1
Q
Composition of Human Blood (What does human blood do; overview of components)
A
- Transports substances to tissues:
- O2, nutrients, hormones, leukocytes, red cells, platelets, antibodies
- Carbon dioxide and nitrogenous waste products of cell metabolism to the excretory organs of the body
- Volume of blood: about 5 quarts, but varies by individual
- Half of blood is cellular components suspended in plasma
2
Q
Composition of Human Blood (cellular elements)
A
- CBC= Complete Blood Count
- Cellular elements are:
- RBC: for oxygen delivery
- WBC: for immune defense
- Platelets: clotting
- RBCs: WBCs: Platelets ~=600:1:15
- 2-3x1013 RBC in the body at any time
- About 2x1011 new RBCs are made each day
3
Q
Composition of Human Blood (hemoglobin)
A
- RBCs are filled with hemoglobin (>90% dry weight)
- Hemoglobin binds heme (porphyrin) groups that are complexed with iron (oxygen binding)…can chelate ions
- Hemoglobin A (adult) is a tetramer of 2 alpha, 2 beta
- Hemoglobin F (fetus) is a tetramer of 2 alpha, 2 gamma
- Fetus needs to take oxygen away from maternal blood stream, gamma has higher affinity than beta for oxygen
4
Q
Anemia (Definition, Most common cause)
A
- Deficiency of RBC or hemoglobin blood. Never normal/always sign of a disease
- Most common cause is an insufficient supply of iron, B12/cobalamin or folate
5
Q
Kinetic approach to classifying anemia
A
- Decreased RBC production
- Lack nutrients
- Bone marrow disorders/suppression
- Low levels of trophic hormones (EPO)
- Increased RBC destruction
- Bone marrow unable to keep up with replacing 5% of RBC mass/day
- Blood loss
6
Q
Morphologic approach to classifying anemia
A
- Macrocytic (large RBC) anemia
- Folate or B12 deficiency
- Lack of intrinsic factor (glycoprotein that binds B12 for adsorption in ileum) in autoimmune disease, pernicious anemia
- Drugs that interfere with DNA synthesis (hydroxyurea, zidovudine)
- Microcytic (small RBC) anemia
- Decreased hemoglobin content within the RBC
- Iron deficiency (hypochromic microcytic anemia) most common
- Disorders of heme biosynthesis
- Reduced hemoglobin production: thalassemia
- Normocytic anemia
- Renal disease, cancer
7
Q
Sites of iron, B12 and folic acid absorption in SI
A
- Iron=duodenum
- Folic acid=jejunum
- Cobalamin/B12+intrinsic factor=ileum
8
Q
Iron Absorption
A
- Iron absorption in intestine
- Inorganic iron via DMT1
- Heme iron via HCP1
- Transported to blood by ferroportin or complexed with apoferritin and stored as ferritin
- Iron transported to erythroid precursors for synthesis of hemoglobin
- via transferrin
- transferrin-iron complex binds to transferrin receptors in erythroid precursors and hepatocytes and is internalized
- Release iron and recycled to plasma membrane
- transferrin then released from receptor
- Release iron and recycled to plasma membrane
- Iron storage in liver
- stored as ferritin
- Iron reclaimed from RBC
- By macrophages
- Export it or store it as ferritin
- Heme converted to bilirubin and excreted in bile
- Take up iron and store it as ferritin via ferroxidase
- By macrophages
9
Q
Folate and B12 Metabolism
A
- Folates in diet are polyglutamates
- Enzymatically converted into folate monoglutamates (via folate reductase in jejunum)
- Folic acid is absorbed two-fold better than folates
- Folate needed for synthesis of TMP
- Folate for neural tube development
- B12 methyl donor to convert homocysteine to methionine
- activated to make SAM, which methylates DNA, proteins, metabolites
- B12 released via HCl and proteases in stomach
- Free B12 combines with intrinsic factor (a glycoprotein from stomach’s parietal cells) and results in complex that undergoes absorption within the distal ileum of small intestine
- Pernicious anemia=immune destruction intrinsic factor
- Liver main storage site B12. Can store 3 yrs worth.
