Molecular Disease Mechanisms 1 Molecular Disease Mechanisms 2 Adaptive Immunity

A Few Notes Hematopoiesis

Molecular Disease Mechanisms 3 Adaptive Immunity Molecular Disease Mechanisms 4 Adaptive Immunity

Innate vs. Adaptive Immunity Cardinal Features of Adaptive Immune Response Main Stages of B Cell Development 2 Antwort 1 Antwort

• production of 1011 cells per day Hi there, fellow biologist!

I made these cards for the exam of the spring semester 2018 and thought I could improve my karma a bit by sharing them. They are probably not com- plete/won’t entirely cover the next iteration of the course but they should be hella helpful. If you are wondering how i made these beauties, it’s all made with LATEX- which I would highly recommend you to take a look at. And if you want to expand or change them or just for telling me what a wonderful person I am for sharing my hard work, send me an email at [email protected] and I’ll send you the original LATEXfiles

Cheers and best of luck,

Pia

PS: They have a lot of typos, sue me.

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• Specificity: mediated by specific receptors expressed on B cells (BcR) and Innate Immunity Adaptive Immunity T cells (TcR) lag time between exposure and max- immediate response (min, h) imal response (+/- 7 days) • Diversity: B and T cell receptors have great variability due to DNA rear- limited specificity - can distinguish highly antigen-specific rangements between different types of pathogens exposure results in no immunologic exposure results in immunologic • Clonal Expansion: B and T cells that recognize antigen become acitvated memory - no difference between pri- memory - secondary response faster and proliferate mary and secondary response and stronger • Self limitation: response wanes elimination of antigen

• Memory: second response is faster and stronger than first

• Self/Non-Self Discrimination Molecular Disease Mechanisms 5 Adaptive Immunity Molecular Disease Mechanisms 6 Adaptive Immunity

Development of B cell after antigen encounter Recogniton of Antigens by T and B cells & Clonal Selection & Epitopes

Molecular Disease Mechanisms 7 Adaptive Immunity Molecular Disease Mechanisms 8 Adaptive Immunity

Antigen Presenting Cells (APCs) - & TcR MHC I & MHC II Protein Composotion 6 Antwort 5 Antwort

• B cells recognize epitopes of tertiary structure proteins 1. antigen binds to membrane IgM

• T cells recognize processed antigens - peptides - presented by self MHC 2. cell gets activated and divides many times → clonal expansion

• epitope: or antigenic determinant - discret site on antigen that gets rec- 3. B cells mature into plasma cells → secret antibodies - up to 2000 anit- ognized, immunologically active regions on antigen that bind B- or T-cell bodies/sec/cell receptors Clonal Selection

• only cells that are able to recognize foreign antigens due to a fitting B or T cell receptor get activated and proliferate

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• denritic cells (DCs), macrophages (MΦ), B cells

• express MHC class I and class II

• foreign antigen binds to their MHC → activate naive T cells

• are infected by or internalize antigen → process antigen by disgestion → display peptides of antigen in context with MHC on cell surface

• TcR (T-cell receptor) recognizes small peptides (8-20 aa) of proteins presentend by major histocompatibility complex (MHC) on APC

• CD4 T helper (Th) cells interact with MHC II, CD8 cytotoxic T cells (CTL) interact with MHC I Molecular Disease Mechanisms 9 Adaptive Immunity Molecular Disease Mechanisms 10 Adaptive Immunity

MHC diversity and inheritance Antigen Processing and Presentation &

Molecular Disease Mechanisms 11 Adaptive Immunity Molecular Disease Mechanisms 12 Adaptive Immunity

Presentation of Antigens on MHC I to CD8 T-Cells Presentation of Antigens on MHC II to CD4 T-Cells 10 Antwort 9 Antwort

1. fragmentation of protein into peptides • human: HLA genes, mouse: H2 genes

2. association of peptide with MHC molecule • diversity of MHC exists at the level of the species

3. transport to cell surface for expression • stems from polymorphism (influence of more than one gene) and polygeny (more than one allele in population) 4. different cellular pathways for association of peptide with MHC class I and class II molecule • diversity mainly affects peptide-binding cleft

• MHC II: recognizes exogenous antigens & interacts with Th cells that secret • MHC genes expressed from both inherited alleles → co-dominant expression cytokines • peptides are bound with different affinities by MHC • MHC I: recognizes endogenous antigens & interacts with CTL cells that then • the more different the MHC genes of parents, the better protected are the chil- kill APC dren from infectious disease → greater chance to recognize diverse pathogen structures → HLA smell

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• endocytic processing pathway: antigen ingested by endocytosis or phago- • derived from self or infection with viruses and bacteria cytosis → transport to endosome → fusion with lysosome → recognition by MHC II in vesicle → transport to cell surface and presentation 1. partly folded MHC I α chains bind calnexin until β2-microglobulin binds

• MHC II α and β chains are produced and complexed with polypeptide - 2. MHC class I α : β2m complex is released from calnexin, binds a complex invariant chain (Ii) - that blocks its peptide-binding cleft → Ii facilitates of chaperone proteins (calreticulin, Erp57) and binds to TAP via tapasin export of MHC II from ER to golgi → fusion with late endosome → breakdown of Ii leaving only small fragment called CLIP - maintains blockage of binding 3. cytosolic proteins are degraded to peptide fragments by the proteasome cleft → CLIP is removed and binding of peptides with higher affinity is allowed 4. TAP delivers a peptide that binds MHC I and completes its folding → folded MHC I is released from TAP complex and exported Molecular Disease Mechanisms 13 Adaptive Immunity Molecular Disease Mechanisms 14 Adaptive Immunity

Handling of Cytosolic, Intravesicular and Extracellular Self MHC Restriction Pathogens

Molecular Disease Mechanisms 15 Adaptive Immunity Molecular Disease Mechanisms 16 Adaptive Immunity

Types of Grafts & T-Cell Maturation Host vs. Graft (and vice versa) Disease 14 Antwort 13 Antwort

• T-cells recognize foreign antigen associated with self MHC → T cells associ- ated with foreign MHC are not recognized

• self MHC restriction occurs in thymus

• a particular TcR is specific for both antigenic peptide and self MHC-molecule → only then killing occurs (otherwise poor killing)

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• T-cell generation in bone marrow and fetal liver • Allograft: allotransplantation - grafts between two members of the same species • occurs in thymus • Isograft: graft between members of species with identical genetic makeup • 96% of cells die here without inducing any inflammation by apoptosis (e.g. identical twins)

• thymic macrophages phagocytose apoptotic thymocytes • Graft vs. Host: immunocompromised host grafted with foreign immuno- competent lymphoid cells → T-cells of graft recognize foreign antigens of host • maturation involves change of expression of TcR-associated molecules and → damage host tissue co-receptors → can be used as markers for stage of maturation • Host vs. Graft: immunocompetent host recognizes foreign antigens of grafted tissue → immune response → rejection of transplant → Allograft Rejection Molecular Disease Mechanisms 17 Adaptive Immunity Molecular Disease Mechanisms 18 Adaptive Immunity

Process of Self MHC Restriction in Thymus Mechanism for Induction of Central T-Cell Tolerance

Molecular Disease Mechanisms 19 Adaptive Immunity Molecular Disease Mechanisms 20 T-Cell Response

Summary T-Cell Selection in Thymus T-Cell Propagation & Activation 18 Antwort 17 Antwort

• AIRE (autoimmune regulator) Transcription Factor: master regulator of • thymus accepts T-cells that fall into a narrow window of affinity for MHC ectopic (abnormal location) expression of peripheral tissue-restricted antigens molecules: interaction too weak or too strong → apoptosis in stromal cells of thymic medulla • positive selection: T-cells with TcR recognizing are retained • allows for expression of antigens to which T cells are negatively selected ◦ peptide is a partial agonist → thymocytes receive a partial signal and is • AIRE-/- mice exhibit wide spread organ-specific autoimmunity, such as ovary, rescued from apoptosis retina, testis, stomach → defect in AIRE gene leads to defect in thymic se- lection and autoimmunity ◦ selection for survival and maturation • negative selection: retained T-cells with TcR recognizing self peptide as- sociated with self MHC are eliminated

