Exam 2 week 2 Flashcards
Describe components of fibrin and clot formation
**Intrinsic vs extrinsic coagulation system
Identify and understand the tests appropriate for monitoring the different coagulation pathways
- aPT
- PT/PTT
- INR
Describe the fibrinolytic system, its components and regulators
- what is lytic state
BAckground
- Fibrinolytic system restricts clot expansion and degrades fibrin during wound healing
- Plasmin is a nonspecific protease that degrades fibrin, fibrinogen and other clotting factors
- Plasmin action on fibrinogen yields fibrinogen degradation products (FDP) which inhibit further conversion of fibrinogen to fibrin
- Plasminogen & Plasmin bind fibrin at a lysine rich binding site
- (tissue-plasminogen activator (t-PA) is released from endothelial cells at a site of injury, tPA binds to fibrin via lysine binding sites at the amino terminus and activates bound plasminogen 300x faster than it activates circulating plasminogen
Natural regulation of fibrinolysis
- a. Damage releases tissue plasminogen activator (t-PA) from endothelium;
- b. t-PA inactivated by circulating plasminogen activator inhibitor-1 (PAI-1)
- c. α2-antiplasmin binds covalently to circulating plasmin at a lysine rich binding site causing inactivation;
- occurs only with circulating plasmin
- d. LYTIC STATE: circulating plasmin > capacity of α2-antiplasmin
- Ideal thrombolytic drug would degrade only desired thrombi and not old fibrin deposits
Summarize MoA of drugs
Anticoagulants vs antiplatelts vs thrombolytic
ANTICOAGULANTS; inhibit fibrin formation (prevent blood clotting)
Drugs; Heparin, LMW-Heparin, Warfarin, Fondaparinux, Argatroban, Dabigatran
ANTIPLATELETS; inhibit platelet aggregation (you already had a heart attack)
Drugs; Aspirin, Dipyridamole, Clopidogrel, Cangelor, Abciximab& Eptifibatide
THROMBOLYTICS/FIBRINOLYTICS; dissolve formed fibrin clots
Drugs; Streptokinase, Alteplase, Anistreplase& Tenecteplase
Anticoagulant Drug - Heparin
- MoA; what has reversible vs irreversible binding
- Therapeutic uses; can you use in pregnancy? mode of administration? what happens if you give IM?
- Clinical test; what do you monitor?
- Adverse effects
- Toxicity
- Antidotes
- *HEPARIN** (Unfractionated contains molecules with MW 15,000-30,000; mean 12 kDa or 40 monosaccharide units; fractionation due to action of endo–D-glucuronidase)
1. Mechanism of action - a. Accelerates (1000 fold) inactivation by Antithrombin III (AT-III) of intrinsic and common pathways including: thrombin (II), IXa, Xa
- b. Thrombin, IXa, Xa bind irreversibly to Arg-Ser site on AT-III
- c. Heparin binding to AT-III is reversible; heparin binding site is specific pentasaccharide sequence that contains a 3-O-sulfated glucosamine residue which recognizes AT-III require minimum of 18 monosaccharide units to bind AT-III and thrombin
- Therapeutic Use
- a. Anticoagulant activity In vivo (when injected into body)
- b. Anticoagulant activity In vitro (when added to blood in test tube)
- c. Venous thrombosis
- d. Pulmonary embolism
- e. Patency of IV cannulas
- f. Anticoagulant in pregnancy, (discontinue 24 h prior to induction of labor)
- g. Administer by injection, Large polar molecule, not absorbed orally
- -Immediate effects if given IV
- -Onset delayed onset (1-2 hr) with s.c.
- -Intramuscular Contraindicated (induces painful hematoma)
- Other effect Lipid clearing effect heparin activates lipoprotein lipase (cleave TG from VLDL)
- Monitoring heparin
- -Activated partial thromboplastin time (aPTT), monitors common and intrinsic pathway
- antithrombin III has poor activity against coagulation factor VII (PT time, extrinsic pathway
- Adverse effects
- a. Bleeding
- 1) Thrombocytopenia Type I (HIT-I) – nonimmune occurs within 2 days of initiating therapy. Mediated by platelet –heparin interaction
- 2) Type II (HIT-II) – immune mediated, more severe. Heparin therapy needed for 5-10 days. Antibodies form against the heparin-platelet factor 4 complex and bind on platelet surface causing aggregation.