10
Q
Hemolytic Anemia
A
Hereditary
- Abnormal shape: hereditary spherocytosis
- Abnormal hemoglobin: hemoglobin S replace A (sickle-cell anemia)
- Defective hemoglobin synthesis: thalassemia minor and major; globulin chains are normal but synthesis is defective
- Enzyme defects: G6PD deficiency predispose to acute episodes of hemolysis (allergy to sulfa, fava, anti-malarial)
- Variation in population; some mild and others severe
11
Q
Sites of hematopoiesis
A
- Fetus:
- Starts in yolk sac and liver
- Adult:
- Primarily in bone marrow
12
Q
Sustaining Hematopoiesis
A
- Sustained by multipotent hematopoietic stem cells
- Stem cells self-renew or commit to one or another hematopoietic lineage by proliferating and differentiating, under the control of cytokines, growth factors, and TF
- Single multipotent SC can repopulate entire hematopoietic system
13
Q
Sequential steps of blood cell development are directed by cytokines
A
Committed stem cell→Cytokine A→Cytokine B→Cytokine C→Differentiated and Functional blood cell
14
Q
The development of T-lymphocytes and red cells
A
- IL=interleukine (general name for hematopoietic growth factors)
- SDF-1 (stromal cell Derived factor)
- FLT-3 Ligand
- SCF (stem cell factor) effects both neural crest and hematopoietic cell development. Binds C0kit tyrosine kinase receptor, mutation of which has near identical phenotype as SCF mutations
- Tpo (thrombopoietin)
- Epo (Erythropoietin)
- GM-CSF (granulocyte macrophage stimulating factor [sargramostim])
- G-CSF (granulocyte stimulating factor [filgrastim])
- Interleukin-11
15
Q
Erythropoietin (EPO)
A
- Stimulates erythroid proliferation and differentiation in red cell progenitors in bone marrow
- Feedback control of RBC production is through EPO
- Necessary to prevent death and promote proliferation of committed precursor
- Shifts non-committed progenitor cells into the erythroid lineage
- Feedback control targets CFU-Es by upregulating their production as well as stabilizing these cells
16
Q
HIF-1 control of EPO expression in response to O2 levels
A
- HIF-1 is a heterodimeric TF that binds hypoxia-response elements (HREs) that are associated with a broad range of transcriptional targets, particularly Epo
- In normoxia, hydroxylation can cause HIF-1alpha to be:
- Degraded by the proteasome
- Blocked from recruiting a transcriptional coactivator, p300
- Prolyl hydroxylase domains (PHDs) downregulate and inactivate HIF subunits
- HIF-1alpha is stabilized in hypoxia and can activate Epo production
- HIF hydroxylases are inactive, allowing transcription to be active
17
Q
JAK-STAT signaling in hematopoiesis
A
- Hematopoietic factors interact with membrane receptors of the cytokine receptor super family
- Binding of cytokines (like EPO) to their receptors (like EPOR) phosphorylates and activates JAK
- Activated JAKs phosphorylates STAT proteins, which dimerize and translocate to the nucleus
- STATs are transcription factors that bind to regulatory elements in the genome to activate gene transcription
- JAK-STAT pathway negatively regulated by:
- SOCS (suppressor of cytokine signaling) proteins
- Protein tyrosine phosphatases
- SH2 domain containing PTP1, SHP2, CD45 and T-cell PTP (TCPTP)
- Ubiquination and degradation of JAK
- PIAS (protein inhibitor of activated STAT)
18
Q
Therapeutic Uses of Erythropoietin
A
- Treat anemia caused by chronic kidney disease
- Target hematocrit 30-36%
- Used previously to treat anemia caused by cancer treatments
- Since 2008, studies shown increased risk of death/tumor growth. Not enough evidence for clinical benefit
- Used previously to treat anemia in critically ill patients
- Didn’t change the # of blood transfusions needed
- Benefit must be weight against the 50% increase in thrombosis
19
Q
Blood Doping
A
- Use in endurance sports
- Pro athletes are already optimized; might not actually see a benefit in them
20
Q
Adverse effects of erythropoietin
A
- Increased risk of HTN in pts with kidney disease if used to rapidly increase Hb to above 13.0 g/dl
- Often to treat anemia in chronic kidney disease, only when Hb levels <10 g/dl
- Recommended caution in using EPO in cancer patients
- Risk of stroke and increased death related to malignancy may outweigh any potential benefits of erythropoietin or darbopoietin in absence of severe anemia
21
Q
Myeloid Growth-Factors: GM-CSF & G-CSF
A
- GM-CSF stimulates the proliferation/differentiation of myeloid cell lineages
- Monocytes, macrophages, neutrophils, eosinophil
- Secreted by macrophages, T-cells, mast cells, endothelial, and fibroblasts
- Recombinant GM-CSF (sargarmostim) is produced in yeast.
- Identical to endogenous GM-CSF except for glycosylation at leucine position 23
- G-CSF enhances phagocytic and cytotoxic activities of neutrophils but has little effect on monocytes, macrophages, and eosinophil
- More specific than GM-CSF and is therefore better tolerated
- Produced by endothelial cells, macrophages, and a number of other immune cells
- Recombinant G-CSF (filgrastim) is produce in E. coli
- Not glycosylated and carries an extra N-terminal methionine
22
Q
Therapeutic Uses of GM-CSF, G-CSF
A
- Primarily used to treat neutropenia
- Low number neutrophils (normally 50-70% circulating WBC)
- Neutropenia observed in:
- Cancer patient after chemo
- AIDS
- Bone marrow transplant
- Used in preparation for autologous stem cell transplant
- Before chemo, HSC are mobilized from bone marrow into peripheral blood by treatment with GM-CSF/G-CSF and collected with apheresis
- Re-infuse HSC after chemo
23
Q
Adverse effects of GM-CSF, G-CSF
A
- GM-CSF can cause fevers, malaise, arthralgias, myalgias, and a capillary leak syndrome characterized by peripheral edema and pleural or pericardial effusions.