◦ peptide is agonist → thymocyte receives a powerful signal and undergoes apoptosis

• self MHC-restricted T-cells are released

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• CD4+ and CD8+ cells leave thymus and enter circulation → in resting state: Property Positive Selection Negative Selection G0 of cell cycle Site Cortex Medulla Stromal cells in- Macrophages and dendritic Epithelial cells • naive cells circulate between blood and lymph system every 12-24h volved cells Elimination of thymocytes • CD4:CD8 T-cell ratio in lymphoid organs ≈ 2:1 Survival of thymocytes Selection mecha- bearing high-affinity receptors bearing receptors for nism for self-MHC alone or self- • activation of naive T-cells results in primary response: self-MHC antigen + self-MHC  after 24h: initiation of repeated rounds of cell division and differentiation Immune conse- Self-MHC restriction Self-tolerance into: quence

 Effector cells: cytokine producers, cytotoxic killers

 Memory cells: long lived, response faster and strong in secondary re- sponse Molecular Disease Mechanisms 21 T-Cell Response Molecular Disease Mechanisms 22 T-Cell Response

T-Cell Response to Antigen Contact T-Cell Activation Factors & Two Signal Model

Molecular Disease Mechanisms 23 T-Cell Response Molecular Disease Mechanisms 24 T-Cell Response

T-Cell Growth Factor IL-2 CD4+ helper (Th) Cells 22 Antwort 21 Antwort

• promotion and inhibition of stimulation by CD28 family 1. Recognition & Activation

◦ CD28 and ICOS positive signals mediated by specific TcR - signal 1 - and costimulatory receptor - signal 2

◦ CTLA-4 and PD-1 negative signals

• CD28 family interact with B7 family members (e.g. CD80, CD86)

• members of Tumor Necrosis factor (TNF) receptor and ligand superfamily (e.g. CD40L) essential for crosstalk between APCs and T-cells 2. Proliferation & Differentiation • Two Signal Model: for B-cell activation: T-helper cells express CD40L clonal expansion of activated cells and differentiation into functional effector after activation and costimulate B-cells by triggering CD40 cells (several days)

3. Effector Function

antigen elimination → apoptosis or development into memory T-cell

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• help B-cells in T-dependent antibody response • resting T-cell: express IL-2R β and γ chains but no α chain or IL-2 → low affinity a) B-cell activation by antigen meditated crosslinking of B-cell receptor → results in upregulation of MHCII and B7 molecules - CD80/CD86 • activated T-cell: after antigen contact transcription factor NFAT binds promoter of IL-2Rα chain gene → α chain converts low affinity IL-2R to high b) antigen presentation to T-cell affinity form

c) T-cell activation, upregulation of CD40L, production of cytokines • growth factor IL-2 leads to T-cell expansion d) CD40 and cytokine-meditated activation of B-cell

• recruit + act. innate immune cells - macrophages, neutro-, eosino-, basophils

• promote survival of CD8 memory T-cells Molecular Disease Mechanisms 25 T-Cell Response Molecular Disease Mechanisms 26 T-Cell Response

Th Subsets Th Subsets (1/2) (2/2)

Molecular Disease Mechanisms 27 T-Cell Response Molecular Disease Mechanisms 28 Tolerance Mechanisms

Regulation Factors of Th Subset Development Mechanism of T-Cell Tolerance 26 Antwort 25 Antwort

•◦ associated with Helminth (parasitic worm) infection and allergies • Th1:

◦ promote clearance of infection with gastrointestinal nematodes by production of IL-4 and ◦ protection from intracellular microorganisms: protozoa, bacteria, fungi & viruses IL-13 ◦ IFN-γ & TNF-α: essential for macrophage activation → ROS & nitric oxide (NO) pro- • Th17: IL-17A, IL-17F duction

◦ promote clearance of bacterial infections at mucosal surfaces - e.g. intestines and lung - ◦ IL-2; IL-21: survival & rapid expansion after secondary infection of memory CD8+ T-cells and skin ◦ IFN-γ: anti-viral and anti-bacterial anitbody response → IgG2a, IgG2b ◦ IL-17A recruits neutrophils and activates epithelial cells to secret anti-microbial factors • Th2:

Treg Th1 Th2 Th17 inhibits in- defense against clearance of gastroin- Benefits anti-bacterial flammation microorganisms testinal nematodes inhibits clearance tissue damage asthma & allergies, Pathology autoimmunity of chronic by ROS fibrosis pathogens

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Central Tolerance • cytokines: IL-4, IL-12, IFN-g, IL-6, TGF-β

• negative selection of autoreactive T-cells in thymus • strength of T-cell stimulation

Peripheral Tolerance • type of APC • PAMPs - pathogen associated molecular patterns • unresponsiveness of peripheral T-cells • microenvironment → tissue ◦ suppression - or dominant regulation • epigenetics ◦ anergy - absence of immune response to antigen • diet → vitamins, lipids, oxidants ◦ ignorance - immune privilege

◦ negative and anti-inflammatory signals

• mediated by: Treg, anti-inflammatory cytokines, dendritic cells, co-stimulatory & inhibitory surface receptors Molecular Disease Mechanisms 29 Tolerance Mechanisms Molecular Disease Mechanisms 30 Tolerance Mechanisms

T-Cell Ignorance & Anergy & Anti-Inflammatory and Immunoregulatory Treg Microenvironment

Molecular Disease Mechanisms 31 Tolerance Mechanisms Molecular Disease Mechanisms 32 Autoimmunity

Scurfy Mice & IPEX Syndrome & Autoimmunity Treg Functions 30 Antwort 29 Antwort

Anergy Reasons for ignorance:

• naive T-cells need co-stimulatory signals to become activated: expression is restricted → • antigen concentration is too low: lymphocytes have a threshold for receptor occupancy to most tissues lack B7.1/B7.2 (CD80/CD86) or CD40 or both trigger a response

• most cells also lack MHC II • antigen sequestration (hiding away): at immunologically privileged sites - e.g. eye, testis, placenta - immune response is restricted to prevent damage from inflammation • no co-stimulation → T-cell becomes refractive to later encounter with same antigen Anti-inflammatory and immunoregulatory microenvironment Treg • anti-inflammatory factors: e.g. Transforming Growth Factor β (TGF-β), Interleukin 10 • express surface markers CD4+ and CD25+ and transcription factor Foxp3 (IL-10)

• from thymus or generated in periphery • negative signals: e.g. cell surface molecules CTLA4 & PD-1 interfere with T-cell response

• inhibit proliferation and cytokine production of other T-cells • expression of death receptors on T-cells: induction of cell death by apoptosis - e.g. Fas- ligand binds to Fas-receptor (CD95) • can limit disease development and pathology

• inhibitory signals mediated by cytokine secretion (IL-10, TGF-β) and/or receptors

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Autoimmunity a chronic inflammatory disease that results from a breakdown • Scurfy mutation leads to useless Foxp3 → i.a. production of autoantibodies of tolerance • IPEX: immune dysregulation, polyendocrinopathy, enterophathy, X-linked • incidences of autoimmune disease increasing in industrialised countries syndrome

• no cures → many treatments allow near normal life-spans • usually fatal in boys during early childhood

• periods of flares and remissions typically, lasting from days to months and • i.a. skin inflammation, anemia, type 1 diabetes, severe diarrhea occasionally years Foxp3+ Treg • flares can be caused by stress and infections • suppresses autoimmunity, colitis, allergies, spontaneous fetus abortion, or- • more common in women gan transplant rejection, response to (persistent) pathogens, recognition & rejection of tumor cells • concordance rate - chance of both monozygotic twins being affected- for com- mon autoimmune disease (e.g. MS, diabetes, arthritits) only at 20-40% • inhibit CD4 and CD8 non-specifically via APCs - cell contact is required