- b. Osteoporosis if given for more than 6 months
6. Treatment of excess hemorrhage
- a. Administer plasma or blood containing coagulation factors
- b. Protamine sulfate: Heparin-protamine complex cannot bind to AT-III so therby disable anticoagulant activity. DO NOT USE in protamine allergy (diabetics, NPH insulin); FISH ALLERGY - DO NOT USE
7. Contraindications
- -Bleeding disorder
- -Pre-existing bleeding sites
Anticoagulant Drug - LOW MOLECULAR WEIGHT HEPARIN (ENOXAPARIN, DALTEPARIN and TINZAPARIN
- MoA; specific for what factor?
- Therapeutic uses (can you use in pregnancy?)
- Contraindication and adverse effects
- advatages over heparin
Description and Mechanism of Action
- a) Low molecular weight heparins fractionated from heparin MW 1000-10,000 daltons; mean, 4500 daltons, or 15 monosaccharide units
- b) Higher specificity for Enhanced Antithrombin III inactivation of Xa; >5400 kDa or 18 monosaccharide units required to bind simultaneously AT-III and thrombin
- Use
- a) Prophylaxis and Acute Deep vein thrombosis, Pulmonary embolism, Orthopedic, Abdominal surgery
- b) Unstable angina or non-Q-wave MI
- c) Administered sub-cutaneous
- d) Monitor anti-Xa activity
- e) Anticoagulant in pregnant women (discontinue 24 h prior to induction of labor)
- Contraindications and adverse effects
- a) Bleeding
- b) Contraindicated in presence of bleeding disorder or active bleeding site
- Current perception of advantages for unfractionated heparin
- Longer interval between doses (outpatient, once a day dosing)
- Slight increase or no change in activated partial thromboplastin time
- Does not require monitoring; could monitor anti-Xa activity
- Better predictability of response to a given dose
- Less thrombocytopenia, reduced binding to platelets
Drug - Synthetic pentasaccharide binds to ATII to accelerate only Factor Xa inactivation
- MoA
- Use (pregnancy?)
- Adverse effect
- Contraindications
FONDAPARINUX
- Mechanism of Action
* -Synthetic pentasaccharide binds to ATII to accelerate only Factor Xa inactivation - Use and administration (mostly SC, IV for acute coronary syndrome)
- -deep vein thrombosis acute and postoperative (hip or knee replacement)
- -pulmonary embolism
- -can be used in pregnancy (discontinue 24 h prior to induction of labor)
- Adverse effect BLEEDING
- Contraindications
- active bleeding
- severe renal impairment (<30 ml/min)
Identify oral anticoagulant
- Only works in vivo
- Inhibits hepatic synthesis of biologically active Vitamin K-dependent clotting factors, Protein C and S
- What are the 4 coagulation factors this drug inhibits
- Use?
- how to monitor levels?
- Adverse reaction and contraindication
- genetics
- Treatment of overdose
WARFARIN
Therapeutic Use
- a) Warfarin, drug of choice for oral anticoagulant
- b) Prophylaxis to prevent Venous thromboembolism and pulmonary embolism
- c) Used in individuals with Prosthetic Heart Valves
- d) Arterial thromboembolism prophylaxis in atrial fibrillation
- e) Delayed therapeutic effect, initial effect occurs in 24 hr and maximal effect takes 5- 7 days
- f) Warfarin is effective as an anticoagulant only when administered in vivo
- Monitoring Warfarin
- a. INR laboratory standardized Quick one-stage prothrombin time
- -citrate plasma incubated with tissue thromboplastin & calcium
- -rate of fibrin generation is dependent on Factors I,II, V, VII and X
- -Monitor extrinsic pathway with prothrombin time
-
b. Difference in thromboplastin sources and/or lots cause inter and intra laboratory variability
- FYI INR International Normalized Ratio standardizes reagent thromboplastin to an International Reference Preparation (IRP) of thromboplastin
- c. Goal for INR is 2.0-3.0 prophylactic for heart valves INR 2.5-3.0
4. Adverse Rxns and Contraindications
- a. Hemorrhage
-
b. Adverse rxns more likely to occur if:
- 1) changes occur in absorption or metabolism of warfarin or Vitamin K
- 2) alterations occur in synthesis or catabolism of coagulation factors