- Allergic reaction
- G-CSF is more frequently used because it is more specific than GM-CSF (better tolerated)
- G-CSF can cause bone pain, but this clears when the drug is discontinued
24
Q
Megakaryocyte Growth Factors: IL-11
A
- IL-11 stimulates the proliferation and differentiation of megakaryocyte and platelets
- Modulates antigen/antibody responses
- Recombinant IL-11 (oprelvekin) is produced in E. coli
- Not glycosylated like endogenous form, short by one AA
25
Megakaryocyte Growth Factors: Tpo
* Thrombopoietin stimulates the proliferation and differentiation of megakaryocytes
* Also regulates downstream production of platelets
* Recombinant is **not approved for therapeutic usage**
* Alternatives: Romiplostim (IgG fusion with thrombopoietin fragment), Eltrombopag (Tpo receptor agonist)
26
Therapeutic uses of IL-11
* Treatment of thrombocytopenia
* Normal: 150,000-450,000 per mm3
* \<100,000
* Have a high risk of hemorrhaging
* Thrombocytopenia is often observed in
* **VitB12 or folic acid deficiency**
* **Leukemia or myelodysplastic syndrome**
* Liver failure resulting in Tpo loss
* Dengue fever resulting in infection of megakaryocyte
* Sepsis/systemic viral or bacterial infection
* **Myelosuppresion induced by chemotherapy agents**
* Idiopathic thrombocytopenia purpura
27
Adverse Effects of IL-11
* Platelet transfusion is commonly used to treat thrombocytopenia
* Some patients fail to reach the expected increase in platelet count
* Often a high frequency of fever and anaphylactic reactions
* Recombinant IL-11 can cause:
* Excessive fluid retention with edema and cardiac decompensation
* Renal impairment
* Allergic reactions
28
Summary table of common hematopoietic growth factor in clinic
29
What happens upon infection?
* Integumentary system (skin, mucus membranes, mucus)
* First line of defense
* Physical barrier
* Dead skin cells are constantly sloughed off; makes it hard for invading bacteria to colonize
* Sweat and oils contain anti-microbial chemicals, including some antibiotics.
* Physiological changes in environment
* Regulated pH and temperature (Fever)
* Mucus contains lysozymes
* Destroy bacterial cell walls
* Flow of mucus washes
* Cilia mucus out of the lungs
* Phagocytes are several types of white blood cells
* Macrophages and neutrophils
* Seek and destroy invaders/damaged cell
* Phagocytes attracted by inflammatory response of damaged cells
* Inflammation is signaled by mast cells
* Release histamine
* Fluids collects around an injury to dilute toxins
* Swelling
* Increased temp to kill temperature-sensitive microbes.
* Fever
* Destroy many types of microbes
* Helps fight viral infections by increasing interferon production
* High fevers can be dangerous
* Some doctors recommend letting low fevers run their course
30
Organs of the immune system
* Bone marrow
* Source of blood meeting
* Thymus
* Maturation of T cell
* Spleen
* Upper left of abdomen
* Hematopoietic organ, site of macrophage antigen presentation, antibody response
* Splenic macrophages remove old RBC and acts a reservoir for platelets
* Lymph nodes and vessels
* Lymphatic system is the source of lymphocytes (NK, T/Bcells)
* Clumps of lymphoid tissue especially in gateways to body
* Tonsils, adenoids, and appendix
* Lymphatic vessels closely parallels body’s veins/arteries
* Cells/fluid are exchanged between blood and lymph
31
Cells of the immune system
* Lymphocytes
* B cell
* Antibodies
* T Cell
* Cytokines
* Large granular lymphocyte
* Cytokines
* Phagocytes
* Mononuclear phagocytes
* Cytokines
* Complement
* Neutrophil
* Eosinophil
* Auxiliary Cells
* Basophil
* Inflammatory mediators
* Mast Cell
* Inflammatory mediators
* Platelets
* Inflammatory mediators
* Other
* Tissue Cells
* Interferon
* Cytokines
32
Innate vs. Adaptive Immunity
* Innate Immunity
* Epithelial barriers
* Phagocytes
* Dendritic cells
* Complement
* NK cells, ILCs
* 0-12 hours
* Adaptive Immunity
* B lymphocytes to make Ab
* Start on Day 1, takes about 7 days
* T lymphocytes to effector T-cells
* Start on Day 1, takes about 7 days
33
Comparing Innate vs. Adaptive Immunity (Summary Table)
* Specificity
* Diversity
* Memory
* Noreactivity to self
* Cellular and chemical barriers
* Blood proteins
* Cells
NK T cells and T cells are cytotoxic lymphocytes that straddle the interface of innate and adaptive immunity
34
Innate Immunity:
* Detection of microorganisms and first-line defense
* Physical barriers
