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CHAPTER 14 UNSTOPPABLE BLEEDING BLOOD COMPONENTS AND HAEMATOPOIESIS loodisaconnectivetissuemadeupofcellularelementssuspendedinanECFmatrix-plasma.Plasmamakesupa¼ofthe B ECF, and acts as a buffer between the cells and the external environment....
CHAPTER 14 UNSTOPPABLE BLEEDING BLOOD COMPONENTS AND HAEMATOPOIESIS loodisaconnectivetissuemadeupofcellularelementssuspendedinanECFmatrix-plasma.Plasmamakesupa¼ofthe B ECF, and acts as a buffer between the cells and the external environment. The composition is; 2L cells, 3L plasma = 5L blood. BloodisaconnectivetissuemadeupofcellularelementssuspendedinanECFmatrix-plasma.Plasmamakesupa¼ofthe E CF, and acts as a buffer between the cells and the external environment. The composition is; 2L cells, 3L plasma = 5L blood Role of blood Transport: gases, nutrients, waste, hormones, growthfactors Regulation: temperature, pH, water Protection: prevent blood loss, infection control omposition of blood C L>M>E>B. Lymphocytesmediatetheacquiredimmuneresponseofthebody,20-35%ofWBC.Thereare3subtypes;Tcells,Bcells,NK cells. Monocytesmatureandenlargeinresidenttissue;theyaretheprecursortomacrophages.Theycantransittotissuesin8h, lifespan can be months/years. They engage in phagocytosis and secrete pro-inflammatory signal molecules (cytokines). NeutrophilsarethemostabundantWBC(50-70%),shortlifespan(1-2days).Theyhave2-5lobularsegmentsofnucleusand engage in phagocytsosis by ingestion of bacteria and release a variety of cytokines and pyrogens (cause fever). Eosinophils are pink stained granules in cytoplasm, and are associated with allergic reactionsandparasiticdiseases.They have a cytotoxic cell type: release substances from granules that kill pathogens. Basophilsarelarge,darkbluestainedgranulesincytoplasmandreleaseinflammatorymediators-histamine,heparin,other cytokines. latelets and platelet production; platelets are small, round and colourless with no nucleus. They contain mitochondria, P smoothERandvesiclesfilledwithcytokines,growthfactorsandclottingfactorsandarealwayspresentintheblood,lifespan of 7-10 days. They play a major role in haemostasis and thrombosis, but also have immune function. T he process of haematopoiesis is orchestrated by pluripotent hematopoietic stem cells, which possess the ability to differentiate into various subtypes ofbloodcells.Thisdevelopmentaljourneycommencesearlyinembryonicdevelopment and persists throughout an individual's life. Hematopoiesis predominantly transpires within the bone marrow, initially in bonesuntiltheageof5,andsubsequentlyinspecificadultlocationssuch as the pelvis, spine, ribs, cranium, and proximal ends of long bones. Approximately25%oftheproducedcellsareredbloodcells(R BCs),while the majority, constituting 75%, are white blood cells (W BCs) with a shorter lifespan (due to the shorter lifespan, there needs to be more production). This process is primarily regulated by cytokines, including colony-stimulating factors, interleukins, and stem cell factor for white blood cells, erythropoietin for red blood cells, and thrombopoietin for platelets. Notably, immature blood cells undergo maturation within the bone marrow before entering the circulation. The head of long bones serves as a critical site for hematopoiesis due to its extensive blood supply, facilitating the immediate release of newly formed blood cells into the bloodstream upon production. This dynamic and finely tuned systemensuresacontinuoussupplyoffunctionalbloodcellsessentialfor various physiological processes throughout an individual's life. E rythropoiesis,theprocessofredbloodcell(RBC)production,isahighly regulated and essential physiological mechanism,generatingapproximately200 billion cells per day. Control over erythropoiesis lies in the hands of the glycoprotein erythropoietin (EPO) and certain cytokines, with EPO being primarilyproducedbythekidneys.ThereleaseofEPOistriggeredbyhypoxia,a conditioncharacterizedbyalackofoxygenreachingthebody'stissues,whichcan beinducedbyfactorssuchasexerciseorexposuretohighaltitudes.EPOisakey targetinblooddopingpractices,asitsadministrationstimulatestheproduction of RBCs, leading to an increase in haematocrit and circulating haemoglobin levels in the blood. Within the bone marrow, committed progenitor cells transform into nucleated erythroblasts, initiating the process. As these cells progress, their nucleus shrinks from 20 micrometers to 7 micrometers. The pivotal step in erythropoiesis is enucleation, wherethenucleusispinchedoffandsubsequentlyphagocytosedbybonemarrowmacrophages.Thisstep,limitingtherate of RBC production, ensures the removal of the nucleus and other organelles, including mitochondria. The final immature stageinthebonemarrowisthereticulocyte,whichlacksanucleusandswiftlyleavesthebonemarrow.Within24hours,the reticulocyte differentiates into a mature erythrocyte (RBC) in the bloodstream, fully equipped to perform its vital role in oxygen transport and gas exchange throughout the body. Stem cell → erythroblast (nucleated) → reticulocyte (enucleated) → mature RBC latelets, essential components of the blood responsible for clot formation and wound healing, originate from a unique P process in the bonemarrow.Megakaryocytes,largeanddistinctcells,undergoaspecializedformofcelldivisionknownas endomitosis.Duringendomitosis,themegakaryocytesreplicatetheirDNAuptoseventimeswithoutundergoingcytokinesis, resulting in a polyploid state (N=128). The growth and maturation of megakaryocytes are carefully regulated by thrombopoietin(T PO),aglycoproteinthatplaysacrucialroleinplateletproduction.Asmegakaryocytesundergomaturation and reach asufficientsize,plateletsbegintoformascellfragmentsthatbudofffromthesurfaceofthemegakaryocytes. Thisprocessofplateletreleaseinvolvesthebuddingofsmallfragmentsintothebloodvesselswithinthebonemarrow.Once releasedintothebloodstream,theseplateletscirculateandplayapivotalroleinrespondingtoinjuries,formingbloodclots, and maintaininghaemostasis. PHYSIOLOGY OF HAEMOSTASIS aemostasisis the process to stop bleeding when injuryoccurs to a blood vessel. There are three major steps in the process; H steps 1 and 2 are primary haemostasis (prevented by antiplatelets), step 3 is secondary haemostasis (prevented by anticoagulants). T hefirststepinvolvesvasoconstriction,atemporaryreductioninbloodflowwithinthedamagedvessel.Thisconstrictionis inducedbyparacrinemoleculesreleasedfromtheendothelium.Applyingpressureachievesasimilareffect,contributingto the overall mechanism of minimizing blood loss. The second step, formation of a platelet plug, commences with platelet adhesion to the damaged site, followed by activationandaggregation.Plateletsinthebloodbindtocollagenintheinjuredendotheliumthroughintegrins.Asplatelets adhere,theyreleasegranulescontainingsubstanceslikeADP(providingenergy),calcium,serotonin,andplatelet-activating factor (PAF),initiatingapositivefeedbacklooptoattractandactivatemoreplatelets.Thisaggregationofplateletsformsa plug that temporarily seals the breach