- 3) change in fibrin degradation
- 4) change in platelet function or number
-
c. Genetic predisposition
- 1) Genetic variants of CYP2C9 and VKORC1 genes use less warfarin
- -CYP2C9 CYP2C9*2 (30%↓) CYP2C9*3 (90%↓) 10%Caucasian 2% African/Asian
- -VKORC1 A allele synthesize less VKORC1 carried by 37% Caucasian 14%African
- 2) FDA approved pharmacogenetic test in 2007
- 1) Genetic variants of CYP2C9 and VKORC1 genes use less warfarin
- d. Contraindicated in patients with bleeding disorder or existing bleeding site
- e. Contraindicated in pregnant women (Pregnancy Category X)
- congenital abnormalities occur if fetus exposed first, second or third trimester
- greater incidence of neonatal and fetal hemorrhage
- Treatment of Overdose
- a. Administer whole blood or plasma
- b. Administer Vitamin K1– (phytonadione)
Identify drugs that increase vs decrease warfarin response
Drugs or Conditions that Increase Warfarin Response
- Vitamin K deficiency
- Aspirin (decrease platelet aggregation)
- Hepatic disease
- Erythromycin
- Thyroid hormones
- Cephalosporins
- Cimetidine
- Ketoconazole
Drugs or Conditions that Decrease Warfarin Response
- Cholestyramine
- Rifampin
- Oral Contraceptives (estrogen/progesterone combination only)
- Excess ingestion of Vitamin K enriched foods
Identify 2 direct thrombin inhibitors
- MoA
- IV vs oral administration
- side effects and drug interactions
- which has no P450 interaction
- which has no antidote
DIRECT THROMBIN INHIBITORS (ARGATROBAN and DABIGATRAN ETEXILATE)
- Mechanism of Action
- -Both are Direct thrombin inhibitors, blocks active site of thrombin
- -Active on free and fibrin bound thrombin (Action independent of ATIII)
- ARGATROBAN
- -IV administration
- -Treatment and prophylaxis in thrombosis with heparin induced thrombocytopenia
- -monitor with aPTT
- DABIGATRAN
- -Oral administration
- -Prodrug converted by esterases to Dabigatran (active)
- -Converted to 4 Different Glucuronides (active)
- -NOT A P450 Substrate (so fewer drug interactions)
- -Substrate for P-glycoprotein transport, drug interactions rifampin induces P-glycoprotein decrease dabigatran blood levels
- -Venous thrombosis prophylaxis
- -Prevent stroke with atrial fibrillation
- -Use with caution in diminished renal excretion (NO ANTIDOTE)
- Side Effects and Drug Interactions
- -Bleeding, no available antidote
- -P-glycoprotein substrate
Antiplatelet drugs
- goal of therapy
- how are they used?
- examples
ANTIPLATELET DRUGS
A. Background
- The goal of therapy is to diminish platelet function. These drugs are used primarily in Arterial Thrombotic Disease such as:
- 1) transient ischemic attacks;
- 2) unstable angina
- 3) history of Myocardial infarction
- Examples
- Aspirin
- Dipyridamole
- Clopidogrel
- cangelor
- Abciximab
- Eptifibatide
Antiplatelet drug -
- MoA
- Adverse effects
*
ASPIRIN
Mechanism of Action
- a. Inhibit platelet aggregation
- b. Irreversible inhibitor of cyclo-oxygenase (acetylates the enzyme)
- c. Platelet prostaglandin formation (TXA2) inhibited by 160 mg/day aspirin
Adverse effects
- a. GI bleeding, pain and peptic ulcer
- b. Bleeding
Identify antiplatelet drug
Mechanism of Action
-Inhibits phosphodiesterase (↑ platelet cAMP)
Therapeutic Use
- Used with other agents, very little benefit if given alone
- Given with Warfarin for prevention of thromboembolism in patients with prosthetic heart valves
DIPYRIDAMOLE
Identify antiplatelet drug
P2Y12 receotor inhibitor (3)
- MoA
- Use
- Major adverse effects
Mechanism of Action
- a. ADP binding at the purinergic P2Y12 receptor. ADP release stimulates platelet purinergic receptors P2Y1 and P2Y12. P2Y1 activation causes TXA2 release, increase PI hydrolysis and a rise in intracellular Ca. P2Y12 activation inhibits cAMP formation. A full response to ADP requires activity of both P2Y1 and P2Y12 receptors. Inhibition of either P2Y1 or P2Y12 receptors is sufficient to impair ADP action on platelets.