• environmental factors crucial: pathogens, commensal bacteria, diet • can suppress Th1, Th2, Th17 Molecular Disease Mechanisms 33 Autoimmunity Molecular Disease Mechanisms 34 Autoimmunity

Induction of Autoimmunity & Molecular Mimicry Link of HLA and Autoimmunity

Molecular Disease Mechanisms 35 Autoimmunity Molecular Disease Mechanisms 36 Autoimmunity

Lyme Disease & Multiple Sclerosis MS Symptoms & Therapies 34 Antwort 33 Antwort

Molecular Mimicry when microorganisms display similar molecular structures • failure of any mechanism of tolerance to self molecules (→ mimicry), the immune system - cross-reactive T-cells or antibodies - mistakenly attack sef • pathogens may trigger autoimmunity → molecular mimicry • modification of self antigens • antibodies raised against Treponema pallidum can cross-react with some ery- throcyte blood group antigens → anemia • release of antigen form immunoprivilieged sites • antigens common to Trypanosoma cruzi and some Streptococcus A bacteria cross-react with human cardiac muscle → usually more than one factor involved in initiation of disease

• antigens from Borrelia burgdorferi mimic self protein, which is expressed on HLA link the surface of T- and B-cells, as well as APCs. • significant correlation of HLA alleles with increased risk for various disease

• penicillin can bind to erythrocytes → anti-penicillin antibodies can target + − • relative risk calculated by: RR = (Ag /Ag )disease erythrocytes → haemolysis and anaemia (Ag+/Ag−)control

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• Muscle: loss of balance, spasms, numbness in any area, problems moving arms or legs, Lyme Disease walking, coordination, tremor and/or weakness in one or more arms and legs • chronic arthritis induced by infection with Borrelia burgdorferi • Bowl and Bladder: constipation and stool leakage, difficulty beginning to urinate, fre- quent need to urinate, incontinence • OspA of Borrelia b. mimics self-molecule LFA-1 (CD18) expressed by leucocytes

• Eye: double vision, uncontrollable rapid eye movement, vision loss (usually one eye) • HLA-DR1 allele presents peptides of both OsP - Outer Surface Protein A - and LFA-1

• Brain and Nerve: decreased attention span, poor judgment, memory loss, difficulty rea- • individuals that encode HLA-DR1 have shown to develope arthritis soning and solving problems, dizziness and balance problems, hearing loss Multiple Sclerosis • increased risk associated with HLA-DR2 on chromosome 6 • caused by immune response against myelin • Glatirameracetat - Copraxone: random polymer of four aas found in MBP reduces fre- quency of relapses • symptoms vary → location and severity of each attack can differ

• Dimethylsulfat induces anti-oxidative response by activation of transcription factor Nrf2 • can last for days, weeks, or months, usually followed by periods of reduced or no symptoms → remission, but disease commonly returns → relapse • IFN-β - a cytokine • fever, hot baths, sun exposure, and stress can trigger or worsen attacks Molecular Disease Mechanisms 37 Autoimmunity Molecular Disease Mechanisms 38 Autoimmunity

Experimental Model for MS Dilated Cardiomyopathy

Molecular Disease Mechanisms 39 Autoimmunity Molecular Disease Mechanisms 40 Autoimmunity

Model for Development of Autoimmune Myocarditis & Rheumatoid Arthritis Bystander Activation 38 Antwort 37 Antwort

• prevalent cause of human heart disease Experimental Autoimmune Encephalomyelitis EAE:

• often associated with viral myocarditis, especially Coxsackie Virus B3 (CVB3) • commonly induced in susceptible animals by immunization with proteins found in myelin sheath - MBP (Major Basic Protein), PLP (Proteolipid • development of autoimmune myocarditis is prevented in mice lacking: IL-1, Protein), MOG (oligodendrocyte protein) - together with adjuvant - immune IL-6, IL-23, GM-CSF, RORγ, CD40 or CD40L response enhancer - like Complete Freunds Adjuvant (CFA)

• chronic stages of disease appear to be mediated by T-cell mediated autoim- • animal models suggest key role of Th17 cells mune response against heart muscle myosin • finding: IL-6 and IL-23 essential for developement of MOG-induced EAE

• finding: RORγ KO mice are protected from EAE due to absence of IL-17- producing CD4+ (Th17) cells

• finding: impact of dietary fatty acids and melatonin

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• progressive debilitating disease of connective tissue 1. infection with CVB3 leads to damage/death of heart muscle → cardiomy- ocytes • most common sites affected are joints 2. release of α-myosin - sequestered self-antigen • immune complexes (autoantibodies + soluble self antigen) are deposited in joints of skeletal system 3. damaged/dead cells and α-myosin are taken up by dendritic cells (DC) which have been activated by CVB3 → bystander activation • formation of immune complexes initiates and amplifies inflammatory response, causing synovial membrane - lining joint capsule and tendon sheaths - damage 4. DC presents myosin and secrete pro-inflammatory cytokines and cell lysis 5. activates autoimmune CD4+ T-cells → differentiate into pathogenic Th17 • inflammatory response characterized by complement fragements C3a and cells C5a, Mast cells, monocytes, T- and B-cells Bystander Activation inflammatory cytokines or microbial products may non- specifically activate DC presenting self-antigen → self-antigen, which would normally be ignored - activates autoreactive T-cells leading to disease Molecular Disease Mechanisms 41 Autoimmunity Molecular Disease Mechanisms 42 Autoimmunity

Graves Disease Hashimoto’s Thyroiditis

Molecular Disease Mechanisms 43 Autoimmunity Molecular Disease Mechanisms 44 Autoimmunity

Spontaneous Autoimmune Disease Models Therapies for Autoimmune Diseases 42 Antwort 41 Antwort

• hypothyroidism caused by apoptosis/necrosis of thyroid cells • unregulated overproduction of thyroid hormones → hyperthyroidism

• characterized by intense cellular infiltrate into thyroid • mediated by antibodies specific for Thyroid Stimulating Hormone (TSH) re- ceptor • mediated by autoreactive T-cells • unlike TSH, autoantibodies are not regulated and overstimulated by thyroid • inflammatory cell infiltrate leads to gland enlargement - goiter - and eventu- ally gland fibrosis • can be transferred with IgG antibodies

• inflammatory process leads to MHC II expression on thyroid cells, possibly • babies born to mothers with Graves’ can show transient symptoms of hyper- leading to increased T-cell expansion thyroidism

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• conventional therapeutic approaches: • Non-obese diabetic (NOD) mice:

◦ cortisone and methotrexate (antagonist of folic acid) ◦ develope insulin-dependent diabetes mellitus

• new therapies: ◦ more prevalent in females (60-80%) compared to males (20-30%)

◦ reagents blocking activity of TNFα, IL-6, IL-1 with antibodies ◦ can be prevented by single injection of mycobacterial adjuvants - CFA - or BCG (Bacille de Calmette et Gu´erin)vaccine ◦ depletion of B-cells with anti-CD20 (treatment for Lupus) ◦ susceptibility to IDDM is polygenic • experimental approaches: ◦ incidence of disease linked to microbiome ◦ antbodies against pro-inflammatory cytokines (IL-6R, IL-17) • New Zealand Black (NZB) mice: display autoimmune abnormalities in- ◦ vaccination cluding hemolytic anemia, elevated levels of anti-DNA antibodies

◦ altered peptide ligands • F1 hybrids of NZB and NZW are widly used as a model form human systemic lupus erythematosus Molecular Disease Mechanisms 45 Danger & Inflammation Molecular Disease Mechanisms 46 Danger & Inflammation

PRRs - Pattern Recognition Receptors Defense against Pathogens in Drosophila

Molecular Disease Mechanisms 47 Danger & Inflammation Molecular Disease Mechanisms 48 Danger & Inflammation

