl; iNOS generated NO) or independent (lysozyme, lactoferrin, defensins) • Responding leukocytes cause pain and loss-of-function via prostaglandins, enzymes • Complete resolution; fibrosis, organization or scarring; abcess formation; progression to chronic inflammation

4. Outcomes of Acute Inflammation • Resolution of tissue structure and function with elimination of stimulus • Tissue destruction and persistent inflammation • Abscess • pus-filled cavity (neutrophils, monocytes and liquefied cellular debris) • walled off by fibrous tissue and inaccessible to circulation • tissue destruction caused by lysosomal and other degradative enzymes • Ulcer • loss of epithelial surface • acute inflammation in epithelial surfaces • Fistula • abnormal communication between organs or an organ and a surface • Scar • Causes distortion of structure and sometimes altered function • Chronic inflammation • Marked by replacement of neutrophils and monocytes with lymphocytes, plasma cells and macrophages • Accompanied by proliferation of fibroblasts and new vessels with scarring

5. Causes of chronic inflammation • Persistent infections • Organisms usually of low toxicity that invoke delayed hypersensitivity reaction • M. tuberculosis and T. pallidum causes granulomatous reaction • Prolonged exposure to potentially toxic agents • Exogenous agents include silica which causes silicosis • Endogenous causes include atherosclerosis caused by toxic plasma lipid components • Autoimmunity • Auto-antigens provoke self-perpetuating immune responses that cause chronic inflammatory diseases like RA, MS • Responses against common environmental substances cause chronic allergic diseases, such as bronchial asthma

6. Granulomatous inflammation • Focus of chronic inflammation encountered in a limited number of conditions • Cellular attempt to contain a foreign body or an offending agent that is difficult to eradicate (i.e. Tb) • Microscopic aggregation of macrophages that are transformed into epithelioid cells, surrounded by a collar of lymphocytes and occasionally plasma cells • Epithelioid cells have a pale pink granular cytoplasm with indistinct cell boundaries, often merging as giant cells • Foreign body epitheloids have dispersed nuclei • Infectious body epitheloids have marginal or horse-shoe nuclei • Enlarged granuloma with central necrosis is an abcess • Enlarged granuloma on a surface is an ulcer

7. Patterns of Inflammation • Serous Inflammation • Marked by outpouring of thin fluid • From blood serum, e.g. burn blisters • Effusion from mesothelial cells lining the pleural, peritoneal and pericardial cavity • Fibrinous Inflammation • A feature of pericardial and peritoneal inflammation • Vascular permeability allows larger molecules like fibrin to pass or procoagulant stimulus exists in the interstitium (e.g. cancer cells) • Suppurative Inflammation • Characterized by production of large amount of pus composed of neutrophils, necrotic cells and edema fluid • Involves pyogenic bacteria e.g. Streptococci and Staphylococcus aureus • Abscesses are focal localized collections of purulent inflammatory tissue caused by suppuration. • Ulcers • Local defect or excavation of the surface of an organ or tissue by sloughing of inflammatory necrotic tissue • Acute stage - intense polymorphonuclear infiltration and vascular dilation in margin • Chronic stage - margin and base develop fibroblastic proliferation, scarring and accumulation of lymphocytes, plasma cells and macrophages

8. Systemic inflammatory response • Acute Phase Response • Fever • Acute-phase protein secretion from liver • Leukocytosis • Tachycardia, increased blood pressure • Shivering, chills • Anorexia, somnolence, malaise • Septic shock

9. Acute Phase Proteins • Secretion of Acute Phase proteins by the liver • C-reactive Protein (CRP) • Serum Amyloid A (SAA) • Serum Amyloid P (SAP) • Complement • Fibrinogen • Prothrombin • Ferritin • Ceruloplasmin • α1-antitrypsin • α2-macroglobulin • Acute phase proteins bind: • Microbial constituents, acting as opsonins to fix complement • Chromatin, aiding early clearing of necrotic cells

10. Autonomic and Behavioral Responses • Autonomic • redirection of blood flow from cutaneous to vascular bed • increased pulse and blood pressure • decreased sweating • Behavioral • Rigors (Shivering) • Chills • Anorexia • Somnolence • Malaise

11. Sepsis • Systemic Inflammatory Response Syndrome involves two or more of the following • temperature >38.3ºC or <36ºC • heart rate >90 beats/min; <32 mm Hg • respiratory rate >20 breaths/min, PaCO2 or need for mechanical ventilation • WBC count >12,000/uL or <4,000/uL or >10% immature forms (bands) • Sepsis is defined as SIRS associated with suspected or confirmed infection--positive blood cultures are not necessary • Severe sepsis is sepsis complicated by a predefined organ dysfunction • Septic shock is cardiovascular collapse (hypotension) related to severe sepsis despite adequate fluid resuscitation