- b. Clopidogrel prodrug
- c. Clopidrogrel thiol metabolite is irreversible inhibitor of P2Y12 receptor.
- d. Cangrelor is a reversible inhibitor, IV administration
- Therapeutic Use
a. Clopidogrel
- -Reduce risk of thrombotic events in predisposed individuals
- -Clopidogrel conversion to active mediated by CYP2C19
- -Normal Platelet function returns when drug is stopped after new platelets are made (7-14 days)
b. Clopidogrel
* -Reduce Thrombotic events following MI, stroke, unstable angina or peripheral arterial disease
c. Cangrelor IV administration
- -half-life 3-6 minutes, quickly dephosphorylated to inactive metabolite
- -therapy until oral agent or during surgery
- Major adverse effects
- a) Bleeding due to diminished platelet
- b) Thrombocytopenia purpura
antiplatelet drug
Glycoprotein IIB/IIIA inhibitors
- MoA
- Use
- adverse effects
- contraindiactaion
GLYCOPROTEIN IIB/IIIA INHIBITORS ABCIXIMAB, EPTIFIBATIDE & TIROFIBAN
- *Platelet Glycoprotein IIb/IIIa Receptor Antagonists
- Bind to platelet IIb/IIIa receptor to prevent platelet aggregation And crosslinking with fibrinogen**
- Abciximab Monoclonal antibody Fab fragment and Eptifibatide (peptide)
- Tirofiban nonpeptide inhibitor
2. USE - Acute coronary syndrome (unstable angina)
- Prevent acute Adjunct with Angioplasty
- Administered IV (bolus plus infusion)
3. Adverse effects - Bleeding
- Increased bruising
4. Contraindications - History of hemorrhagic stroke
- Active internal bleeding or GI/genitourinary bleeding in past 6 weeks
- Thrombocytopenia
- Major surgery or trauma in past 6 weeks
-Cannot use concurrent with warfarin
Fibrinolytic drug (Use, adverse effect, contraindications)
- Protein derived from beta-hemolytic streptococci; antigenic
- Mechanism of action
a. Complexes stoichiometrically (1:1) with plasminogen
b. Conformational change of plasminogen exposes catalytic site for conversion of a second
plasminogen molecule to plasmin c. Acts on fibrin bound and circulating plasminogen; lytic state may occur
Lytic state: circulating plasmin > capacity of α2-antiplasmin
STREPTOKINASE
Use
- a. Reperfusion of occluded coronaries following acute MI
- b. Pulmonary emoblism
- c. Arterial thrombosis
- Adverse effects
- a. High antigenic activity; fever occurs in 33% of patients
- b. Bleeding
- -lysis of fibrin at sites of vascular injury
- -systemic lysis of fibrin, fibrinogen and clotting factors
- -provide plasma to replenish clotting factors and fibrin
- Contraindications
- a. Surgery or trauma in past 10 days
- b. Pre-existing bleeding disorder or episode
- c. Intracranial trauma
- d. Diastolic blood pressure >110
Identify fibrinolytic
- Streptokinase complexed with human lys-plasminogen. The active catalytic center is blocked by
an acyl group. - Designed to provide more specific binding to thrombi
- Acyl group removed by plasma enzymes
- Similar uses and side effects to streptokinase
ANTISTREPLASE
Identify fibrinolytic
MoA
- a. Activates fibrin bound plasminogen more selectively than circulating plasminogen
- b. Lytic state is less marked than observed with streptokinase
- Therapeutic Use
- -reperfusion of coronary arteries in acute MI
- -pulmonary embolism
- -thrombotic stroke
ALTEPLASE Reteplase; TISSUE-TYPE PLASMINOGEN ACTIVATOR (rt -PA)
not as effective
Identify fibrinolytic
- Genetically engineered derivative of alteplase
- (compared to alteplase), this drug has longer half-life, greater fibrin specificity than alteplase and slower inactivation by PAI-1
- Approved for MI
TENECTEPLASE
Identify fibrolytic INHIBITOR
- Inhibitor of Plasminogen activation
- Lysine analog, compete for lysine binding site on plasminogen and plasmin
**AMINOCAPROIC ACID**
- Inhibit fibrinolysis
- inhibit interaction of palsmin and fibrin
1) A 25 year old pregnant female develops deep vein thrombosis.