TLRs - Toll-Like Receptors TLR Signaling (I/II) 46 Antwort 45 Antwort

• Toll and Imd pathways confer host defense against pathogens • expressed mainly by innate immune cells

• Toll pathway regulates production of antimicrobial peptides against fungi and • recognize Pathogen Associated Molecular Patterns (PAMPs) and endogenous stress molecules Gram-positive bacteria • Membrane-bound PRRs

• Peptidoglycan Recognition Protein (PGRP-SA) is essential for activation of ◦ Toll-like receptors (TLRs) & scavenger receptors Toll pathway in response to Gram-positive bacteria ◦ C-type lectin receptors (e.g. Dectin-1, DC-SIGN, Mannonse receptor) • Persephone is involved in the activation of Toll pathway in response to fungi • Cytoplasmic PRRs • PGRP-LC recognizes the invasion of Gram-negative bacteria & is required ◦ NOD-like receptors (NLRs): NODs (nucleotide-binding oligomerization domain) & NALPs for activation of Imd pathway → mediates response against Gram-negative bacteria ◦ RNA helicases: RIG-I, MDA5

• Secreted PRRs

◦ collectins (e.g. mannan-binding lectin MBL) & pentraxins (e.g. SAP, CRP)

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• myD88: TLR signaling adaptor protein, used by all TLRs except TLR3 • humans have 10 functional TLR genes (mice have 12)

• interacts with IRAK (interleukin-1 receptor-associated kinase) family • TLR4 interacts directly with bacterial LPS

• which interacts with TRAF6 (tumor-necrosis factor-receptor-associated fac- • also bind endogenous danger molecules tor 6) TLR Ligand affects • which leads to activation of NF-κB (nuclear factor-κB) and mitogen-activated 1 + 2 lipopeptides B. cell wall protein (MAP) kinases and JNK (Jun N-terminal kinase) 3 dsRNA V. genomes 4 LPS B. cell wall • pathway leads to production of pro-inflammatory cytokines such as IL-1, 5 flagellin B. cell wall IL-6, and TNF-α (tumor-necrosis factor α) 2 + 6 lipopeptides B. cell wall • myD88 also activates IRF7 (interferon (IFN)-regulatory factor 7) which in- 7 uncapped ssRNA V. genome duces IFN-α (interferon-α) 8 ssRNA V. genome 9 CpG DNA B. + V. genome • TLR2/4 signaling recruits second adaptor MAL that seems to be a bridging 10 unknown adaptor protein inducing IFN-β 11 (only in flagellin, proflilin flagellae, pilli mice) Molecular Disease Mechanisms 49 Danger & Inflammation Molecular Disease Mechanisms 50 Danger & Inflammation

TLR Signaling TLR Signaling Pathways (II/II)

Molecular Disease Mechanisms 51 Danger & Inflammation Molecular Disease Mechanisms 52 Danger & Inflammation

NF-κB Pathway Toll-IL1-Receptor Superfamily 50 Antwort 49 Antwort

• TLR3 signals through TRIF (Toll/IL-1 receptor-domain)

• which interacts specifically with TBK1 (TRAF-family-member-associated NF-κB activator-binding kinase 2)

• which leads to IRF2 activation and IFN-β production

• TRIF also interacts with RIP1 (receptor-interacting protein 1)

• which leads to activation of IκB (inhibitor of NF-κB) kinase 1 (IKK1)- IKK2-NEMO complex

• TRAM seems to be a bridging adaptor for TRIF recruitment, especially for TLR4

• TLRs that interact directly with MyD88: 5, 7, 9

• TLRs that interact with MyD88 with adaptor; 1, 2, 3, 4, 6

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• proteins with TIR domains can be subdivided into: • key factors involved in regulation: members of IκB (inhibitors of κB) fmaily and IκB kinase (IKK) complex - consisting of IKKαB-IKKκB heterodimers ◦ Immunoglobulin Domain Subgroup: immunglobulin domains extra- and scaffold protein IKKα/NEMO cellularly and include receptors that bind specifically endogenous ligands • variety of stimuly activate IKK complex → phosphorylation of IκB proteins ◦ Leucine-Rich-Repeat Subgroup: TLR subgroup, have leucin-rich re- → IκB polyubiquitination → degradation by 26S proteasome → liberation of peats extracellularly NF-κB proteins → translocate to nucelus → bind promoter region of NF-κB- responsive genes to increase gene expression ◦ Adaptor Subgroup: involved in signaling transduction

• TLRs on cell surface or in endosome Molecular Disease Mechanisms 53 Danger & Inflammation Molecular Disease Mechanisms 54 Danger & Inflammation

NOD-Like Receptors NOD1 and NOD2 NLRs

Molecular Disease Mechanisms 55 Danger & Inflammation Molecular Disease Mechanisms 56 Danger & Inflammation

NLR Family Interleukin-1 (IL-1) 54 Antwort 53 Antwort

• recognize bacterial petidoglycans • NOD: Nucleotide Oligomerization Domain

• NOD1 binds meso-diaminopimelic acid (DAP) found mainly in gram negative • cytoplasmatic proteins that regulate inflammatory responses and cell death pathways and some gram positive bacteria (Listeria m. and Bacillus spp. • more than 20 of these proteins in mammalian genome - 2 major (phylogenetic) subfamilies: NODs and NALPs (=NLRPs) • NOD2 binds muramyldipeptide - minimal motif found in all gram postive and negative bacteria • or division into 4 subfamilies based on type of N-terminal domain: NLRA, NLRB, NLRC, NLRP • NOD1/2 bind to RICK/RIP2 leading to activation of NF-κB • recognize microbial or endogenous danger molecules • link between NOD2 polymorphisms and Crohn’s disease • form oligomers that activate inflammatory caspases → cleavage and activation of inflamma- tory cytokines - e.g. IL-1β and IL-18 - and activation of NF-κB signaling • Crohn’s disease: disturbance in normal immunological unresponsiveness to components of intestinal microflora → hyperresponsiveness to components → • NALPs key for activation of inflammatory caspases 1 and 5 → secretion of IL-1β, IL-18, inflammatory respone via Th17 and Th1 and IL-33

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• general name for two distinct proteins: IL-1α and IL-1β • members share a three-part domain

• typically produced during inflammatory response • C-terminal leucin-rich-repeat (LRR) domain → involved in ligand recognition

◦ mainly by dendritic cells and macrophages • central NACHT (aka NOD) domain to self-oligomerization and ATPase ac- ◦ but many other cells can produce them tivity

• function: • N-terminal domain composed of protein-protein interaction cassettes, such as CARDs or pyrin domains ◦ immune system: defense against bacterial infection

◦ cell migration: IL1α/β upregulate adhesion molecules on endothelial cells → transmigra- tion of leucocytes (neutrophils, monocytes, B- and T-cells

◦ bone formation and remodeling

◦ fever induction

◦ appetite regulation & insulin secretion Molecular Disease Mechanisms 57 Danger & Inflammation Molecular Disease Mechanisms 58 Danger & Inflammation

Activation of IL-1β & NLRP3 Uric Acid

Molecular Disease Mechanisms 59 Danger & Inflammation Molecular Disease Mechanisms 60 Danger & Inflammation

Cholchicine & Autoimmune Disease Caused by Atherosclerosis NALP3 Mutations 58 Antwort 57 Antwort

• generated in breakdown of purine → danger signal of dying cells • PAMP activates TLR → activates NF-κB → transcription + translation of pro-IL-1β → cleaved by inflammasome (Casp1 + CARD) → IL-1β function- • potent anti-oxidant ally active → inflammation • eliminated over kidney ◦ K+ efflux → bacterial pore-forming toxins, Pannexin1 activated by extracel- • if not eliminated: forms uric acid crystals (MSU - monosodium ureate) lular ATP through P2X7

• Gout ◦ cytosolic mitochondrial DNA → mitochondrial damage ◦ one of the most painful forms of arthritis ◦ Cathepsin B → lysosomal protein → lyosome rupture ◦ occurs when too much uric acids builds up in body ◦ NLRP3 activation leads to its association with inflammasome cleavage of pro- ◦ sharp uric acid crystal deposits in joints, often in big toe Caspase1 → active Caspase