12. Septic stimuli • Gram-negative bacteria • LPS, endotoxin • Binds to LPS binding protein (LBP) • Binds to CD14 opsonin receptor • TLR-4 binds LPS and LPS-LBP • Stimulates release of TNF, IL-1, IL-6 • Gram-positive bacteria • Exotoxins, superantigens • Bind Vb regions of TCRs and/or to MHC-II • TLR-2 binds cell wall components • Stimulates release of IFN-g, TNF, IL-1, IL-6

13. Progression of sepsis • Cytokine release and amplification • Vasular response and neutrophil migration • Coagulation cascade • Short arm, extrinsic pathway, activated by expression of Tissue Factor VIIa Xa thrombin fibrin • high plasma levels of plasminogen-activator inhibitor type-1 (PAI-1) suppress plasmin and fibrinolysis • disseminated intravascular coagulation in 30-50% cases • Counter-inflammatory response • Apoptosis of Th and B-cells • Systemic acute phase response • increased cortisol production and release of catecholamines • upregulation of adhesion molecules • release of prostanoids and platelet-activating factor (PAF) • Organ failure

14. Multiple organ failure • Neutrophils damage tissue directly by releasing lysosomal enzymes and superoxide-derived free radicals • TNF-α induces nitric oxide synthase • nitric oxide causes further vascular instability • contributes to direct myocardial depression • Widespread vasodilation • Decreased production of vasopressin (ADH) and glucocorticoids • Circulatory collapse and tissue hypoxia

15. Findings of shock at autopsy • Congestion of lung • may also have fibrinous casts lining alveolar spaces • Petechial or ecchymotic hemorrhages on serosal and endothelial surfaces • Necrosis • proximal tubular epithelium in kidneys • entrilobular hepatocytes

18. Restoration of Structure and Function • Occurs if connective tissue structure relatively intact • Surviving parenchymal cells must have the capacity to regenerate • Labile Cells • Actively divide throughout life • cells of the epidermis and gastrointestinal mucosa • cells lining surface of the genitourinary tract • hematopoietic cells of the bone marrow • Stable Cells • Undergo few divisions normally, but can be activated from G0 cells when needed • hepatocytes • renal tubular cells • parenchymal cells of glands • mesenchymal cells (smooth muscle, cartilage, connective tissue, endothelium, osteoblasts)

19. Regeneration • Proliferation of cells and tissues to replace lost structures • Whole organs and complex tissues rarely regenerate after injury • Compensatory growth rather than true regeneration • Liver hypoplasia and kidney hypertrophy • Continuously renewing tissues regenerate after injury if tissue stem cells are not destroyed

20. Stem Cells • Characterized by self-renewal properties and capacity to generate differentiated cell lineages • obligatory asymmetric replication • one daughter cell retains its self-renewing capacity • the other enters a differentiation pathway • stochastic differentiation • stem cell divisions generate either two self-renewing stem cells or two cells that differentiate • Stimulation for either outcome is conjecture—seemingly random • embryonic stem cells (ES cells) are pluripotent • adult (somatic) stem cells are restricted by niche • skin, gut lining, cornea, hematopoietic tissue

21. ES cells and KO/transgenic mice • KO mice have specific gene deletion or inactivation • Transform cultured ES cells • Transformants injected into blastocysts • Blastocyst transplanted to surrogate dam • Mouse develops in utero • Transgenic mice have specific human gene insertion or replacement • Transformed ES cells injected into blastocysts • Continued development in surrogate dam

22. Somatic cell cloning • Reproductive • Transfer of adult nucleus into enucleated oocyte restores pluripotency • Transfer of resulting embryo to surrogate dam • Production of cloned individual • Therapeutic • Transfer of adult nucleus into enucleated oocyte restores pluripotency • Induced to differentiate into various cell types in vitro • Injected into damaged organ

23. Induced Pluripotent Stem Cells • Mouse ES cell pluripotency depends on the expression of Oct3/4, Sox2, c-myc, Klf4, Nanog • Human fibroblasts from adults and newborns have been reprogrammed • Oct3/4, Sox2, c-myc and Kfl4 • Oct3/4, Sox2, Nanog, and Lin28 • Generated cells from endodermal, mesodermal, and ectodermal origin • c-myc and Kfl4 are oncogenes