- What agent can cause teratogenicity?
- What agent would you prescribe?
2) A 63 year old male underwent mitral valve replacement 1 year ago and has been successfully regulated on warfarin for the past 8 months. He develops a sore throat and upper respiratory infection. The patient is placed on a 10 course of erythromycin for his infection.
- effect of erythromycin on INR
- Should additional tests be done to monitor the effectiveness of the warfarin?
.3. what induce fever
1.
- Warfarin - cause teratogenicity
- Heparin should be prescribed in DVT
2.
- Increase INR ( erythromycin increase warfarin response)
- Clearance of eythromycin will increase?
3. Streptokinase - cause fever
- Describe clinical entities associated with Epstein Barr Virus (EBV) infection
- EBV initiate infections by infecting cells of the respiratory system and regional lymph nodes associated with therespiratory system.
- EB virus causes a primary infection in the RS and then spread to secondary sites of infection, which leads to the obvious signs and disease symptoms. These viruses infect and are transported by lymphocytes. EBV, for example, infects cells of the B cell lineage. HHV 6,7 and 8 will also be briefly discussed
- Herpesviridae: Subfamily Gammaherpesvirina
- α = HSV, VZ; β =CMV; Gamma = EB
- Double-stranded DNA wound around protein core
- Icosahedral capsid composed of capsomers
- Envelope with glycoprotein spikes on its surface
Properties of EB virus
- Morphologically same as other herpes viruses, Smaller size DNA genome
- Virus replication
- Found in lymphoblastoid cell lines,
- In vivo infects primarily B cells and certain types of epithelial cells in nasopharynx that are CD21 (complement factor C3d) positive cells
- CD 21 acts as receptor for EBV on B cells
- Also binds to MHC II
-
Primary viral antigens found in EBV infected cells; EBNA - DNA binding proteins, VCA - capsid,MA - membrane, EA- early Ags,(EA- R and EA- D)
- Ags not related to other herpesviruses
- In total EBV genome can encode over 70 viral proteins
- Non-structural virus proteins: EB Nuclear Antigens-EBNA 1,2,3A, 3B and 3C; Latent Proteins (LP), Latent Membrane Proteins( LMPs)1 and 2
- Note: In addition to EBNA (see above) LPs are DNA binding proteins
- Small viral RNAs (EBER) 1 and 2; detected by in situ hybridization techniques, very senstive approach
- Host Range – limited, humans and some non-human primate
Know the three types of EBV infections as it relates to different cells
**which is most common worlwide
EB (Epstein-Barr) Virus, 2 subtypes, Type 1 most common world wide
EBV interactions with cells
-
Permissive cells such B cells and types of epithelia cells support EBV replication, Permissive cells (non-memory resting B cells in tonsils).
- Immediate early, early and late pahses (gp350/220 are glycoproteins) of cell cycle
- Latent infection of memory B cells when competent T cells are present
- Immortalization of B cells, enhance proliferation, especially B cells in tissue culture
Describe in detail the interactions of EBV with cells
1) Replication of EBV in permissive cells (non-memory, resting B cells in tonsils):
EBV infection can cause the synthesize up to 70 viral proteins. Immediate early, early and late phases of the cell cycle. (See previous herpesvirus lecture)
Late proteins gp350/220 are glycoproteins that are located in the viral envelop enable attachment of the to specific receptors on the surface of cells (CD21). B Cell entry via fusion, gp42 interacts with MHC class II molecules to promote fusion.