bybindingtofibrinogen/fibrin.VonWillebrandFactor(vWF)isanadhesiveprotein that helps platelets stick to damaged blood vessel walls when injured. Thus, the platelet is essential but temporary to strengthen the plug. The third step, coagulation, leads to the formation of a stable blood clot. Fibrinogen is converted to fibrin, an insoluble biopolymerthatformsanetwork,trappingbloodcells.Platelets,bindingtofibrinorfibrinogen,contributetoplatelet-platelet aggregation. Three pathways—the extrinsic, intrinsic, and convergence to the common pathway—lead to thrombin activation, ultimately resulting in fibrinogen conversion to fibrin. Positivefeedbackloops,whereincreasedthrombinlevels enhance intrinsic and extrinsic pathways, amplify the clottingresponse.Withinthecoagulationcomponent,thereiseither extrinsicorintrinsicpathways,andbothoftheseconvergetothecommonpathway-needtomemorisethis.Thesepathways use calcium dependent enzymes. The intrinsic pathway is instigated by contact activation i.e. FXII gets activated by collagen, setting off a cascade. The extrinsic pathway is instigated by cell injury i.e. damage exposes tissue factor (FIII) leading to FVII activation. These two pathwaysconvergewhichresultsinthrombinactivation(FII),whichthenleadstofibrinogenconversiontofibrin (FI) (biopolymer), which then leads to cross linking of fibrin (via FXIII). Intrinsic begins within the blood vessel (slower), whereas extrinsic begins outside the blood vessel (faster). oagulation factors can get disrupted - haemophilia typeis dependent on the factor that is affected: C Haemophilia A:FVIIIaffected, 80-85% of cases → unstableclots, bleeding Haemophilia B: FIXaffected, 10-15% of cases → bleeding lot retraction and dissolution; after the platelet plug and coagulation forms aclot,theclotbeginstoshrinkandfibrinis C broken down to fragments → fibrinolysis. Plasminogen is activated by tissue plasminogen activator (tPA), which forms plasmin - plasmin breaks down fibrin polymers into fibrin fragments (in fact, tPA is given to breakdown clots following strokes). T heroleofendothelium;healthyendotheliummaintainshaemostasisinequilibriumandregulatesmanybiomoleculesthat actively inhibit thrombosis. As such there are various structural components that play different roles; S tructural components tPAis produced by the endothelium and forms plasmin- fibrinolysis Heparansulphate-formsglycocalyxthattrapswaterandpreventsmoleculesandplateletsfrominteractingwiththe endothelium. Forms anti-thrombin factor that inhibits thrombosis Thrombomodulin- activates C-protein to inhibit thrombinand prevent coagulation Nitric oxide (NO) and prostacyclin (PGI2)- inactivateplatelets CD39- inactivates platelets by restricting energythe blood can use (restricts ADP) THROMBOSIS aemostasis vs thrombosis H Haemostasisistheprocesstostopbleedingwheninjuryoccurstoabloodvessel- equilibrium can be restored after haemostasis. It involves a balance between bleeding and thrombosis. Thrombosis is the pathologicalformationofbloodclot-uncontrolledorexcessive coagulation or platelet activation. This is due to inappropriate activation of the coagulationsystemorwhenregulatorymechanismsthatpreventclotformationare disrupted.Thiscaneitherbehereditary(e.g.haemophiliaissexlinked)oracquired (anticoagulant/antiplatelet agents). T hrombosis- occurs via Virchow Triad (at least oneof the following): Abnormal vessel wall: endothelial dysfunction/disturbances/damage, contact pathway activation (intrinsic), tissue factor release (extrinsic) Abnormal blood flow: venous obstructions/stasis, AF,aortic stenosis, mechanical valves Abnormalbloodconstituents:abnormalitiesinthrombingeneration,platelets/coagulationandfibrinolyticpathwaysorother cellular components/microparticles A combination of these three creates an environment that promotes the formation of blood clots. T hrombosis occurs in various waysbased on its locationand aetiology; Arterial thrombosis:high shear force due to dysfunctionin endothelial cell Venous thrombosis:low shear force due to valve involvement(within veins) Medicaldevicethrombosis:highandlowshearforceduetobloodclot+fibrin+plateletcausesthrombosisonthemedical implant surface rterial thrombosis poses a significant risk, asitcanleadtosevere A consequences such as heart attacks or strokes. This type of thrombosis often stems from arterial narrowing caused by the buildup of lipids and cholesterol. The heightened blood flow, or shear stress, in the narrowed region triggers the activation of plateletsandvon Willebrand Factor (vWF). vWF binds to collagen, unfolding and exposing receptors. Platelets roll along these receptors and are arrested through the integrin GPIb-V-IX, initiating adhesion through αIIbβ3 and α2β1 integrins. This cascade signals further platelets, leading to their aggregation. Fibrinogens circulating in the blood attach to platelets, promoting the formation and propagation of the thrombus. Downstreamofthegrowingplateletclot,disturbedflowandcoagulationcontributetotheformationofawhiteclot,richin platelets.Refer to image below for more information. enous thrombosis, specifically deep vein thrombosis, occurs V due to blood flow stasis downstream of a vein valve. Reduced blood flow and stasis (low shear stress) impede the mass transport of coagulation factors downstream, leading to the accumulationofcoagulationfactorsandactivationoftissuefactor (TF). This process results in the formation of a red clot, characterized by its richness in fibrin and red blood cells. edical device thrombosis is triggered by contact with M foreign materials in medical devices, leading to protein adhesion and the activation of pathways that induce thrombosis. The flow conditions depend onthemedical device geometry and blood flow, with examplesranging fromlow-flowshearsituations(e.g.,venouscatheter)to high-flow shear scenarios (e.g., arterial mechanical valve). Certain medical devices, such as artificial hearts, may experience both high and low flow conditions. E mbolisms occur when blood clots break off and lodge downstream, causing further complications. Arterial embolisms, suchascerebralembolism,canresultinstrokes.Venousembolisms,includingpulmonaryembolisminthelungsorsystemic embolism in areas like the legs, present additional challenges. Medical device embolisms depend on the location ofthe device in the body and can lead to diverse complications based on their specific contexts. ANTITHROMBOTICS ntithrombotics: prevent formation of clots - these include antiplatelets and anticoagulants as these both contribute to A thrombosis. Antiplatelets:prevent platelet activitiy - coagulationactivity could still theoretically occur Anticoagulants:prevent coagulant activity - plateletactivity could still theoretically occur latelet activation, adhesion, aggregation - Begins with damage to the endothelium which exposes collagen and vWF P (activation),allowingbindingofplateletstoGPreceptors(integrins)(adhesion).Plateletsthenrecruitotherplateletsbythe release of other cofactors: COX-1 → produces thromboxane A2 (TxA2) → recruits more platelets ○ Aspirin