* Pattern recognition receptors (TLR)
* Regulation of inflammation
* Swelling, redness, heat and pain
* Serum proteins: Complement system, C-reactive protein, lectins such as mannose-binding lectin and ficolins
* Activation and instruction of adaptive immune response
* Phagocytes (neutrophils, monocytes, macrophage) use lysozyme
* Macrophage, mast cell, NK cells release cytokines and inflammatory mediators
35
Adaptive Immunity:
* Specifically respond to variety of antigens
* Discriminate between foreign antigens and self antigens
* Response to a previously encountered antigen in a learned way by initiating a vigorous memory response
* Causes production of antibodies (humoral immunity), activation of T-cells (cell-mediated immunity)
36
PRRs recognize PAMPs (Innate Immunity)
* Tissue macrophage and dendritic cells express:
* **Pattern Recognition Receptors**
* TLRs
* C-type lectin receptors (CLRs)
* Nucleotide-binding Oligomerization Domain-like receptors (NLRs)
* To detect:
* **Pathogen Associated Molecular Patterns** (evolutionarily conserved)
* Unmethylated CpG DNA
* Flagellin
* Double-stranded RNA
* Peptidoglycans
* **LPS from membranes** (used to assay for sepsis/infections)
* Stimulate release of proinflammatory cytokines, chemokines, and interferons
* Success=ingested, degraded, and eliminated pathogen
* Bacterial endotoxins (LPS from Gram -) dangerous in drug formulations/need to be tested for
37
The Complement Cascade (Innate Immunity)
* Modifies membranes and promotes inflammatory response
* Primary Functions:
* **Chemotaxis**
* C3a/C5a act as chemoattracts to draw in macrophage and neutrophils
* **Opsonization**
* C3b coats bacteria
* **Cell Lysis**
* MAC complex (C5b, C6, C7, C8, C9)
38
Complement Cascade: Membrane Modification
* Complement fragments deposit on unwanted materials
* In \<5min, millions C3b fragments coat
* Opsonization
* MAC
39
Complement Cascade: Promotion of Inflammation
* Release peptides known as anaphylatoxins
* Directed movement of motile cells (chemotaxis and chemokinesis)
* Release of mediators, such as histamine from mast cells
* Activation of cells like epithelial and endothelial cells
* Contraction of smooth muscle
* Dilation of blood vessels
* Exudation of plasma and cells
40
Two Arms of Adaptive Immunity: Humoral immunity
* Microbe: Extracellular microbe
* Responding lymphocytes: B lymphocytes
* Effector mechanism: Secreted antibody
* Transferred by: Serum
* Functions: **Block infections and eliminate extracellular microbes**
41
Two Arms of Adapative Immunity: Cell-Mediated Immunity
* Cell mediated immunity (1)
* Microbe: phagocytosed microbes in macrophage
* Responding lymphocyte: Helper T cell
* Effector Mechanism: Bind to macrophage
* Transferred by: Cells (T lymphocytes)
* Functions: **Activate macrophages to kill phagocytosed microbes**
* Cell mediated immunity (2)
* Microbe: Intracellular microbes (virus) replicating w/i infected cell
* Responding lymphocyte: Cytotoxic T cell
* Effector Mechanism: Bind to infected cell
* Transferred by: Cells (T lymphocytes)
* Functions: **Kill infected cells and eliminate reservoirs of infection**
42
Graph of Adaptive Immunity Response
* Primary exposure to antigen X
* Naïve B cells create a response, peaking around 2 weeks
* Created plasma cells to release serum Ab
* Generate memory B cells at end of response, around 4 weeks
* Secondary exposure to antigen X
* Get a very fast and large antibody response, creating plasma cells
* Memory B cells made at end of response
43
Antibody Structure
* 2 heavy chain/2 light chains
* Variable region formed by V(D)J recombination
* Recognize antigen like lock/key
* Stem of Ab participates in the immune defenses
* Identical in all Ab of the same class
* 5 isotypes
44
V(D)J Recombination
* Mechanism for diverse binding selectivity in the B-cell receptor (antibodies) and T-cell receptors
* Limited genetic info to make million of antibodies
* Genes spliced from DNA in two different chromosomes
* Heavy chain is where the binding of antigens occurs, so much genetic variation is involved in its assembly
* 1 of 400 possible **variable gene segments (V)**
* 1 of 15 **diversity segments (D)**
* 1 of 4 **joining segments (J)**
* 24,000 possible combination for DNA heavy chain
* Light chain is (VJ)
* Occurs in B-cell receptor/antibodies and T-cell receptor
* 107 to 109 antigenic determinant can be discriminated
45
Five Immunoglobulin isotypes
* IgG and IgM in plasma