EBV binding to CD 21 initiates the activation and replication of B cells. Expression of certain viral proteins (see full listing above) help maintain the infectious process. Virus replication occurs and progeny virus particles are produced to can be found in high amounts in the infected patient’s saliva. Lytic infection initiates with the immediate early protein, ZEBRA, acting as a transcription factor that furthers the transcription of immediate early genes that then stimulates early gene transcription. Following viral genome synthesis in the nucleus of the infected cell late capsid and envelop proteins are synthesized. Progeny virus is then formed. (The more detailed replication of herpesviruses was covered previously)
2) Latent infections require a different type of EBV and B cell interactions. The lytic infection (in permissive cells) can give rise to latent infections depending on the differentiation state of B cells. Memory B cells are conducive to the formation of latent infections.
In latent infections viral genomes circularize and resemble a plasmid. This form of viral DNA replicates in tandem with cell division via cell DNA polymerase. EBNA-1 is important in maintaining latency
Therefore, only specific genes are expressed from the circular genome.
Viral antigens include EB Nuclear Antigen-1, and latent
proteins (LPs), both EBNA and LPs are DNA binding proteins).
3) B cell immortalization: Latent Membrane proteins 3(LMPs) stimulate the replication of B cells and promote their immortalization.
Clinical features of EB virus
- cell type
- primary symptoms
Disease - Infection at early age –no disease, Delayed exposure to virus until adolescence or adulthood causes more severe disease, ie IM
Virus found in saliva, eg. spread by kissing; replication in regional lymph nodes and epithelial cells in the respiratory system
Infected B lymphocytes transmit virus to other lymph nodes and spleen
- Atypical lymphocytes (Downey Cells) in blood correspond to large T lymphocyte that form in response to EBV infected B cells
- Some disease symptoms associated with IM result from the immunological attack by T lymphocytesagainst EBV infected B lymphocytes, cytokine production is present
- Synthesis of a IL-10 analog by EBV during the infection thwarts the TH1 CD4 T cell immunological response
three (3) primary symptoms
- lymphadenopathy (enlarged lymph nodes/glands),
- splenomegaly (enlarged spleen),and
- exudative pharyngitis
- plus headache, chills, high fever, malaise
Understand the significance of heterophile antibodies and the use of the “monospot test
Distinguish between EBV and CMV mononucleosis
Immune Response: Several responses
-
Heterophile antibodies - short lived ab - not virus specific; infected B cells differentiate and produce polyclonal antibodies some of which are heterophile and autoantibodies
- Heterophile Antibodies agglutinate RBCs of non human origin, horse or sheep RBCs
- 60-80% positive sera for heterophile Ab
- Specific antibodies to EB virus proteins
- T lymphocyte response to EBV infected B cells
Lab diagnosis of EB virus
1) Atypical lymphocytes (T lymphocytes= Downey cells), large, basophilic, foamy (vacuolated) cytoplasm
2) Heterophile antibodies (IgM) - “monospot test” - patients serum is treated with GP-RBC to remove nonspecific factors ——> Horse (sheep) RBC——-> Agglutination = + test;
- some sera from patients are heterophile - negative, must differentiate from heterophile negative CMV mononucleosis,
- use antibody detection to VCA (IgM),EA, MA, EBNA
3) Best: virus neutralization, immunofluoresence, CF for viral antibodies
4) Time course VCA (IgM) —> VCA (IgG) —-> EA + EBNA Abs,
antibodies peaks few weeks after infection - last for years
antibodies for EBNA in combination with VCA (IgG) indicate a past infection
EB virus
- Epidemiology and control
- Treatment
- vaccine
- Epidemiology and control
- 90% of adults have antibody
- Spread orally - large dose required in saliva “Kissing Disease”, share toothbrush etc
- Common in institutions and colleges, asymptomatic primary disease of young children, more severe primary infection for adolescence and adults(college students)
- Treatment – symptomatic, limited approval antiviral drugs; Valacyclovir for severe cases + steroids
- Vaccine: Experimental subunit vaccine
What 3 conditions can EB virus cause
EBV May act as a cofactor rather than the sole cause of malignancies
- Infectious mononucleosis
- Associtaed with Burkitt’s lymphoma (Africa);
- Geographical area (africa also where there is malaria)
- activation of MYC protooncogenes may cause lymphoma
- Development of lymphoblastoid cell lines that grow in tissue culture - EB viral DNA exist as plasmid in cells from EB patient and imparts increased cell growth
- Associated with nasopharyngeal carcinoma (china)
Predilection for B lymphocytes
- Possibly Burkitts lymphoma and other types of lymphomas,Hodgkins Infectious mononucleosis (heterophile antibody +)nasopharyngeal carcinoma, gastric cancers,oral hairy leukoplakia in AIDS patients, autoimmune disease links
What is the role of EBV in initiating various cancers?