inhibits COX1 → inhibits TxA2 → inhibits platelet recruitment ADP released from the cell acts on P2Y1/P2Y12 receptors on nearby platelets to recruit and adhese to them ○ Clopidogrel, prasugrel, ticagrelor inhibit the P2Y1/P2Y12 receptor Finally, platelets aggregate and form a haemostasis(aggregatrion). S imultaneously to platelet adhesion and aggregation, blood coagulation occurs, beginning with endothelial dysfunction, exposing tissue factor (activates FVII), and a cascade of interactions of factors. This activates thrombin (FIIa), converts fibrinogen to fibrin, fibrin causes cross linking to strengthen the clot. WarfarininhibitsFII, FVII, FIX, FX(old TV channels- it’s a Sydney thing :) ). Direct oral anticoagulants (DOAC) and heparininhibitsFXa, FIIa i.e. directly inhibits the common the pathway. ntithrombotic drugs A Oralanticoagulants:da bigatran(directthrombininhibitor),apixaban/rivaroxaban(FXainhibitors),Warfarin(VitKantagonist- interferes with clotting factors in the liver). Injectable anticoagulants:heparin, low molecularweight heparin, fondaparinux (FXa inhibitor) Antiplatelets: aspirin, clopidogrel/ticagrelor/prasugrel(P2Y12 inhibitors), dipyridamole I nternational normalised ratio (INR) quantifies the length of time it takes for blood to clot. This is only for patients on warfarin. INR2-3isnormal,butptswithprostheticvalveswillneedhigherINR2.5-3.5-bloodneedstotakelongertoclotbecausethey have prosthetic valves → increased risk of clotting. arfarin mechanism of action; R-warfarin inhibits vitaminKreductasefromworking,inhibitingtheproductionofclotting W factors. Vitamin K reductase is a reduced version of Vitamin K, which operates to convert clotting factors from being non-functional to functional i.e. activates clotting factors from their inactivated form e.g. FVII → FVIIa (‘a’ stands for activated). In their activated form, clotting factors are known asfunctional zymogens.Refer to diagramfor more: eparins and fondaparinux; antithrombin is synthesised in the H liver - it is an endogenous anticoagulant. It inhibits clotting factors 2a (thrombin) and 10a. Unfractionated heparin (snake looking) wraps around the anti-thrombin and thrombin molecules, and increases its effect as an anticoagulant (the chain has to be >18 molecules in length) Low molecular weight heparin - shorter than unfractionated heparin binds to 10a moreso than 2a (less so tothrombin,but moreso to antithrombin). Fondaparinuxbinds to antithrombin, only 10a, not2a Summary: Unfractionated - both 2a and 10a Low weight heparin - 10a predominantly, but also a little bit 2a Fondaparinux - only 10a Indications for anticoagulants or antiplatelets: atrial fibrillation, venous thromboembolism or pulmonary embolism, coronary artery disease, valvular heart disease, stroke Dental implications - need to balance the risk of bleeding with clotting Medical/medication history, including doses/indications Other medications such as NSAIDs and complementary/alternative medicine (can have an antithrombotic effect) Discuss with clinician managing patient Temporary interruption vs continuation of antithrombotic Patient related bleeding risk factors: increased blood pressure, decreased kidney/liver function → decreased ability to metabolisedrugs→prolongedeffectofmedication→increasedriskofbleeding,priorstroke,historyofbleeding,bleeding isorder, poor anticoagulant control - INR is fluctuating and poorly managed, increased age or frailty, NSAIDs, increased d alcohol consumption Procedure relatedbleeding risk factors Timing of cessation if indicated -only with consultationwith prescriber Warfarin - 5 days prior to procedure DOAC - 24-72 hours (1-3 days) prior to procedure, depending on renal function and specific DOAC Antiplatelet - 5-7 days prior to procedure L ocal haemostatic measures Non-pharmacological: pressure to the wounds, decreased tissue trauma, cellulose and collagen (if indicated), sutures → closure of the wounds (if indicated) Pharmacological: transexamic acid mouthwash 4.8% - antifibrinolytic agent - apply topically just before surgery, after the procedure,givethepatientforhomeusex2days(10mLrinsedinmouthfor2mins,thenspitout,4timesdailyfor2days).If the mouthwash can’t be compounded, crush 500mg tablet of transexamic acidanddispersein10mLwater.Thisworksby inhibiting the breakdown of fibrin clots which are formed during the process of blood coagulation (blocks plasmin - the enzyme that breaks down fibrin). INTRODUCTION TO LEUKAEMIA AND LYMPHOMA L ymphoma and leukemia are both types of cancersthataffectthebloodandimmunesystem,particularlylymphocytes, but they differ in their primary locations and manifestations. L ymphomainvolvesprimarymalignancyinlymphnodes.Lymphomasarecancersoflymphocytes,whichareatypeofwhite blood cell. The hallmark feature is the presence ofabnormallymphocytesinthelymphaticsystem,leadingtolymphnode enlargement and other symptoms. It is important to note that myeloid leukemias, which involve cells from the myeloid lineage, may also affect lymph nodes, but these cases are not classified as lymphomas. L eukemia involves primary malignancy in the blood and bone marrow. Leukemias affect white blood cells, and these abnormal cellscirculateinthebloodstream.Therearedifferenttypesofleukemias,includingchroniclymphocyticleukemia (CLL). In CLL, there is an increase in abnormal lymphocytes (white blood cells) in thebloodandbonemarrow,andlymph nodes may become enlarged. However, the primary manifestation is the presence of these cells in the blood. Insummary,thekeydistinctionliesintheprimarylocationofthemalignantcells.Lymphomasprimarilyinvolvelymphnodes, while leukemias involve the blood and bone marrow. ellular origin of human B cell lymphomas and C leukaemias - begins with genetic deformation of the precursing B cell (don’t have to understand this chart, just knowthatlymphomascanbeeitherBcellorTcell dominant (B cell is more common). L ymphoma:80%fromBcellsand20%arefrom T cells (T cell ones are worse) Leukaemia: 50% B cell and 50% T cell derived Starts off with B cell precursor in bone marrow (can developallthisdisorders)→canalsogotolymphnode (germinalcentre)tomature-caneitherbecomeplasma or memory B cell - can develop into various diseases throughout. aematopoiesis: begins as long term haematopoietic stem cell (LTHSC) → differentiates to short term HSC (STHSC) - H subtypes: Common lymhoid progenitor: T cells, B cells, NK cells Common myeloid progenitors: MEP (megakaryocyte, erythrocytes), GMP (granulocytes (WBCs), monocytes (macrophages)) Types of lymphocytes- defined by surface antigens,function, and the type of illness when lacking: B cells - humoral immunity, responsible for antibody production T cells - cellular immunity, cytotoxicity against virus, fungi, provide help for B cells → Most lymphomas are of B cell type. Primarylymphoidtissue-lymphocytematuration-primarylymphoidtissueisthesiteatwhichBandTcellsmaturei.e.B cells in the bone marrow and T cells in the thymus. Secondarylymphoidtissue-lymphocytesbroughtintocontactwithantigen-secondarylymphoidtissueisthesiteatwhich