* IgG in extracellular fluid around body
* IgE in mast cells
* IgM activates complement system
* IgA transported across epithelium
* Fetus get IgG
* Brain has none
46
The Action of Antibodies
* Agglutination
* Activation of complement
* Opsonization
* Marking target cells for destruction by macrophages, eosinophils, NK cells (Ab dependent cellular cytotoxicity)
* Neutralizing bacteria, virus or toxin
* Therapeutic uses (Herceptin/Trastuzumab to treat Her2+ breast cancer)
47
MHC Class I and II: Comparison Table
* Class I
* Comprised of an MHC-encoded alpha chain and a ß2 microglobulin chain
* **Present on most cells**
* **Bind endogenous antigens synthesized in a cell**
* Present antigen to CD8 cytotoxic T cell
* Bind CD8 adhesion molecules on cytotoxic T cells
* **Presence of foreign or over abundant antigens targets cell for destruction**
* Class II
* Comprised of MHC-encoded alpha chain and a ß chain
* **Present only on antigen-presenting cells**
* **Bind exogenous antigens**
* Present antigen to CD4 helper T cell
* Bind CD4 adhesion molecules on helper T cells
* **Presence of foreign antigens induces antibody production, and attracts immune cells to area of infection**
48
MHC Class I
* Present cytoplasm-derived peptides that are representative of the normal components for the same cell or of intraceullar parasites (viruses)
* Degraded in proteasome, peptide transferred to ER, assembled MHC/antigen complex transported to cell surface
* MHC-1 present to CD8 cytotoxic T-cell
* Induce apoptosis or release cytotoxic proteins
* Since virus attack any cell, MHC-1 on al cell (except RBC)
* High levels on APC (dendritic cells, macrophages, B lymphocytes, vascular endothelial)
49
MHC Class II
* Noncovalently associated heterodimer of alpha-beta chain
* Bind peptides from exogenous proteins
* Endocytosed, fragmented, and put on cell surface
* Present to CD4 helper T-cell
* Expressed constitutively only on the surface of interstitial dendritic cells, macrophages, and B cells
* Epithelial/vascular endothelial cell MHC-2 upregulated by proinflammatory cytokines (IL-2, IFN-gamma)
50
MHC and HLA
* Major histocompatibility complex
* Human leukocyte antigen is human version
* HLA important in immune system
* **Involved in the processes of identifying self and non-self**
* Very variable, **barcode for self**
* **Responsible for tissue rejection in organ transplants**
* Genetic inheritance, basis for autoimmune disease
* Certain subtypes associated with disease
* Certain subtypes impact drug efficacy
51
Antibodies and T-cell Receptors are Antigen receptors
* B-cell uses ab-receptor to bind matching antigen, engulfs, processes
* Becomes large plasma cell
* Cannot kill pathogen, only can mark
* Helper T-cell only recognize antigen in MHC II
* Sees antigen in MHCII on APC
* Helper T-cell confirms with CD4 protein
* Becomes activated helper T, aka commander of immune response
* Orders to increase # specific Ab-producing plasma cells and cytotoxic killer cells
* Killer T cells only recognize antigen in MHC I
* Recognizes virus fragments from macrophage
* Binding process and an activated helper T cell activate cytotoxic T-cell
* Goes around and kills diseased cells with same antigen in MHC I
52
Antigen Receptors
* B cell receptor on outer surface and is a sample of antibody it is prepared to manufacture
* T-cell receptor only recognize antigen after is has been processed and presented in MHC
* Ensure that T-cell only act on precise targets at close range
53
T-cell activation (2 required signal steps)
* Signal 1: MHC + TCR
* Dendritic cell present antigen on MHC
* TCR engages MHC + antigen
* Signal 2: Co-stimulatory molecules and receptor
* Activates T Cell
* CD80/86 (APC)→CD28 (T-Cell)
* CD40 (APC)→CD40L (T-Cell)
* ICAM-1 (APC)→LFA-1 (T-cell)
* Negative regulation to shut off T-cell
* CD80/86→CTLA-1 (T-cell)
* If the second signal is absent, T-cell either stops responding (anergic) or undergoes apoptosis
54
Cell mediated and humoral immunity
55
Immunopathology
* Hypersensitivity: allergic reaction to harmless antigens
* Immunodeficiency: unable to stop an infection
* Autoimmune disorder: immune system targets self-antigens
56
Immunosuppression
* Immunosuppressive drugs are used to
* Prevent organ rejection
* Activated T cells are main mediators of immunologic rejection, attack donor organ
* Treat GVHD after allogenic HCT
* Allogenic is genetically similar but could be a total stranger
* Crohn’s or RA