nasopharyngeal carcinoma vs chronic EBV mononucleosis
EBV May act as a cofactor rather than the sole cause of malignancies
1) Nasopharyngeal carcinoma
- Nonlymphoid cells, EB genome present in epithelial cells
- Males of Chinese extraction Lymphoproliferative disease
- If T cell deficiency exists in patient, EBV induces B cell leukemia-like or lymphoma may develop. Shows the importance of T cells keeping EBV infected B cell proliferation in check.
2) Chronic EBV mono - possible association
Fatigue, fever, headaches, mental lapses, numbness and depression, symptoms last months to years
- Chronic mononucleosis or chronic viral fatigue syndrome most common, different symptoms per patient, fatigue is common, woman 2X affected compared to men, 1 year average duration
- Other possible causative agents include Human herpesvirus 6 or a retrovirus
- what viruses cause roseola vs karposi sarcoma
Roseola is caused by HHV 6 and 7 while Kaposi’s Sarcoma involves HHV 8
1) Human herpes virus 6(HHV6B); not antigenically related to other herpesviruses:
- Causes Exanthem subitum (roseola) and infect large numbers of children.
- Infects B and T cell (lymphotropic) and can infect epithelial and endothelia cells as well as neurons.
- Cause latent infections in T cells and monocytes and can reactivate in transplant patients and immunosuppressed patients.
- Found at high levels in saliva as a result of replication in the salivary glands
2) HHV 6 and 7 isolates also causes roseola which is characterized by a high fever (103-105 F)and a disseminated rash (trunk and face first and then spreads) follows as the fever resides, rash last 24 to 48 hrs
3) HHV 8 has been associated with Kaposi sarcoma and a rare type of B cell lymphoma. B cells are primarily infected by HHV8 but other cell types can be infected. HHV8 encodes proteins with growth factor characteristics and ability to slow apoptosis of infected and uninfected cells, eg. Interleukin-6 homolog
- Kaposi sarcoma found at elevated levels in AIDS patients.
Understand the structural features of human immunodeficiency virus and how its genome RNA differs in the number and types of genes compared to retroviruses discussed previously, ie. Chronic leukemia viruses and acute leukemia/sarcoma viruses.
HIV - Virus Structure – basically similar to other retroviruses, especially other Lentiviruses.
- It is considered to be a complex retrovirus and has genome that possesses genes in addition to gag,pol and env genes, which found in the genomes of the simpler retroviruses. The additional genes are TAT, REV, VIF, NEF and VPU, and VPR, all of which encode accessory proteins that seem to play a regulatory role during HIV infection.
Review: GAG proteins are the core and capsid structural proteins
- POL proteins are part of a enzymatic complex which includes reverse transcriptase, DNA polymerase, RNAse H, protease and integrase
- ENV proteins constitute the spikes on the envelop of the virus, gp 160 = gp120 + gp 41
Identify accessory genes that encode accessory proteins in HIV
- transcription transactivator
- Regulates transport of viral mRNAs from nucleus to cytoplasm
- key component in enabling a limited HIV infection to progress to full blown HIV replication.
- blocks cellular protein (APOBEC-3G)
- enhance release of virus from infeceted cell
- arrect infected cells in G2 phase - enhance replication?
Accessory Genes encode Accessory Proteins
- TAT = Transcription Transactivator for both viral and cellular control regions of genes, ( Remember from our previous discussion of retroviruses that the LTRs located at each end of the provirus has promoters and enhancers that bind regulatory proteins such as TAT)
- REV = Regulates transport of viral mRNAs out of the nucleus and into the cytoplasm, also regulates splicing of viral RNAs that leads to the formation of viral mRNAs
- NEF = Diminishes the expression of CD4 and MHC 1 which changes T cell signaling and is required to achieve a high virus load. NEF is a key component in enabling a limited HIV infection to progress to full blown HIV replication.