B and T cellsactontheantigeni.e.atlymphnodes-APC(dendriticcells)presenttheantigentoTcells,whichtheneither help B cells (create Ig), or become cytotoxic in nature and killtheantigene.g.adenoids,tonsils,spleen,appendix,Peyer’s patch Anatomy of lymph nodes Follicle centres contain predominantly B cells Cortical regions contain predominantly T cells Lymphoma Leukaemia riginates in lymphatic system - includes lymph nodes, o o riginates in bone marrowandaffectsproductionofblood spleen, thymus, and lymphoid tissue cells (RBC, WBC, platelets) Involveuncontrolledgrowthandaccumulationofcancerous Involves overproductionofabnormalWBC(lymphocytesor lymphocytes (B, T, NK cells) in lymph node/tissue myeloid cells) in bone marrow Subtypes:Hodgkin lymphoma and non-Hodgkin lymphoma S ubtypes (based on the type of WBC affected): acute lymphoblastic leukaemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukaemia (CLL), and chronic myeloid leukemia (CML) → Remember these are both types of cancers of the blood that affects the immune system - a lot of overlap, making diagnosis hard. WHO Classification System Non-Hodgins lymphoma - either B or T cell neoplasms Hodgkins lymphoma Plasma cell diseases Immunodeficiency-associated lymphoproliferative disorders B cell Non-Hodgkin Lymphoma Lymphoma/leukaemia Indolent nodal or extranodal lymphoma (slower) Aggressive nodal or extranodal lymphoma (faster) E.g. of extranodal is gastric/CNS Clinical features of lymphoma Lumps in neck, under arms, in groin (lymphadenopathy) Lymphadenopathy noted during examination for otherreason(asymptomatic)duringcheck up Abnormal blood findings unusual (compared with leukaemia - usually abnormal blood findings) Mycosis fungoides (type of cutaneous T cell lymphoma) - rash on skin; brown spots when cutaneous but once it becomes blood borne, known as sezary syndrome (cutaneous T cell lymphoma) Spleen involvement iagnosis of lymphoma; requires a large tissuebiopsy(histologicalassessmentisthegoldstandard).Ifsizeofthetissueis D limited, then formalin fixing is the priority. Flow cytometry is a way of looking at surface protein expression of cells - cytogenetics (refer to diagram). Tissue biopsy → requires a large biopsy Histopathology of lymphoma H and E staining of different lymphomas, nucleus = purple colour Classified as either nodular (small nucleus) or diffuse (cannot differentiate b/c nuc or cells) If nodular/follicular (mostly small cells) → indolent If diffuse (mostly large cells) → aggressive 80% of lymphoma canbedividedinto2groups;follicularmakeup40%ofHLandtheother40%isdiffuselargeBcellNHL ( aggressive). Cancerisaclonaldisorder-thismeansthatifonecellmutates,itclonesandbecomesthepredominanttypeofcellwithin thecellularpopulationitisapartof,andcausesmonoclonality.Immunoglobulin(IG)expressedonthesurfaceofBcellsand IG made up of2heavyand2lightchains-lightchainscanbekappaorlambda(KandLonpic)2/3arekappaand1/3are lambda(ratio2:1).Inmalignancy,oneismorethantheother(moreoftentheLcells),andthispresentsonthegraph(right graphismalignant).Thisisduetolackofapoptosis,causingincreasedclonalityofthecelltype.CD20isamarkerforBcells; usingcytometry, we can see the ratio of L:K cells. Gene translocation in lymphoma; different types ofgenes that are altered can lead to different lymphomas. L ymphoma staging Stage 1 - early disease- cancer is found only ina single lymph node region or one organ or area outside the lymph node Stage 2 - locally advanced disease- the cancer isfound in two or more lymph node regions on one side of the diaphragm Stage 3 - advanced disease- the disease involveslymph nodes both above and below the diaphragm Stage 4 - widespread disease - lymphoma has advanced beyond the lymph nodes andspleenANDhas spread to one or more organ - bone, bone marrow, skin, liver Diagnosis of staging based on glucose labelling that emits a small amount of radiation → PET SCAN: Brain lights up normally Black spots are lymphoma though Figure is stage 4 lymphoma - diffuse large B cell lymphoma Treatment strategies Grow slowly → treat slowly (indolent): Watch and wait Local radiation for local disease Start with simple chemotherapy and move to more complex chemotherapy Grow fast → treat fast (aggressive) Aggressive multiagent chemotherapy, often with radiation therapy to sites of bulky disease Consider autologous or allogeneic stem cell transplantation Redo PET scan to see if it has gone down Repeat in 5 years Common anti-lymphoma drugs Chlorambucil CHOP: cyclophosphamide, doxorubicin, vincristine, prednisone Fludarabine Bendamustine Monoclonal antibodies ○ Anti CD20 (expressed by B lymphocytes) ○ Anti CD52 (B and T lymphocytes, monocytes dendritic cells) Outcomes of therapy Indolent: No cure, median survival 8 years, 25% alive in 10 years Aggressive: cure 30-40%, 50% dead in 2 years → indolent eventually dead, but no cure. Aggressive can be cured in 30-40%, but 50% dead in 2 years. Acute Leukaemia Chronic Leukaemia T imeline: rapidly progressive if untreated → severe bone T imeline: slower course, even non-progressive, marrow marrowfailureandnoopportunityforbodytocompensate failure often not present at diagnosis and physiological progression days/weeks → survival weeks/months → compensation possible for some symptoms→progression weeks/months → survival months/years Presenting features:bone marrow failure, bone pain resenting features: non-specific complains (often picked P up on routine blood tests, symptoms related to splenomegaly or lymphadenopathy athobiology: deregulation of differentiation and P athobiology:resultsfromtheneoplastictransformationof P maturation programs → characterised by abundance of a haematopoietic stem cell, some blast cells present, but immature ‘blastic’ cells that grow uncontrollably and lack more mature cells that can carry out their normal functions normal differentiation Summary:mature cells > blast cells Summary:blast cells > mature cells athobiology:clonaldisorderthatresultsfromhaematopoieticprogenitors(eitherlymphoidormyeloidpathways,orfrom P primitive stem cells, decreased apoptosis. L ack of normal haematopoiesis can cause bone marrow failure: Anaemia→ fatigue, shortness of breath Neutropaenia→ infection and mouth ulcers Thrombocytopaenia→ bleeding and bruising E xcess malignant blood cells: High WBC in blood → hyperviscosity →stroke Increase in size of secondary lymphoid tissue → splenomegalyandlymphadenopathy Bone pain cute Myeloblastic Leukaemia (AML)is: A Acute → high blasts, low mature cells Myelo → myeloid cells (RBC, platelets, monocytes, granulocytes) Leukaemia → WBC E.g.64yearoldmanwith5dayhistoryoffever,shortnessofbreathandbruisingoftheleg.Thebloodtestbelowshowsthat he is anaemia (Hb = 84), neutropaenic (0.1), low myeloblasts and low platelets. Anaemic + neutropaenic + thrombocytopaenic → pancytopaenia; low in all 3. cute Lymphoblastic Leukaemia (ALL)is: A high blast cells, neutrophil normal range Acute → high blasts, low mature cells Low platelets Lympho → lymphoid cells (T and B cells) Leukaemia → WBC Other features: cervical and supraclavicular lymphadenopathy, large mediastinal mass, heavy infiltration of CD2+ blast cells E.g. 39yo male presents with swelling in neck for 1 week, terminaldeoxynucleoidtransferasepositivity(TDT+)tellsusthat it is T-cell ALL. Lymphocytes would be high in a blood test. hronic lymphocytic leukaemia is a disease of the elderly - C median age of onset is 65. It involves the accumulation of functionally incompetent mature lymphocytes in blood, bonemarrow,spleen,lymphnodes.Lymphocytesaremonoclonal; almost always a B cell disorder (B-CLL) with associated complications. linical presentation is predominantly asymptomatic, but raised lymphocyte count onbloodcountandlymphadenopathy. C Others include immune dysfunction (viral infections e.g. shingles), and other symptoms of marrow failure; anaemia - tiredness, SOB, low platelet count - easy bruising, bleeding, low neutrophil count ( → bacterial infection). linical course follows a slow progression, markedbyagradualincreaseinthewhitebloodcellcountprimarilyduetothe C accumulation of abnormal lymphocytes. As the disease advances, there is notableenlargementoflymphnodes,liver,and spleen.Progressivebonemarrowfailurebecomesevident,leadingtocompromisedhematopoiesisincludingimmuneparesis and B cell immunosuppression,contributingtoanincreasedsusceptibilitytoinfections.Additionally,patientswithCLLmay exhibitthesecretionofaparaprotein,anabnormalproteinproducedbythecancerouslymphocytes. This can lead to a complication known as autoimmune haemolytic anemia, where the immune system attacks and destroys red blood cells, leading to anemia. Another complication is immune thrombocytopenia purpura, a condition characterized by low platelet counts, which can result in excessive bruising and bleeding. T he aetiology of chronic lymphocytic leukaemia (CLL) remains largely unknown, with multiple factors contributing to its development. Immune regulation plays a role in CLL, and approximately 80% of cases involve somatic gene mutations. Notably, mutations in tumour suppressor genes, such as ATM and p53, are prevalent in CLL. One significant molecular alteration observed in CLL is the elevation of the anti-apoptotic protein bcl-2, attributed to the deletion of inhibitory icroRNA. This dysregulation of apoptosis, or programmed cell death, contributes to the survival and accumulation of m abnormal lymphocytes characterizing CLL. There is also an element of inherited risk associated with CLL. Individuals with first-degreerelativesaffectedbyCLLhaveanincreasedrisk,althoughthereisnoconsensusonasingulargeneresponsiblefor thedevelopmentofthedisease.Theinterplayofgeneticfactorsandenvironmentalinfluencesunderscoresthecomplexityof CLL's aetiology, T he diagnosis of chronic lymphocytic leukaemia (CLL) involves specific criteria and diagnostic procedures to identify the characteristicfeaturesofthisbloodandbonemarrowdisorder.OneessentialcriterionforCLLdiagnosisisthepresenceofa peripheral blood monoclonal B-cell population, exceeding a count of >5x10^9/L, persisting for at least fourweeks.The characteristic B-cellpopulationinCLLco-expressesCD19,CD5,andCD23.Additionally,thereistypicallyweakexpressionof CD20,CD79B,andsurfaceIgMontheseBcells.WhileabonemarrowbiopsyisnotmandatoryfordiagnosingCLL,itcanbe beneficial in certain cases. Bone marrowbiopsyisparticularlyusefulwhenthereisaneedtodetermineifcytopenias(low blood cell counts) arearesultofbonemarrowinfiltrationbyCLLcells.Thisprocedureprovidesvaluableinformationabout the extent of CLL involvement in the bone marrow and aidsinassessingtheimpactonhaematopoiesis.Otherdiagnostics include WBCcountisveryelevated(lymphocytecount-althoughnormalneutrophilcount),plateletcountisretained,flow cytometry shows more kappa and less lambda chains (express CD20+CD5+CD23 B lymphocytes). S taging systems in CLL are used to classify the severity and progression of CLL. Rai staging is based on the presence or absence of certain clinical findings and blood counts, whereas Binet staging is based on the extent of lymphadenopathy (enlarged lymph nodes) and presence of anaemia/thrombocytopaenia. Treatment begins at RAI stage 2-3 or Binet stage C. rognosisfollowsoneoftwopaths,dependingonthemutatedstatus-thiscanbedeterminedbyobservingtheexpressionof P immunoglobulin (Ig) variable (V) heavy chain (H) gene segments (IgVH). These mutations occurs in somatic cells (non- reproductive). Mutated -gone through germinal centre Unmutated -hasn’t gone through germinal centre lone IgVH region gene differs fromgermlinesequenceby C C loneIgVHgeneinCLLcellsdiffersfromgermlinesequence more than 2% = mutated → means somaticmutationhas by less than or equal to 2% (same as saying Clone IgVH occurred in this gene (changes the sequence by >2%) region gene shows >98% homology (similarity)) oodprognosis(goodchanceofrecoveringfromadisease) Bad prognosis G and account for50%of CLL cases rogression requiring treatment after10-20years→more P P rogression requiring treatment after 2-5 years → more indolent (slow progressing disease - doesn’t require aggressive and faster progression of disease → requires treatment for longer period of time) treatment a lot sooner hronic Myeloid Leukaemia (CML) is a type of leukaemia, a cancer of the blood and bone marrow, characterised by the C abnormal and uncontrolled growth of myeloid cells. Pathophysiology:massive myeloid hyperplasia refersto the excessive production of myeloid cells in the bone marrow. Accumulation of immature and mature myeloid cells in both the blood and bone marrow contributes to the disease. Epidemiology:CML can affect individuals of both sexes,the most common age of onset is between 40 and 60 years. RiskFactors:priorirradiationhasbeenassociatedwithanincreasedincidenceofCML,howeverCMLisnotsignificantlymore frequent in monozygotic twins, suggesting that genetic factors alone may not be the primary cause. Disease Phases: Chronic Phase:This is the initial phase and is characterisedby slow, asymptomatic growth of abnormal myeloid cells. Accelerated Phase: In this phase, the abnormal cells start to grow and divide more rapidly, leading to symptomatic manifestations. Blast Crisis (Acute Phase): This is an aggressive phase wherethemajorityofcellsinthebonemarrowareimmatureblast cells. It resembles acute leukaemia and progresses rapidly. Genetic Mutation: The primary cause of CML is a specific genetic mutation known as the Philadelphia chromosome (Ph chromosome). The Philadelphia chromosome results from a reciprocal translocation between chromosomes 9 and 22 [t(9;22)],leadingtothefusionoftheBCR(breakpointclusterregion)geneonchromosome22withtheABL(Abelsonmurine leukemiaviraloncogenehomolog1)geneonchromosome9.ThisfusiongeneiscalledBCR-ABL1.Inlightmicroscopy,dividing cells in 95% of cases. BCR-ABL1Oncogene:TheBCR-ABL1fusiongeneproducesaconstitutivelyactivetyrosinekinasethat promotes uncontrolled cell proliferation and inhibits apoptosis. This aberrant kinase activity is a key driver of CML. F indings on FBC, blood film and special tests Findings on FBC: neutrophil