◦ deposits of uric acid (tophi) that look like lumps under the skin & kidney stones ◦ MSU ◦ 1-3% of population develop gout ◦ NLRP3 inflammasome iniciates obesity-induced inflammation and insulin re- sistance and steatohepatosis

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• chronic inflammatory disease affecting arterial vasculature • inhibits NLRP3 activation

• lipid deposition and oxidation in artery wall • isolated from Autumn Crocus

• cholesterol crystals induce IL-1α and IL-1β production • interferes with mitosis (metaphase)

• oleate selectively triggers IL-1α • used to treat rheumatism, arthritis, and gout

• induces foam cell formation → promotes atherogenesis ◦ Muckle-Wells syndrome (MWS) - spontaneous IL-1β release in monocytes

◦ Familial Cold Autoinflammatory Syndrome (FCAS, FCU)

◦ Chronic Infantile Neurological Cutaneous and Articular Syndrome (CINCA, NOMID)

◦ treatable with IL-1 receptor antagonist (IL-1Ra) - Anakinra Molecular Disease Mechanisms 61 Intestinal Immunology Molecular Disease Mechanisms 62 Intestinal Immunology

MALT - Mucus-Associated Lymphoid Tissue Small Intestine

Molecular Disease Mechanisms 63 Intestinal Immunology Molecular Disease Mechanisms 64 Intestinal Immunology

Large Intestine Oral Tolerance 62 Antwort 61 Antwort

• absorbs 90% of all ingested protein, fat, and carbohydrates • found in:

• aa’s, sugars, Vit. C, Vits B, Fe, Ca, and Mg carried through blood to to liver ◦ nasal assocaited lymphoid tissue - NALT- i.e.tonsils, adenoid

• FA, cholesterol, Vit. A, D, E, and K go into lymph system then into blood ◦ bronchial associated lymphoid tissue - BALT and liver ◦ gut associated lymphoid tissue - GALT - e.g. intestine • Duodenum: absorption of Fe, Ca, Mg • may be present either as single lymphoid follicle embedded in the wall of the • Jejunum: lined with villi and microvilli, absorption of simple sugars, wa- tissue or as aggregated follicles such as Peyer’s patches in the intestine ter soluble vitamins (except C and some Bs), aa’s to blood, fat to lymph capillaries

• Ileum: absorption of aa’s, fat-soluble vitamins, FA, cholesterol, Na, K, Vit. B12

64 Antwort 63 Antwort

• systemic tolerance occurs after oral administration of any soluble protein • divided into cecum, colon, and rectum given in absence of adjuvant • absorbs fluids and salts in colon • likely occurs through entry of proteins directly into bloodstream and subse- quent dispersal throughout lymphoid organs → presentation by local toler- • colon soaks up 1 to 1.5 L of H2O every day gonic DC in absence of pro-inflammatory signals • 30% of weight of feces is bacteria ◦ possible mechanisms: • bacteria in colon make Vit. K and B12 ◦ deletion of specific T-cells

◦ anergy of specific T-cells

◦ induction of specific regulatory T-cells

• regulatory factors:

◦ antigen dose, form/nature of antigen, cytokine milieu, age Molecular Disease Mechanisms 65 Intestinal Immunology Molecular Disease Mechanisms 66 Intestinal Immunology

Intestinal Microflora Physical Defense Mechanisms & Types of Intestinal Epithelial Cells

Molecular Disease Mechanisms 67 Intestinal Immunology Molecular Disease Mechanisms 68 Intestinal Immunology

Organization of the GALT & Antigen Entry into GALT Peyer’s Patches (I/II) 66 Antwort 65 Antwort

• stomach acid • 1014 bacteria

• epithelial cell layer (enterocytes and tight junctions) • 500 - 1000 species

• enzymes & anti-microbial peptides (lysozyme, defensins produced by Paneth • dominated by just 2 (out of ¿ 30) phyla of bacteria - Fermicutes and Bac- cells) teroides - and only one member of Archaea - i.e. Metanobrevibacter

• mucus (produced by goblet cells) • most are cultivatable

Cell Type Function • characterized by 16S sequencing Enterocytes absorption of nutrients • genetically obese mice show an increase in Firmicutes and decrease in Bac- Goblet cells mucus production teroides and transmission to WT germ-free mice leads to adiposity Paneth cells anti-microbial Microfold (M) cells antigen uptake • microflora leads to ignorance rather than tolerance Tuft cells pathogen sensing Enteroendorine hormone production • DC presenting commensal bacterial antigens can traffic only as far as mesen- teric lymph nodes → barrier between mucosal and systemic immune system

68 Antwort 67 Antwort

(a) antigen enters through M cells in FAE and • Peyer’s patches (PP) and mesenteric lymph nodes (MLN) are involved in the induction of immunity and tolerance (b) after transfer to local DCs, might be presented directly to T-cells in Peyer’s patch • effector sites - scattered lymphoid cells - are scattered throughout lamina (c) OR: antigen or antigen-loaded DCs from PP might gain access to draining lymph propria and epithelium of the mucosa (d) with subsequent T-cell recognition in MLNs • Villus lamina propria and PP are drained by afferent lymphatics that go to (e) similar process of antigen or APC dissemination to MLNs might occur if antigen enters MLNs through epithelium covering villus’ lamina propria • Peyer’s patches (f) then: no future possibility that MHC II positive enterocytes might at as local APCs

in all cases: antigen-responsive CD4+ T-cells acquire expression of α4β7 integrin and ◦ PP are overlaid by specialized epithelium called follicle-associated epithe- chemokine receptor CCR9 → leave MLN in efferent lymph → lium (FAE)

(g) enter blood thru thoracic duct → exit into mucosa thru vessels in lam. prop. - antigen ◦ FAE is composed of enterocytes and specialized M cells recognizing T-cells in MLN may spread from blood throughout periph. immu. system ◦ Goblet cells are infrequent → FAE is not covered by a thick layer of mucus (h) OR: antigens gain access to blood from gut & interact w T-cells in peripheral lymph. tiss. → forms a direct contact with luminal contents Molecular Disease Mechanisms 69 Intestinal Immunology Molecular Disease Mechanisms 70 Intestinal Immunology

Antigen Entry into GALT M Cells (II/II)

Molecular Disease Mechanisms 71 Intestinal Immunology Molecular Disease Mechanisms 72 Intestinal Immunology

Intraepithelial Lymphocytes - Antibodies of Mucosa IEL 70 Antwort 69 Antwort

• specialized epithelial cells overlying lymphoid tissues

• lack glycocalyx or microvilli

• take up luminal antigens by endocytosis → lymphocytes sit within ”pockets” at the base of M cells

• M cells then release luminal antigens to underlying DC which gon on to prime an immune response by activating naive T and B cells within PP

72 Antwort 71 Antwort

• antibody responses are central to effective mucosal immunity • a network of lymphocytes lies underneath the intestinal epithelium

• main isotype is secretory IgA and - to a lesser extent - IgM and IgG • largely CD8αα+, αβ+TCR or γδ+TCR, which:

• normal intestinal mucosa contains at least 20 times more IgA+ than IgG+ B ◦ express limited TCR diversity & seem to arise indep. of thymic selection cells ◦ are thought to largely bind non-classical MHC molecules (such as MIC- • IgA normally exists as a polymer of between 2-4 IgA monomers, joined by A/B, MHC 1B (Qa2) and TL) which ”present” conserved pathogenic lig- J-chain ands or are expressed by stressed cells

• polymeric IgA is passed across the epithelial layer into gut lumen ◦ appear to play a role in maintaining epithelial integrity by killing dam- aged/stressed cells • IgA molecules do not cause inflammation → not recognized by phagocyte nor do they fix complement