- VIF promotes virus assembly and blocks the action of a cellular protein (APOBEC-3G) that normally acts as an antiviral protein
- VPU enhances the release of virus from infected cells and inhibits the cell surface expression of CD4 on infected cells
- VPR arrests infected cells in G2 phase of cell cycle, which is optimal phase for HIV replication and enhances the transport of proviral DNA (provirus) from the cytoplasm into the nucleus
HIV structure - components and functions
- fusion protein?
- what are the major antigens of the protein?
- Genomic RNA exists in two identical copies in direct association with two proteins; p 9, p 7.
- Genome RNA is housed within a cylindrical core composed of p24 - reverse transcriptase is located inside core.
- An outer envelope surrounds core. Underlying the envelope is a layer of p 17 protein.
- The envelope possesses glycoprotein spikes extending outward from its surface. Each spike is composed of 3 or 4 identical polypeptides, each polypeptide is designated gp 160. (glycoprotein 160,000 mw)
- Each gp 160 is usually proteolytically cleaved into two subunits; distal gp 120 (gp 120,000 mw) and proximal gp41.
- gp 120 and gp 41 are held together by noncovalent bonds.
- gp 160 and gp 120-gp41 are the major antigens of the virus, which change antigenically within a given population or even in a particular individual infected with the virus (see M tropic àT tropic HIV above)
- gp 120 important for attachment of virus to receptor on cell surface. gp41 possess amino acid sequence necessary for viral envelope fusion with cell membranes or fusion between an infected cell and uninfected cells. (Remember the HA protein of influenza virus and its role in cell membrane- envelop fusion)
Know the various stages of HIV replication and the role that accessory genes play in the replication cycle.
HIV provirus: Diagram below is not drawn to scale and represents the proviral form derived from the viral genome.
LTR = Long terminal repeat located at each end of the provirus
Note: TAT and REV mRNA are synthesized as a result of multiple splicing events. Segments 1 + 2 + 3 = mRNA; 1,2, and 3 represent different sequences for TAT and REV
Recombination (copy choice) between two copies of genomic
RNA is a source of genetic variation in addition to variations caused by point mutations. Reverse transcriptase can
jump from on 35S RNA to another to yield the final form of the provirus. May be a mechanism to repair breaks in the template genomic RNAs when it is copied into a provirus.
Recombinational changes assume that the two genomic copies within a virus vary in ribonucleotide sequence and are not the same.
Understand presumptive and confirmatory diagnostic tests for AIDS. Understand the time course for the development of AIDS and the progression from the initial infection to the disease AIDS
- HIV infection diagnostics: Detection of HIV specific antigens and antibodie__s specific for HIV proteins; screened in human serum.
- CDC/APHL algorithm which includes antigen-antibody combination assay as part of a screening process to detect HIV infections; 1 out of 1,000 to 3000 yield false positive results
A few examples: Abbott Architect HIV Ag/Ab Combo, Bio-Rad GS HIV Combo, Siemens Advia Centaur HIV AG/Ab Combo, etc. Based on immunoassays
- If combo test is positive an antibody test can differentiate between HIV-1 and HIV-2, Again an immunoassay, Bio-RAD Geenius HIV 1/2 Assay
- If discrepant results are found between initial antigen- antibody screen and the HIV 1/2 Assay then a nucleic acid test, Hologic Aptima HIV-1RNA Qualitative Assay, is employed
Positive RNA test indicates acute HIV infection
- Nucleic acid testing is based on Reverse Transcriptase (RT) PCR is used to determine the HIV load in blood. This is reported in RNA copies per ml and is used as an indicator of the extent of HIV replication.
- HIV genomes can be sequenced to detect specific mutations in viral genes that encode key enzymes, such as Reverse Transcriptase. The mutations have been shown to impart drug resistance to HIV, such as resistance to reverse trans- scriptase inhibitors.
- What is confirmatory test for HIV?
- Presumptive test?
- Confirmatory test (picture below); Test for antibodies for presence of HIV viral proteins
- Detection of HIV specific antigens and antibodie__s specific for HIV proteins; screened in human serum.
- Presumptive test
- OraQuick Advance HIV (1/2); fingerstick whole blood, oral fluid; moderate complexity with plasma. Result in 20 minutes. Screens for HIV 1 and HIV 2. Store at room temp.
- Collect oral fluid specimens by swabbing gums with test device