* Asthma
* Allergic rhinitis
* Psoriasis and eczema
57
Mechanisms of action of immunosuppressive drugs
* Main pathways in immunoregulationT cell activation
* MHCII and B7 (CD80/86)→calcineruin→IL-2
* Target of rapamycin and cyclin-dependent kinase
* Steroids and NF-kappaB
* Ca2+ levels, Calcineurin, NFAT, IL-2 synthesis
* Autocrine stimulation by IL-2 to mTOR and cell cycle
* DNA synthesis and proliferation
58
Immunosuppressant- a timeline
* Four stages in development strategies
* Anti-proliferative agents
* Steroid therapy
* Lymphocyte/depletion/modulation
* Disruption of cytokines
* mTOR inhibitor, calcineurin, IL-2 receptor blockade
59
Azathioprine
* Purine analong
* Its active metabolite (6-MeMPN) blocks the enzyme amido-phosphoribosyltransferase
* Hinders DNA synthesis
* **Inhibits the proliferation of cells, especially fast-growing cells without a method of nucleotide salvage**
* **T cells and B cells are particularly affected by the inhibition of purine synthesis**
60
IMP Dehydrogenase Inhibitors
* Mycophenolate mofetil to mycophenolic acid in liver
* MPA inhibits IMPD that control **rate of de novo guanine (purine) synthesis in the proliferation of B and T cells**
* ****IMPDH catalysis NAD-dependent oxidation of IMP to XMP
* Committed step
* Used in place of azathioprine
* Used in three compound regiment
* Calcineurin inhibitor (cyclosporine or tacrolimus)
* Corticosteroid (prednisone)
61
Corticosteroids
* Endogenous steroid hormone
* Cortisol/corticosterone
* Drugs
* Prednisone, hydrocortisone, fluticasone propionate
* Widely prescribed
* Autoimmune
* Severe allergies
* Arthritis
* Atopic dermatitis
* Long term use associated with osteoporosis, metabolic disease and cardiovascular disease
62
Corticosteroids MOA
* Bind to glucocorticoid receptor that binds DNA GRE
* Alters expression of target genes like anti-inflammatory IkBalpha
* GC-GR also interacts with other TF like NF-kappaB, AP1, CREB, STAT to alter gene expression
* **Glucocorticoids repress transcription of many pro-inflammatory cytokines and chemokine, cell adhesion molecules and key enzymes involved in the initiation and/or maintenance of the host inflammatory response**
* Modulates transcription of genes in a hormone-dependent manner through tethering to other DNA-bound TF
* Regulation of these other TF by extracellular signals
* GR ubiquitously expressed in all cells in the airway
* GC-GR can also bind negative GRE and result in repression of transcription
* GRs can recruit histone deactylases to NF-kappaB-driven proinflammatory gene transcription to decrease transcription
* Suppress production of IgG and cytokines
* Inhibit differentiation, migration and antigen-presentation of dendritic cell and activation of TH17 cells
63
Calcineurin Inhibitors
* Cyclosporine and Tacrolimus
* Macrolide rings
* Bind with immunophilins
* Proteins that have cis-trans peptidyl-prolyl isomerase activities
* Act primarily on T-helper cells
* Cyclosporine:
* Shift TH1 to TH2 response in RA
* Increase TGF-beta, causing renal fibrosis
* Tacrolimus:
* Interferes with IgE mediated histamine/serotonin release
* Minimal effects upon amplification of immune response following activation
* Lack myelosuppressive activity
64
Calcineurin Inhibitors: MOA
* Bind to cyclophilins for cyclosporine, FK binding proteins for tacrolimus
* Calcinuerin is activated by intracellular Ca after stimulation of TCR
* Ca/calmodulin dependent phosphatase
* Drug-receptor complex specifically and competitively binds to and inhibits calcineurin
* **Inhibits translocation of TF NF-AT (not deP) into nuclease leading to reduced transcriptional activation of early cytokines genes for IL-2, TNF-alpha, IL-3, IL-4, CD40L, GM-CSF, IFN-gamma.**
65
mTOR inhibitors
* Kinase
* Sirolimus (Rapamycin) and Everolimus
* **Sirolimus and Everolimus inhibit IL-2 mediated signaling and results in cell cycle arrest at G1-S, blocking activation of T and B cells by cytokines**
* Contrast: tacrolimus inhibit synthesis/secretion of the cytokine IL-2
* Sirolimus also binds FK (like tacrolimus), inhibits mTOR by directly bind to mTOR Complex 1
66
mTOR inhibitors MOA
* Bind to FK binding protein and modulate activity of mTOR
* mTOR inhibits IL-2-mediated signaling transduction, resulting in cell-cycle arrest in G1-S phase
* Block the response of T and B cell activation by cytokines
* Prevents cell-cycle progression and proliferation
67
Anti-lymphocytic antibodies
* Muromonab-CD3 (OKT3)