leucocytosis (elevated WBC count), immature cells mainly myelocytes circulating in the peripheral blood, increase in circulating basophils, anaemia Findings on bone marrow biopsy: hypercellular, increase in myeloid series (bone marrow and blood appear similar), cytogenetic analysis and molecular analysis to detect the BCR-ABL chromosome Blood film:both blast and mature cells (for acute,would not see mature) T reatment;targetedtherapies,suchastyrosinekinaseinhibitors(TKIs),arethemainstayoftreatmentforCML.Thesedrugs specifically target the BCR-ABL1 tyrosine kinase,inhibitingitsactivityandslowingdownorstoppingtheprogressionofthe disease. Imatinibisafirst-generationtyrosinekinaseinhibitor(TKI)thatspecificallytargetstheBCR-ABLkinase,whichisresponsible fortheabnormalgrowthinCML.ItworksbyinhibitingthetyrosinekinaseactivityoftheBCR-ABLfusionprotein.Whilenot considered a cure, imatinib can induce haematological remission (normalising blood counts), cytogenetic remission (eliminationorreductionofleukaemiacellswiththePhiladelphiachromosome),molecularremission(undetectablelevelsof BCR-ABL at the molecular level using PCR). Resistance to imatinib can occur through various mechanisms, including either de novo or acquired: point mutations in BCR-ABL gene preventing imatinib from inhibiting kinase activity, BCR-ABL gene amplification/BCR-ABL independent mechanismsor activation of theSRC family kinases. S econd-GenerationTyrosineKinaseInhibitorsareusedonthosewhodevelopresistancetoorcannottolerateimatinib.The choice of TKI depends on factors such as the patient's response, side effects, and the specific mutations present in the BCR-ABL gene. Regular monitoring and adjustment of treatment are crucial to manage the disease effectively. asatinib: A dual SRC-ABL kinase inhibitor that targets both active conformations of ABL and SRC, which are structurally D similar. About 300-fold higher potency against native BCR-ABL compared to imatinib, making it very effective. Nilotinib: Specifically designed to have a higher binding affinity for BCR-ABL kinase compared to imatinib. Modified from imatinibtoenhanceBCR-ABLkinasebindingactivity.Lesspronetoresistancedevelopmentcomparedtoimatinibduetoits increased potency. An option for CML patients who do not respond well to imatinib. Ponatinib: a multitargeted kinase inhibitorthatisactiveagainstallknownBCR-ABLmutants,includingtheT315Imutation, which is particularly challenging to treat with other TKIs. IMMUNOLOGICAL BASIS FOR TRANSPLANTATION T ransplantationisthemosteffectivetreatmentforendstageorganfailure,althoughtransplantedorgansurvivalislimited byimmunerejection;theshorttermoutcomesareexcellent,andlongtermoutcomeshavechangedverylittleoverthepast few decades - rejection is due to T-cell mediated rejection and/or antibody-mediated rejection. 1954 = first successful living-relatedkidneytransplantbetweenidenticaltwinHerrickbrothers,Dr.JosephMurray’s team 1962 = first cadaveric kidney transplant, Dr. Joseph Murray 1963 = first lung transplant, Dr. James Hardy 1967 = first liver transplant , Dr. Thomas Starz 1967 = first heart transplant, Dr. Christiaan Neethling Barnard Transplanted organ survival is limited by the side effects of long-term immunosuppressive regimens Infection: serious bacterial, fungal and viral infections Malignancies - skin cancer, most common in kidney and liver transplantation Neurotoxicity: apparent months or years Diabetes CVS events Metabolic syndrome - hyperlipidaemia, diabetes, HTN Majority of rejection occurs within the first 6 months of transplantation, as this is when immunosuppression is highest. Recommendation: no elective dental work within these 6 months. Malignancy is the tell-tale sign of immunosuppression - skin cancer is the most common. ejection - the transplant recipient’s immune system attacks the transplanted organover three stages: R Hyperacute rejection: minutes to hours-themostrapidandsevereformofrejection,causedbypre-existingantibodiesin therecipient’sbloodthatreactagainstantigensonthedonororgan;anti-donorantibodiesagainstABO/HLA/otherantigens causes complement activation, thrombosis, leukocyte inflammation, endothelial damage, leading to immediate and irreversible damage to the transplanted organ. This is however rare these days due to extensive pre-transplant testingto identify donors. Acuterejection:daysto12months-occursastherecipient’simmunesystemrecognisesthetransplantedorganasforeign, mounting animmuneresponseagainstit-Tcellmediatedalloresponse.Itischaracterisedbyinflammationanddamageto the transplanted tissue and is often treated with immunosuppressive meds. Chronicrejection:monthsoryearsafterandischaracterisedbyfibrosisandvascularabnormalities.Denovodonorspecific antibodies (dnDSA) is an important risk factor. It involves slow deterioration of the organ’s function - often resistant to treatment with immunosuppressive drugs → can lead to organ failure. T cellsarecentraltotheprocessoftransplantationrejectionthroughallorecognitionofforeignantigens.BothCD4andCD8T cellsareinvolved.BothTcellsandBcellsplayaroleinacuteandchronicrejection.ThesealloreactiveTcellscanrecognise and respond to antigens on transplanted organs or tissues that are different from the recipient’s own antigens. Allorecognition:abilityofanorganismtodistinguishitsowncellsandtissuesfromthoseofanotherfromthesamespecies i.e. how it responds to foreign antigens. onororgan→recipientlymphnode→CD8+Tcell+dendriticcells+CD4+Tcell→releasecytokines→clonalexpansion D andadditionalcytokineandchemokineproduction→differentiatedmacrophages+B-cellantibodyproduction→activatedT cells → acute rejection Organ → T cells → cytokines → B cells → macrophages → acute rejection. T cell activation signals Signal 1: antigen specific, T cell receptors recognise MHC-allo-peptide complex (CD4 binds MHC 2 and CD8 binds MHC1) Signal 2: antigen-non-specific, binding of co-stimulatory molecules (CD28-B7, CD80/CD86, CD40L-CD40 pathways) Signal 3: cytokine stimulation (IFN-gamma) S ignal 2 and 3 are critical in rejection. Signal 1 and 2 are at least needed for T cell activation. T hediversityofthealpha-betaTcellreceptor(TCR)isessentialfortheimmunesystemtoeffectivelyrecognizeandrespond to avast array of antigens. Expression in Human Peripheral Blood T Cells: Over 95% of human peripheral blood T cells express the alpha-beta TCR complex. This complex consists of two protein chains, the alpha chain, and the beta chain. Overall TCR Diversity: the diversity of the TCR is immense, with approximately 2.5 x 10^7 (25 million) different TCRs estimated to exist in humans. Genetic Basis of Diversity: The diversity of the TCR is primarily generated through genetic mechanisms during T cell development.ThisinvolvestherearrangementofgenesegmentsencodingtheTCR.TheTCRgenesarecomposedofvariable (V), diversity (D), and joining (J) gene segments. The combination of different V, D, and J segments contributes to the diversity of TCRs. N-Region Diversity: The diversity is further increased by the addition ofnon-templatednucleotides,knownasNregions, during