• IgA molecules are believed to funciton by coating potentially infectious ma- terial preventing it from adhering to ephithelial cells Molecular Disease Mechanisms 73 Intestinal Immunology Molecular Disease Mechanisms 74 Intestinal Immunology

Migration of Lymphocytes to the Intestines Role of Vitamin A in Intestinal Immune Response

Molecular Disease Mechanisms 75 Intestinal Immunology Molecular Disease Mechanisms 76 Intestinal Immunology

Inflammatory Bowel Disease (IBD) Treatment of IBD & Experimental Models 74 Antwort 73 Antwort

• vitamin A deficient mice show impaired migration of lymphocytes to intestinal mucosa & • different adhesion molecules and chemokines govern the migration of lympho- marked decrease in number of IgA-secreting B cells and CD4 T cells in the ileum cytes to different tissues

• reduced infant mortality from persistent diarrhea in developing countries when vitamin A • T and B cells homing to intestines express α4β7, which binds to MAdCAM was administered on mucosal endothelial cells - note: otherwise activated T and B cells express • Vit. A metabolite retinoic acid (RA) enhances expression of gut-homing receptors (α4β7 α4β1, which binds VCAM and CCR9) • intestinal epithelial cells produce chemokines CCL25 and CCL28, which re- • retinoic acid receptor (RAR) and retinoic X receptor X receptor (RXR) are ligand induced cruit lymphocytes expressing CCR9 and CCR10 transcription factors

+ • RA promotes differentiation of FoxP3 Treg cells and inhibits Th17 cells in the intestines

• RA synergizes with intestinal DC-derived TGF-β, IL-5, and IL-6 to promote IgA production

76 Antwort 75 Antwort

• conventional: broad spectrum immunosuppressive drugs, antibiotics, surgery • chronic and relapsing inflammatory condition which affects the distal small intestine and the colon in in a transmural manner → Crohn’s disease - or only • new: selective blockers of inflam. → antibodies against TNFa, IL-6, IL-23 the colon in a superficial manner → ulterative colitis

• needed: increased understanding of immune dysfunction leading to IBD • affects 1 in 500 people in westernized countries

• treatment with chemicals • results from inappropriate inflammatory innate and adaptive immune re- sponse against intestinal commensal bacteria ◦ e.g. dextran sodium sulfate, DSS • conditional KO mice lacking the transcription factor NF-κB selectively in ◦ causes epithelial cell damage and innate inflammatory immune response intestinal epithelia develop spontaneous IBD due to death of epithelial cells

• T-cell adoptive transfer model • inflammatory response to commensal bacteria cause chronic disease → pro- inflammatory cytokines involved in IBD: IL-6, IL-23, and IL-17 ◦ transfer of CD25neg CD41 (naive T cells) from normal mouse to mouse lacking T- and B- cells • imbalance of Th17 (promoter) and Treg (suppressor) cells promotes IBD + ◦ if mouse also receives CD25 CD4+ (Treg cells): no disease Molecular Disease Mechanisms 77 Obesity Molecular Disease Mechanisms 78 Obesity

Obesity Co-Morbidities Obesity Pathways

Molecular Disease Mechanisms 79 Control of Food Intake and Obesity Molecular Disease Mechanisms 80 Control of Food Intake and Obesity

Hunger, Appetite, & Satiation Role of Hypothalamus in Feeding Regulation & Energy Homeostasis Control 78 Antwort 77 Antwort

• dyslipidemia - abnormal amounts of lipids in blood

• hepatic steatosis

• cardio-vascular diseases

• Type 2 diabetes - insulin resistance

• depression

• cancer

→ lipodystrophy (inability to produce and maintain healthy fat tissues) has the same co-morbidites

80 Antwort 79 Antwort

• Ventromedial hypothalamic nucleus - VMH: satiety center Hunger sensation associated with drive to eat

• Lateral hypothalamus - LH: feeding center Appetite psychological desire to eat

• Arcuate nucleus - ARC: receptors for hormones and neuropeptides that Satiation termination of eating after hunger has been satisfied regulate feeding Energy Homeostasis Control • Paraventricular nucleus - PVN: integration of signals from ARC with thyroid and HPA (hypothalamic-pituitary-adrenal) axis • adiposity signals: fat → leptin, pancreas → insulin

• Vagus nerve: satiety signals to brain stem after ingestion of meal • satiation signals: liver & gastrointestinal tract: CCK, GLP-1, gastric disten- sion (stomach stretch) • Nucleus of solitary tract (NTS) & PVN: connection of brainstem with hypothalamus • signals reach arcuate nucleus (ARC) in hypothalamus

• together with other brain influxes (stress, social situation, time of day, hedo- nics) → regulation of adiposity and plasma glucose Molecular Disease Mechanisms 81 Control of Food Intake and Obesity Molecular Disease Mechanisms 82 Control of Food Intake and Obesity

Satiety & Hunger Signals Integration of Long-Term Homeostatic and Short-Term Satiety Signals

Molecular Disease Mechanisms 83 Control of Food Intake and Obesity Molecular Disease Mechanisms 84 Control of Food Intake and Obesity

Integration of Homeostatic and Reward-Related Inputs Neurocircuits Involved in the Homestatic Regulation of Feeding 82 Antwort 81 Antwort

• regulation of food intake on a meal-to-meal basis is adjusted in response to • satiety: CCK (Cholecystokinin), GLP-1, PYY (in GI-tract), insulin, leptin, changes in body fat content serotonin, norepinephrine

• leptin stimulates POMC neurons and inhibits neurons that express AgRP • hunger: Ghrelin (in GI-tract), AgRP (Agouti Related Protein), NPY (Neu- and neuropeptide Y in the hypothalamus ropeptide Y, in brain)

• neurons project to second-order neurons in PVN and LH → project to NTS - satiety signals processed here

• satiety signals activate vagal afferents that terminate in the NTS to promote termination of meal

• NTS response to satiety response is amplified both by direct input to NTS from leptin and indirectly through action of leptin in hypothalamus

84 Antwort 83 Antwort

• AgRP neurons in the ARC stimulate feeding when activated • LH integrates reward-related input from NAc - Nucleus Accumbens - with information related to energy homeostasis from ARC neurons • AgRP neurons are inhibited by insulin and leptin and activated by ghrelin • LH neurons project to and influence the mesolimbic dopaminergic system • POMC inhibit food intake by binding and activating melanocortin receptors while also influencing satieity perception through projections to hindbrain

• POMC neurons are stimulated by leptin • weight loss lowers plasma insulin and leptin levels while increasing plasma ghrelin levels

• those responses increase the rewarding properties of food and the motivation to eat through either direct effect in the NAc or indirect effect in LH Molecular Disease Mechanisms 85 Control of Food Intake and Obesity Molecular Disease Mechanisms 86 Control of Food Intake and Obesity

Emergency Circuits that Stimulate Feeding Sources of Leptin

Molecular Disease Mechanisms 87 Control of Food Intake and Obesity Molecular Disease Mechanisms 88 Control of Food Intake and Obesity

Relationship of Leptin and Obesity Thrifty Theories & Obesity Statistics 86 Antwort 85 Antwort

• mainly: white adipose tissue • increased feeding

• BAT - brown adipose tissue • increased hepatic glucose production (HGP) → mediated by increased plasma levels of glucagon and corticosterone • ovaries + placenta • conservation of energy by inhibiton of growth and reproduction • skeletal muscle • reduction of metabolic rate through suppression of HPT (hypothalamic–pituitary • mammary epithelial cells –thyroid) axis • bone marrow