* Binds TCR-CD3 complex leading to activation but then apoptosis of T cell
* Protects transplanted tissues from T cells
* Alemtuzumab (Campath, Campath-1H)
* Ab to CD52 (protein on mature lymphocytes)
* Rituximab (Rituxin)
* Ab to CD20 on surface of B cells
* ADCC
* Antibody dependent cell mediated cytotoxicity
* NK cells lyse target cell whose membrane antigens have been bound by Ab
68
Anti-IL2R antibodies (daclizumab and basiliximab)
* Daclizumab is humanized monoclonal Ab to alpha chain of IL-2 receptor
* Roles in T cell activation/proliferation
* **Kidney transplant rejection**
* **Higher specificity than anti-lymphocyte Ab drugs**
* Dose sparing regimens to lower doses of steroids/CNIs
* Children who receive these are at increased risk for growth impairement, HTN, hyperlipidemia, lymphoproliferative disorders, DM, cosmetic changes
69
Targeting CD80/86
* Belatacept (Nulojix) binds to CD80 and CD86 on APC, blocking costimulation of T cells
* Abatacept is a recombinant fusion protein with CTLA-4 fused to IgG
* Shuts down T-cell activity
* Activated T lymphocytes are the prominent mediator of immunologic rejection
70
Rh(D) Immune Globulin (RhoGam)
* Prevents Rh hemolytic disease of the newborn
* Rh factor is a protein on the surface of RBC. Rh+ is more common
* A primary ab response to a foreign antigen can be blocked in specific ab to that antigen is administered passively at the time of exposure to antigen
* Rh(D) Immune Globulin is concentrated hIgG containing more Ab against Rh(D) antigen of RBC
71
Summary of Immunosuppresant Drugs
72
NSAIDs and Acetaminophen
* **NSAIDs have antipyretic (anti-fever), analgesic (pain-relief) effects, anti-inflammatory, and anti-neoplastic properties**
* **NSAIDs inhibit COX enzymes, prostaglandin-endoperoxide synthases**
* **Acetaminophen acts on the same COX enzyme but blocks peroxidase**
* **Doesn’t help with inflammation**
* **Fever/pain relief by inhibiting prostaglandins in CNS**
* NSAIDs useful in pain caused by inflammation
* Rapidly metabolized by liver, excreted through kidneys, some through biliary excretion
* Most NSAID in body is bound to albumin
73
Several chemical classes of NSAIDs
* Different Cox1/2 selectivity and variable half lives in plasma
* Cox-2 inhibitors have a longer half life in plasma
* NSAIDs can be individual specific
74
Why should you care about Lipids?
* Phospholipid membranes produce 5 levels of **signaling** lipid metabolites
* Ether lipids
* **Eicosanoids (Prostanoid, Leukotrienes, Lipoxin)-inflammation**
* Endocanniboids
* **Lysophospholipids-inflammation and immune regulation**
* Free fatty acids
75
Role of cytosolic phospholipase A2 in the activation of inflammation
1. Phosphorylation of cytoplasmic PLA2 can occur by ligand-receptor signaling and/or increased intracellular Ca2+
2. Calcium binding to the C2 domain of phospho-cPLA2 triggers binding to plasma membrane
3. cPLA2 hydrolyses phospholipids to release AA and lysophospholipids
4. AA converted to prostaglandins by COX
* Inhibited by NSAIDs
5. Arachidonate 5-lipoxygenase (5-LOX) converts AA to leukotriene
76
Prostaglandin-endoperoxidase synthases=COX enzymes
* COX1 is housekeeping expressed in almost all tissues and is cytoprotective
* Maintains prostanoid levels for tissue homeostasis
* COX2 is an immediate-early response gene that is highly induced at sites of inflammation and during tumor progression by cytokines and mitogens
77
Adverse effects of NSAIDs
* **Cardiovascular**
* **Gastrointestinal**
* **Renal**
* CNS, Hematologic, Hepatic, Pulmonary, Skin
78
NSAIDs Dual Insult
* Primary insult: direct acid damage
* Secondary insult: Prostaglandin inhibition
* Causes gastric damage in about 20% of chronic users
* Discontinue or switch to Cox-2 inhibitors
79
Cox-2 Selective Inhibitors
* Coxibs more selective for 2 over 1
* Most withdrawn except for celecoxib because increased risks for thrombotic events
* Improved GI events, but more concern for thrombotic events
80
Aspirin aka Acetylsalicylic Acid
* Irreversible, nonselective cox inhibitors
* Acetylates and inhibits platelet COX (lasts 8-10 days)
* Low dose aspirin reduces colorectal cancer risks
* Aspirin’s effects and respective mechanisms vary with dose
* Low doses: cardioprotective, antithrombic
* Intermediate: pain and fever
* High dose: anti-inflammatory
* Rarely used because of adverse GI effects and hearing loss
81
Daily Aspirin Therapy
* Lowers cancer and heart attack risk?