24. Stem Cells in Homeostasis and Healing • Bone marrow • Hematopoietic Stem Cells generate all of the blood cell lineages • Marrow Stromal Cells generate precursors of tissue to which migrated • Liver • Oval cells are bipotential progenitors of hepatocytes and biliary cells • Brain • Neural precursor cells generate neurons, astrocytes, and oligodendrocytes in the subventricular zone and the dentate gyrus of the hippocampus • Skin • Hair follicle bulge, interfollicular areas of the surface epidermis, and sebaceous glands • Intestinal epithelium • crypts are monoclonal structures derived from single stem cells • villus contains cells from multiple crypts • Skeletal and cardiac muscle • satellite cells beneath the myocyte basal lamina generate differentiated myocytes after injury • Cornea • limbal stem cells maintain corneal transparency

27. Proliferative capacity of tissues • Labile tissues • Continuously dividing tissues containing stem cells • Stable tissues • Parenchymal cells of solid organs in G0 • Endothelial cells, fibroblasts, smooth muscle • Limited regeneration after wounding • Permanent tissues • Absolutely nonproliferative • Cardiac muscle, neurons

28. Growth factors • Polypeptides that promote survival and proliferation by signal transduction • Increase in cell size • true growth factors • Increase in cell number • mitogens • Protection from apoptosis • survival factors

29. Signaling mechanisms • Receptors with intrinsic tyrosine kinase activity • Dimeric transmembrane molecules • Ligand binding induces stable dimerization and phosphorylation • 7tm GPCRs • Seven transmembrane proteins • Ligand binding induces association with GTP-binding protein, which swaps GDP for GTP • Gi or Gs protein inactivates or stimulates another effector • Gs activates membrane adenylyl cyclase; GTPGDP • cAMP activates PKA, etc. • Receptors without intrinsic enzymatic activity • Monomeric transmembrane molecules • Ligand binding stimulates interaction with JAKs

32. Growth Factor-mediated Proliferation • Platelet Derived Growth Factor (PDGF) • promotes the chemotactic migration of fibroblasts and smooth muscles • chemotactic for monocytes • competence factor that promotes the proliferative response of fibroblasts and smooth muscles upon concurrent stimulation with progression factors • Epidermal Growth Factor (EGF) • promotes growth for fibroblasts, endothelial and epithelial cells • is a progession factor - promotes cell-cycle progression. • Fibroblast Growth Factor (FGF) • promote synthesis of fibronectin and other extracellular matrix proteins • chemotactic for fibroblast and endothelial cells • promotes angiogenesis • links extracellular matrix components (collagen, proteoglycans) and macromulocules (fibrin, heparin) to cell-surface integrins. • Transforming Growth Factors (TGFs) • TGF-α - similar to EGF • TGF-β - mitosis inhibitor that aids in modulating the repair process. May be responsible for hypertrophy by preventing cell division. Chemotactic for macropahges and fibroblasts • Macrophage-derived cytokines (IL-1 and TNF) • promote proliferation of fibroblasts, smooth muscle and endothelial cells

33. Repair Process • Removal of Debris • begins early and initiated by liquefaction and removal of dead cells and other debris • Formation of Granulation Tissues • connective tissue consisting of capillaries and fibroblasts that fills the tissue defect created by removal of debris • Scarring • fibroblasts produce collagen until granulation tissue becomes less vascular and less cellular • progessive contraction of the wound occurs, resulting in deformity of original structure

34. Factors that Impede Repair • Retention of debris or foreign body • Impaired circulation • Persistent infection • Metabolic disorders • diabetes • Dietary deficiency • ascorbic acid • protein

36. Healing and granulation • Fibroplasia is a response to • Damaged connective tissue • Parenchymal damage exceeds regenerative capacity • Hyperplasia of connective tissue • Neovascularization • Granulation • coordinated proliferation of fibroblasts with a rich bed of capillaries • intensely hyperemic with a roughened or granular, glistening surface • healthy granulation tissue resists secondary infections

37. Healing by First Intention • Clean, surgical incision or other clean narrow cut • Focal disruption of epithelial basement membrane with little cell damage • Regeneration dominates fibrosis • Scabbing with fibrin-clotted blood • Neutrophils migrate to edges • Epidermis becomes mitotic and deposits ECM • Macrophages replace neutrophils • Vascularization and collagen deposition fills gap • Contraction of collagen minimizes epidermal regeneration

38. Healing by Second Intention • Larger area of tissue injury such as abcess, ulcer, infarction that destroys ECM • Large clot or scab with fibrin and fibronectin fills gap • Larger volume of necrotic debris must be removed by more neutrophils and macrophages • Opportunity for collateral damage by phagocytes • Scar tissue formed from vascular cells, fibroblasts, and myofibroblasts • Contraction of myofibroblasts distorts tissue • More prone to infection

42. Keloid—excessive cutaneous fibrosis

44. Granulation at tracheotomy