• pituitary gland

• liver

• mutations of leptin gene leads to obese (ob/ob) mice

88 Antwort 87 Antwort

Thirfty Gene Theory gene predisposing to effective energy storage enabled to • leptin increases metabolic rate/energy expenditure by increasing body tem- survive during time of starvation, however they predispose to obesity perature and oxygen consumption nowadays • decreases food intake by inhibiting appetite through appetite-stimulating Thrifty Phenotype Hypothesis observation that underweight babies develop in- NPY neurons and appetite-inhibiting POMC neurons in ARC sulin resistance and obesity more often compared to normal weight babies • leptin inhibits NPY/AgRP neurons resulting in inhibition of food intake Predictive Adaptive Response Model relative difference in nutrition between pre- and postnatal environments, rather than absolute level of nutrition • defects in leptin lead to obesity determines risk of insulin resistance ◦ absent leptin 2 • 30% of adult population classified as clinically obse (BMI > 30kg/m ◦ regulatory defects:normal leptin levels in adipose tissue, decreased secre- tion/expression • 50% overweight (BMI = 25 − 30kg/m2) ◦ leptin resistance: high leptin levels, in hypothalamus (e.g. db/db mice) • childhood obesity increasing ◦ high fat diet leads to leptin resistance in mice Molecular Disease Mechanisms 89 Control of Food Intake and Obesity Molecular Disease Mechanisms 90 Control of Food Intake and Obesity

Non-Metabolic Factors Regulating Food Intake & Hedonic Mechanisms in Non-Homeostatic Eating Food Reward Mechanism

Molecular Disease Mechanisms 91 Control of Food Intake and Obesity Molecular Disease Mechanisms 92 Control of Food Intake and Obesity

Physiological Components of Reward Psychological Components of Reward (I/II) 90 Antwort 89 Antwort

• hedonic processing integral part of homeostatic regulatory system • environmental cues, rewards, cognitive & emotional factors

• even at satiety and replete energy stores, cortex and limbic system can overpower hypotha- • non-metabolic factors mainly processed in cortico-limibic structures lamus into ingestive mode • food deprivation (fasting) strongly augments reward value • important factor influencing body weight set point: food hedonics, particularly shift towards higher body weight by highly palatable, calorie-dense foods • leptin and insulin inhibit brain reward circuitry by lowering circulating levels • increase availability of palatable , energy-dense foods mostly responsible for obesity epidemic of these hormones

• obesogenic food environment in genetically and/or epigenetically susceptible individuals • energy restriction increases sensitivity of reward circuits leads to higher body weight set point

• VTA (ventral tegmental area) → NAc pathway may promote consumption of palatable food

• LH contains neurons that potently stimulate food intake and is supplied by fibres from striatum, orbitofrontal cortex and ARC

• LH neurons supplying the NTS may diminish response to satiety signals and increase the amount of food consumed during a meal

92 Antwort 91 Antwort

1. Learning • necessary to learn about relationship among stimuli and about consequences of action • learned responses require knowledge: • reward consumption can produce hedonic consequences ◦ relationship between stimuli and actions

◦ reward prediction • individual has to be motivated to learn and act

◦ making anticipatory responses & guidance by cues • can be subdivided into different categories: learning, motivation, and emotion

◦ goal-directed action

2. Reward

• neural manipulation can alter an affective (emotional) or motivational process

• distinction among core processes of reward is between affective consequences of reward (liking) - regulated by NAc and ventral pallidum - and their motivational consequences (wanting) - controlled by ventral tegmentum, amygdala, ventral pallidum, and NAc) Molecular Disease Mechanisms 93 Control of Food Intake and Obesity Molecular Disease Mechanisms 94 Drug and Food Addiction

Psychological Components of Reward Drug Addiction (II/II)

Molecular Disease Mechanisms 95 Drug and Food Addiction Molecular Disease Mechanisms 96 Drug and Food Addiction

Stages of Addiction Cycle & CNS Sampling of Neurotransmitters by in vivo Animal Models for Addiction Microdialysis 94 Antwort 93 Antwort

• relapsing disorder that is characterized by a compulsion to seek and take 3. Affect drugs • core ”liking” and conscious pleasure • loss of control in limiting intake • liking for tastes involves activity in a distributed neural network that have been implicated in drug reward • emergence of a negative emotional state (e.g. dysphoria, anxiety, irritability) when access to drug is prevented 4. Motivation

• often starts with impulsive behaviour, but individuals develop compulsive • wanting generally refers to a conscious or subjective desire drug-seeking behaviour • ”wanting” is used to refer to an underlying implicit and objective motivation process: in- centive salience → chronic relapsing syndrome that moves from an impulse control disorder involving positive enforcement to a compulsive disorder involving negative re- • incentive salience is a motivational rather than an affective component of reward inforcement • incentive salience or ”wanting” - unlike ”liking” - particularly influenced by dopamine neu- rotransmission

96 Antwort 95 Antwort

• sampling of neurotransmitter in conscious animals → correlation of brain 1. preoccupation chemistry with behaviour • exaggerated motivation for drug use • implantation of semi-permeable prob membrane in a specific brain region of 2. binge/intoxication phase interest • downregulation of positive responses, subsequent increase in level of drug intake • substances diffuse across membrane based on concentration gradient 3. withdrawal/negative affect phase • both neurochemical sampling and localized drug delivery possible • negative effects of addictive substance become driving factor for continued drug seek- • studies support the role of dopamine in reward ing and intake

Animal Models

• for withdrawal/negative affect phase: brain stimulation

• for transition to addiction: escalation in drug administration OR drug taking in selected lines of drug preferences Molecular Disease Mechanisms 97 Drug and Food Addiction Molecular Disease Mechanisms 98 Drug and Food Addiction

Reward Transmitters and their Effect on Withdrawal Neurotransmitters Involved in Reward & Marks of Addiction

Molecular Disease Mechanisms 99 Drug and Food Addiction Molecular Disease Mechanisms 100 Drug and Food Addiction

Direct and Indirect Drug Action Withdrawal 98 Antwort 97 Antwort

receptors function brain area • dopamine → dysphoria pleasure, euphoria, Dopamine D1, D2 VTA, NAc mood, motor function • serotonin → dysphoria mood, impulsivity, anx- Serotonin 5HT3 • opioid peptides → pain iety, sleep, cognition Cannabinoids CB1, CB2 pain, appetite, memory • GABA → anxiety, panic attacks Opioid peptides NAc, amyg- (endorphins, κ, µ, δ pain dala, VTA ◦ rodents can be trained to self-administer virtually all addictive drugs includ- enkephalins ing opiates, amphetamines, cocaine, alcohol and nicotine

◦ usually need repeated exposure/prolonged access to intravenous drug to de- velop addiction

◦ addiction marked by escalation of dose, tolerance and resistance to ”extinc- tion” (when behavior no longer delivers drugs but punishment - e.g. foot- shock)

100 Antwort 99 Antwort

• follows a physiological adaptation to the presence of the agent → tolerance • direct: e.g. cocaine: inhibits reuptake of dopamine

• result of abrupt cessation of drug • indirect: e.g. alcohol: binds subreceptor GABAA → dopaminergic activity is increased in VTA by inhibiting GABAergic interneurons • disturbance of autonomic nervous system inhibits GABAergic neurons that project to dopaminergic Alcohol • activation of thalamus neurons in VTA binds to opioid receptor that inhibit GABAergic neurons • release of corticotrophin releasing factor (CRF) → increase of heart rate, Heroin blood pressure, blood glucose, and response to stressors that project to DAergic neurons in VTA block function of dopamine transporter by binding to DAT Cocaine • activation of locus coeruleus (LC) and slowing transport activates cholinergic neurons that project to DAergic neu- Nicotine rons in VTA Molecular Disease Mechanisms 101 Drug and Food Addiction Molecular Disease Mechanisms 102 Drug and Food Addiction

Development of Addiction Long Term Brain Changes due to Drug Abuse & Inheritability

Molecular Disease Mechanisms 103 Drug and Food Addiction Molecular Disease Mechanisms 104 Drug and Food Addiction

Current Causes of Obesity Role of Dopamine in Obesity 102 Antwort 101 Antwort

• decreases in CREB transcription factor in NAc and amygdala • use of drug is increased to maintain euphoria or avoid dysphoria and with- drawal • decrease in metabolism in Orbito Frontal Cortex (OFC) • number of receptors gradually increases to counter for continual presence of • decrease in dopamine D2 receptor binding drug