* Not something that you should start without MD supervision
* Rebound effect, may have to continue on for the rest of life
82
Choice of NSAIDs
* All NSAIDs (including aspirin) are about equally efficacious
* Choice depends on balancing effectiveness, toxicity, and cost
* No single NSAID is right for all patients
83
Rheumatoid Arthritis (RA)
* 70% of join erosions are detectable by radiograph in first 1-2 years of disease onset
* Increased infections, cardiovascular disease, lymphoma, shortened life expectancy
* Cause is unknown but risk factors include:
* Women
* Age \>60 years
* Certain HLA II genotypes/mutations in PTPN22
* Smoking
84
Rheumatoid Arthritis Figure
* Monocytes attracted to joint and differentiate into macrophages/become activated
* Secrete TNF and IL-1
* TNF increases expression of adhesion molecules on endothelial cells (recruit more cells to the joint)
* Induce synovial fibroblasts to express cytokines (such as IL-6), chemokines (such as IL-8), growth factors (GM-CSF) and matrix metalloproteinases (MMPs)
* Contribute to cartilage and bone destruction
* TNF contributes to osteoclast activation and differentiation
* IL-1 mediates cartilage degradation directly by inducing the expression of MMPs by chondrocytes
* Chemokines (MCP1 and IL-8) are also secreted by macrophages and attract more cells into the joint.
85
Disease modifying anti-rheumatic drugs
* DMARDs
* **Methotrexate: First line DMARD**
* Cyclosporine
* DNA/RNA inhibitors:
* Azathioprine: DNA synthesis inhibitor blocks B- and T-cell proliferation, IL-2 secretion
* Mycophenolate Mofetil: DNA synthesis inhibitor blocks B- and T-cell proliferation
* **Leflunomide**: inhibits dihydrorotate dehydrogenase in pyrimidine synthesis and leads to G1 growth arrest. Leads to decreased T-cell proliferation and antibody production by B-cells
* Antibodies/Biologics:
* Abatacept: CTLA-4 fusion binds to CD80/86 and blocks T-cell activation
* Rituximab: Monoclonal Ab targeting apoptosis of CD20 B cells
* Anti-TNFalpha agents
86
Methotrexate
* Lower doses in RA than in chemotherapy
* Inhibits **AICAR transformylase** and thymidylate synthetase
* AICAR competitively inhibits AMP deaminase, leading to accumulation of AMP
* AMP is released and converted extracellularly to **adenosine, which is a potent inhibitor of inflammation**
* Inflammatory functions of neutrophils, macrophages, dendritic cells, and lymphocytes are suppressed
87
TNF-alpha blocking agents
* Second line to MTX
* Prevent signaling to TNFR’s by binding TNF-alpha
* **TNF-alpha normally contributes to osteoclast activation and differentiation (remodeling of bone)**
88
Side Effects of TNF-alpha blockers
* Important for macrophage activation, phagosome activation, differentiation of monocytes into macrophages, recruitment of neutrophils and macrophages, granuloma formation, and maintenance of granuloma integrity
* Acute infusion rxn: anaphylaxis
* Delayed infusion rxn: skin rash, joint pain
* Neutropenia
* Infection: emergence of latent infection (TB)
89
Glucocorticoids
* Like prednisone used in 60-70%
* Into nucleus to repress gene expression of pro-inflammatory genes
* Side effects from long term use: osteoporosis, metabolic disease, cardiovascular disease
* Can be injected into joints
90
Gout
* Build up of urate crystals in joints, bones and soft-tissues
* **Urate is a poorly soluble end product of purine metabolism**
* Hyperuricema is a necessary predisposing factor for gout, but the majority of hyperuricemic patients never develop gout
* Risk Factors:
* Male, old age, ethnicity, obesity, meat/seafood diets, alcohol
91
Pathogenesis for Gout
* Uric acid crystals in joints are taken up by macrophages as a part of innate immune response to urate crystals
* Macrophages produce IL-1beta via intracellular inflammasomes
* IL-1beta helps recruit neutrophils from the bloodstream into the affected joint
* Activation and degranulation of neutrophils contribute to pain associated with an acute gout flare
* ♦♦♦♦♦♦♦♦♦♦♦♦
92
Treatments for Gout
* NSAIDS
* First line treatment
* Colchicine
* Previous first line therapy
* Inhibit microtubule to prevent WBC migration and phagocytosis
* Also causes side effects: nausea, diarrhea, vomiting
* IV admin runs risk of life-threatening SE
* Uricosuric Agents
* Probenecid and Sulfinpyrazone
* Increase uric acid excretion by decreasing readsorption of uric acid in the proximal tubule
* Allopurinol
* Inhibit xanthine oxidase (which produces uric acid)
* Xanthine/hypoxanthine are more soluble
* Standard of care between episodes of gout
93
NSAIDs and Colorectal Cancer
* Risk reduction for colorectal cancer (especially aspirin)
* Induces apoptosis
* COX involved in colonic tumorgenesis
* Potential benefits are outweighed by the risks
* Good screening can prevent/treat colorectal cancer
Phcol 401
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