the gene rearrangement process. This N-region diversity adds extra variability to the TCR sequence. CDR3RegionandDiversity:Thecomplementarity-determiningregion3(CDR3)isahighlyvariableregionwithintheTCR.Itis formedbythejunctionoftheV,D,andJgenesegmentsandtheaddedNregions.TheCDR3regionisparticularlyimportant for antigen recognition, as it directly interacts with the peptide-MHC complex. T he Major Histocompatibility Complex (MHC), alsoknownastheHumanLeukocyteAntigen(HLA)systeminhumans,isa crucial genetic system involved in immune responses, particularly in the recognition and rejection of foreign or non-self tissues. Genetic Loci and Chromosome Location:The MHC is agenomic region located on chromosome 6 in humans. Role in Tissue Rejection: MHC molecules are essential for the immune system's ability to distinguish between self and non-self tissues. Mismatch of MHC molecules between donor and recipient is a significant factor in the rejection of transplanted organs or tissues. PolymorphicCellSurfaceStructures:MHCmoleculesarehighlypolymorphic,meaningthereisasignificantvariationinthe gene sequences that encode these molecules among individuals. This polymorphism contributes to the diversity of MHC molecules, allowing the immune system to recognize a wide range of antigens. MHC Class I and Class II:MHC molecules are categorizedinto two main classes: MHC class I and MHC class II. MHC Class I (HLA-A, B, C):These molecules are foundon the surface of almost all nucleated cells. MHCClassII(HLA-DR,DP,DQ,DO,DM):Thesemoleculesareprimarilyexpressedonthesurfaceofantigen-presentingcells (APCs) such as macrophages, B cells, and dendritic cells. Antigen Presentation: MHC molecules play a crucial role in presenting antigens to T cells. Potential donor siblings Whenconsideringorganortissuetransplantation,thecompatibilityofthedonor'sHumanLeukocyteAntigen(HLA)withthe recipient's HLA is a crucial factor in minimizing the risk of transplant rejection. HLA molecules are highly polymorphic, meaning there is a wide variety of HLA alleles within the human population. The more closely matched the donor and recipient HLA, the lower the likelihood of rejection. Siblings are potential donors because there is ahigherlikelihoodof sharingHLAalleleswithsiblingsduetothegeneticsimilaritiesinheritedfromcommonparents.Ifasiblingisafullmatch oraclosematchintermsofHLAcompatibility,itcanenhancethechancesofasuccessfultransplant.Theprobabilitythat2 siblings will have the same HLA allele is 25% (0.5 x 0.5). irectallorecognitionoccurswhenrecipientTcellsdirectlyrecognisedonorMHCsonthesurfaceofdonorAPCswithinthe D transplanted organ. Donor APCs migrate from the transplanted organ to the recipient’s lymphoid tissues, where T cells recognise the foreignMHCmoleculesonthedonorAPCs.Thisoftenresultsinastrongandrapidimmuneresponseagainst the transplanted organ →acute rejection(the primarypathway for acute rejection). Indirect allorecognition occurs when recipient T cells recognise donor antigens that have already been processedbythe recipient’s APCs (donor antigens are presented as peptides to the TCR). Recipient APCs take up donor antigens after transplantation,processedaspeptides,thenpresentedtorecipientTcells,whichrecognisetheseantigensarenon-self.This is associated withchronic rejection- less immediate,but can be more persistent than direct allorecognition. Semi-direct allorecognition is a hybrid pathway combining direct and indirect. Donor APCs release MHC peptides into circulation, which are taken up by recipientAPCsandpresentedtorecipientTcells.ThisoftenhappenswhendonorAPCs aren’t able to attach to TCRs due to physical barriers. It can contributetobothacuteandchronicrejection→essentially allowsrecognitionandresponsetodonorantigensindirectly,whendirectinteractionsbetweendonorandrecipientAPCsare limited physically. irect: donor APCs + donor MHC + donor antigen → T cells D Indirect: recipient APC + recipient MHC + donor antigen → T cells Semi-direct: recipient APC + donor MHC + donor antigen → T cells Immunosuppressive agents in transplantation(don’thave to know in detail) Deletion/Inhibition of Alloreactive T Cells: ○ Thymoglobulin: An antibody preparation that depletes T cells and other immune cells to reduce the recipient's immune response. ○ Tacrolimus (FK506) and Cyclosporine: Calcineurin inhibitors that inhibit T cell activation by blocking the production of interleukin-2 (IL-2). ○ Basiliximab: A monoclonal antibody that targets CD25 on activated T cells, reducing their activity. ○ Mycophenolate Mofetil (MMF): Inhibits the proliferation of T and B cells by blocking the synthesis of nucleotides. ○ Rapamycin: Inhibits T cell proliferation by targeting the mammalian target of rapamycin (mTOR) pathway. ○ Belatacept(CTLA4-Ig):AcostimulationblockadeagentthatinterfereswithTcellactivationbybindingtoCD80 and CD86 on antigen-presenting cells. Deletion of B Cells: Rituximab: A monoclonal antibody that targets CD20 on B cells, leading to their depletion. B-Cell-T-CellInteractions:Alemtuzumab:AmonoclonalantibodythattargetsCD52,whichispresentonbothBandT cells, leading to their depletion. TNFInhibition:Etanercept:Aninhibitoroftumornecrosisfactor(TNF),whichisinvolvedininflammationandimmune responses. IL-6Inhibition:Tocilizumab:Anantibodythatinhibitsinterleukin-6(IL-6)signaling,whichisinvolvedininflammation and immune responses. Balance between activation and regulation; the goal is long term transplant organ acceptance in the absence of immunosuppressive therapy → transplant tolerance. Transplant tolerance is when the immune system does not attack transplantedgrafts,butalsoremainscapableofrespondingtopathogenicmicroorganismsandotherantigensi.e.thereisa balance between allo-reactive TcellsandregulatoryTcells.AlloreactiveTcellsaretheTcellsthatrecognizealloantigens- presentedonMHC→initiateimmuneresponseagainst transplantedorgan,andregulatoryTCells(TregCells)aretheTcells with immunosuppressive properties. When: Alloreactive T cells > reg T cells → transplant rejection Reg T cells > alloreactive T cells → immunosuppression to all antigens Cell based immune cell therapy in transplantation Bone marrow cells:mixed and full haematopoietic donorchemierism (kidney transplants) Tolerogenic dendritic cells:in kidney transplants Regulatory B cells:diabetic patients Regulatory T cells- the most attractiveFoxp3+ T-regulatorycell therapy - kidney and liver transplantations D4+CD25+Foxp3+ regulatory T cells (Tregs) play a critical role in immune regulation and tolerance. C DevelopmentandInduction:CD4+CD25+Foxp3+TregsdevelopinthethymusasasubsetofregulatoryTcells,andtheycan also be inducedperipherallyfrom conventionalCD4+T cellsin response to certain environmental cues. Immune Homeostasis and Autoimmunity Prevention: Tregs are essential for maintaining immune homeostasis by suppressing excessive immune responses. They play a crucial role inpreventing autoimmunity. Suppressing Transplant Rejection: Tregs are involved in suppressing transplant rejection by inhibiting the activation and functionofalloreactiveTcellsthatrecognizeantigensfromthetransplantedorgan.Theirsuppressiveactivityhelpsdampen the immune response directed against the graft. PromotingTransplantTolerance:Transplanttoleranceinvolvestheimmunesystemacceptingthetransplantedorganwithout mounting an immune response against it. Tregs are critical for promoting transplant tolerance by dampening alloreactive responses and promoting an environment conducive to long-term acceptance of the graft. IPEX Syndrome: Immunodysregulation Polyendocrinopathy Enteropathy X-linked (IPEX) syndrome is asevereautoimmune disorder caused bymutations in the FOXP3 gene,whichleads to dysfunctional Tregs. Experimental Evidence in Transplant Tolerance: Animal models of transplant tolerance have provided evidence for the importance of Foxp3+ Tregs. Depletion experiments showed that the absence of Foxp3+ Tregs abolished kidney graft tolerance. Donor antigen-specific Foxp3+ Tregs were identified as key players in maintaining kidney tolerance in these models. Furthermore, their presence providedspontaneousacceptance of kidney allotransplant tolerance. Methods to Generate and Induce Foxp3+ Tregs: Various methods have been explored to generate and induce Foxp3+ Tregs, including: Direct Antigen-Expanded Tregs: Tregs can be expanded usingantigenspresentedbydonorantigen-presentingcells (APCs) and donor MHC molecules - focuses on antigens from the donor tissue Indirect Antigen-ExpandedTregs:TregscanalsobeexpandedusingantigenspresentedbyhostAPCsandhostMHC molecules - approach is based on antigens from the recipient's own tissues. Polyclonal Expansion with Anti-CD3/CD28Beads:involvesstimulatingTregswithanti-CD3andanti-CD28antibodies attached to beads. Isolation of Specific Tregs Using Tetramers composed of host MHC II and donor MHC CAR-Treg(ChimericAntigenReceptorTregs),HLA-A2specific:CARsaredesignedtopromoteimmunosuppressionand immune tolerance. Insertion of Allospecific TCR (T Cell Receptor): Transgenic TCR expression Donor antigen specific Foxp3+ Tregs FULL BLOOD COUNT, BLOOD FILM AND COAGULATION Normal blood film BC: pale centre and red rim R Neutrophil: RNA is purple and cytoplasm pink (eventhough it looks purple) - has granules T hephysiological production of red blood cells (erythropoiesis)is a dynamic andtightly regulatedprocess that involves various stages of cell differentiation and interaction with the microenvironment. The process begins with amultipotent hematopoieticstem cell, which can give rise to various blood celllineages. When the stem cellcommitsto the erythroid program, it becomesanerythroid progenitor. After commitment to the erythroid lineage, several divisions later, the cells startexpressing the erythroidprogram. This involves the activation of specific genes that drive the cells toward becoming red blood cells. The committed cells undergo further differentiation into erythroid precursor cells. As these cells mature, they eventuallylose theirnucleusduring thereticulocyte stage.Reticulocytesareimmature red blood cellsthat still contain some cellular remnants,such as ribosomal RNA.The loss of the nucleusinreticulocytes is a critical step in the maturation of red blood cells. Mature red blood cells areenucleated, allowing themto have more space to carry oxygen and increasing their flexibility to navigate through narrow blood vessels. During ontogeny (the development of an organism),erythropoiesis occurs in distinct anatomic sites, such as the yolk sac, liver, and bone marrow, at different stages of development. Each of these sites provides a unique microenvironment that influences the development of erythroid cells. The microenvironment includes interactions with stromal cells, hematopoietic accessory cells, and the extracellular matrix. These interactions play a crucial role in supporting and regulating erythropoiesis - and development is influenced by cytokines. Normal bone marrow E rythroid Precursor Cell Compartment and Cellular Dynamics: Erythropoiesis involves the differentiation and maturation of erythroidprecursorcells,ultimatelyleadingtotheproductionof red blood cells. Gene activity during erythroid maturation is dominated by the expression of globin genes, constituting the majority of protein at the reticulocyte stage (95%). RedCellProductionandCellularDynamics:Intheadultmarrow, approximately3x10^9newredbloodcellsperkilogramperday are produced, accounting for 1% of the total red cell mass. Successful red cell production requires an intact microenvironmentandanadequatesupplyofiron,essentialfor haemoglobin synthesis. aemoglobin Synthesis: Haemoglobin synthesis involves three key components:haemsynthesis,globinsynthesis,andthe H availability of iron. Factors Required for Red Cell Production:Erythropoietin,producedbythekidneyinresponsetooxygenlevels,stimulates redbloodcellproductionANDVitaminB12(cobalamin)andfolicacid(folate)areessentialforDNAsynthesisandarecritical for erythropoiesis. Megaloblastic Anaemia and Vitamin B12 Deficiency: megaloblastic anaemia (perniciousanaemia),characterizedbylarger red blood cells (megaloblasts), can result from vitamin B12deficiency.VitaminB12deficiencyleadstotheinabilitytotrap folate within red blood cells, disrupting DNA synthesis and causinglarger, inefficiently maturing cells. Mechanism of Megaloblastic Anaemia: Vitamin B12 deficiency prevents the trapping of folate within red blood cells, hinderingtheconversionofuridinetothymidine→RBCsattempttoinserturidineintoDNA,disruptingsynthesisandleading to the production of larger megaloblastic cells (additional cycles toattempttocorrecterrors)→RBCS(nowmegaloblastic cells) undergo apoptosis within the bone marrow, resulting in megaloblastic anemia and an elevation in bilirubin due to increased RBC breakdown. Neutrophils: Vitamin B12 deficiency can also affect neutrophils, leading to hypersegmentation (additional lobes) in their nuclei E rythropoietin EPO - growth factor for erythroid development. It has been abusedbyathletesasit increases red cell mass and capacity for RBC to carry O2. It is produced mainly in the kidney by peritubularcells.Haem-containingproteinsensesoxygenneed→synthesisofEPO.InteractionofEPO with receptor-bearing cells → increased red cell production. ormal Shape of Red Blood Cells: N Appropriatelyassembledmembraneproteinswiththecytoplasmiccytoskeletongiveredbloodcellsabiconcavediskshape. This biconcave disk shape allows for a high surface area-to-volume ratio, optimizing the oxygen-carrying capacity of haemoglobin.Transmembranecomplexesembeddedinthelipidbilayercontributetotheflexibilityofredbloodcells,which isessentialfortheirabilitytonavigatethroughnarrowcapillariesandpassthroughthespleen.Thisflexibilitydecreaseswith the aging of red blood cells. Clinically relevant blood group antigens are present on these transmembrane complexes. Red Cell Shapes: The majority of red blood cells are disciform, meaning they are disc-shaped. However, a minority may exhibitabowl-shapedmorphology.Theaveragediameterofredbloodcellsisintherangeof7.2to7.9microns.Redblood cells have acentra