• inheritabillity found to range from 40-60% • amount of neurotransmitter gradually decreases through depletion and feed- back inhibition

• reinforcing properties of drug are gradually decreased → tolerance

• need for drug to maintain new homeostasis therefore increased → dependence

• behavioural repertoire is narrowed and eventually other important behaviours are ignored (e.g. familial, financial)

• reward and cognitive systems are compromised → imbalance in impulsive behaviour (e.g. violence, crime)

104 Antwort 103 Antwort

• DA response becomes insensitive at weight gain → overeating → even lower • disproportionate food intake of carbohydrates and simple sugars DA response • subsequent decrease in other foods such as protein, fats, and fiber → poor • people at high risk for obesity have an hypersensitive DA response nutrition

• additional factors: stress, poor nutrition, genetics, environmental toxins • diminished physical activity

• diminished sleep

• medication / medical issues

• convenience, cheap sugar

1 • genetics - accounts for only 3 of variance in obesity Molecular Disease Mechanisms 105 Diabetes Mellitus Molecular Disease Mechanisms 106 Diabetes Mellitus

Insulin Causes for Insulin Insufficiency

Molecular Disease Mechanisms 107 Diabetes Mellitus Molecular Disease Mechanisms 108 Diabetes Mellitus

Etiologic Classifcatin of DM Differnces between Type I and Type II DM 106 Antwort 105 Antwort

Absolute • anabolic hormone → promotes synthesis of carbohydrates, proteins, lipids and nucleic acids • genetic disorder Target Organ Effect • autoimmune damaging of β-cells hypothalamus food intake ↓ • viral damage skeletal muscle glucose uptake ↑

• toxic influence on β-cells liver glucose production ↓ brown adipose tissue thermogenesis ↑ • diseases of pancreatic gland white adipose tissue lipogenesis ↑

Relative

• β-cells

• insulin transport

• receptors (tissue insensitivity)

108 Antwort 107 Antwort

• Type 1: insulin-dependent diabetes mellitus (IDDM) • Type 1: destruction of β-cells → absolute insulin insufficiency

• Type 2: noninsulin-dependent diabetes mellitus (NIDDM) ◦ autoimmune or idopathic (=unknown cause)

• Type 2: resistance to insulin and relativ insulin insufficiency or defect in insulin secretion Type 1 Type 2 Age young (under 35) old, middle • other specific types Beginning of disease acute gradual ◦ genetic defects of β-cell function Duration labile stable Ketosis, ketoacidosis oftern develops rarely develops ◦ genetic defect of insulin action obesity in 89-90% of pa- Body weight decreased or normal tients ◦ pancreatic or endocrine diseases ◦ infections

◦ drug exposure

• getation diabetes Molecular Disease Mechanisms 109 Diabetes Mellitus Molecular Disease Mechanisms 110 Diabetes Mellitus

Who Should Be Screend for Diabetes? Stages of DM Development

Molecular Disease Mechanisms 111 Diabetes Mellitus Molecular Disease Mechanisms 112 Diabetes Mellitus

Diagnostic Criteria for DM Degrees of Severity of DM 110 Antwort 109 Antwort

• prediabetes - risk factors and predispose factors • all patients ≥ 45 years of age

◦ obesity • all patients with BMI ≥ 25 and:

◦ family history ◦ hypertension of ≥ 140/90 mmHg or HDL > 35 mg/dL or triglycerides > 250 mg/dL ◦ clinical insulin resistance (severe visceral obesity, acanthosis nigricans) ◦ people with birth weight > 4kg ◦ history of CVD ◦ women who had children with birth weight > 4kg ◦ gestational DM, or delivery of baby > 4 kg ◦ endocrine disorders ◦ African/Latino/Native/Asian American or Pacific Islander • impaired glucose tolerance (latent DM) ◦ first degree relative with DM

• clincial manifestation of DM ◦ physically inactive

• if testing is normal, repeat screening in 3 years

112 Antwort 111 Antwort

mild moderate severe • a1c (test measuring glucose attached to hemoglobin) ≥ 6.5% compensation can oral hypoglycemic oral hypoglycemic diet be achieved by agents or insulin agents or insulin • FPG (fasting plasma glucose) ≥ 126mg/dL - fasting for ≥ 8h last stages of chronic onl func- chronic and acute not last stages, complications are • 75g sugar, 2h OGTT (oral glucose tolerance test) ≥ 200mg/dL tional complications ketosis can occur present, ketosis is stages • random glucose ≥ 200mg/dL + symptoms of hyperglycemia common Molecular Disease Mechanisms 113 Diabetes Mellitus Molecular Disease Mechanisms 114 Diabetes Mellitus

Clinical Manifestations of DM Diabetes Mellitus Pathways

Molecular Disease Mechanisms 115 Hepatic Steatosis Molecular Disease Mechanisms 116 Hepatic Steatosis

Portal Circulation Non-Alcoholic Fatty Liver Disease NAFLD 114 Antwort 113 Antwort

• 3 P’s (plus 2 additional P’s)

• polyurea - when blood glucose levels are > 9 mmol/L, glucosurea (excretion of glucose in urin) arises

• polydipsia - as more water is secreted, the body requires more water intake

• polyphagia - increased catabolic activity of the body and as its result lack of energy

• pruritus - severe itiching of skin

• paresthesia - ”sensation of tingling, burning, pricking, or numbness of skin”, more generally known as feeling of ”pins and needles” or of a limb falling asleep

116 Antwort 115 Antwort

• fat deposition (steatosis) in liver • hepatic portal systems directs nutrient rich blood from intestines to liver

• not due to excessive alcohol use • liver drains venous blood from pancreas

• related to insulin resistance and metabolic syndrome • highest insulin levels of all organs

• may respond to treatments of T2D, weight loss, metformin, and thiazolidine- diones

• genetic forms exist

• symptoms: hypertension, dyslipidemia, increased waist circumference, insulin resistance, fatigue, dull right-upper-quadrant abdominal discomfort

• but most patients have few or no symptoms Molecular Disease Mechanisms 117 Hepatic Steatosis Molecular Disease Mechanisms 118 Hepatic Steatosis

Risk Factors NAFLD and NASH Sources of Increased TAG Deposition in Liver

Molecular Disease Mechanisms 119 Hepatic Steatosis Molecular Disease Mechanisms 120 Hepatic Steatosis

SREBP Role of Insulin Resistance in Hepatic Steatosis 118 Antwort 117 Antwort

• rate of free fatty acid uptake and synthesis bigger than needed for essential established function • obesity & T2D • impaired VLDL export • dyslipdemia

• increased de nove lipogenesis (DNL) • metabolic syndrome

suspected

• PCOS

• hypothyroidism

• hypopituitarism & hypogonadism

• pancreato-duodenal resection

• sleep apnea

120 Antwort 119 Antwort

• liver main site of insulin action - next to skeletal muscle and adipose tissue • sterol regulatory element-binding proteins

• fatty liver: ability of insulin to inhibit hepatic glucose production is impaired • key regulator in hepatic fat metabolism → hyperglycemia • transcription factor that bind to sterol regulatory element • hyperglycemia and hyperinsulinemia both stimulate DNL by activation of ChREBP & SREBP-1c • basi loop helix transcription factor

• stimulates liver to overproduce trisaccharide rich VLDL in fasting state • SREBP-1 expression produces two different isoforms: SREBP-1a and -1c • isoforms differ in first exon → SREBP-1c responsible for DNL

• SREBP-2 regulates genes of cholesterol metabolism

• mTORC1 activation not sufficient to stimulate SREBP1c in absence of Akt signaling Molecular Disease Mechanisms 121 Hepatic Steatosis Molecular Disease Mechanisms 122 Hepatic Steatosis

Hepatic Steatosis Pathways Two Hit Theory

Molecular Disease Mechanisms 123 Hepatic Steatosis Molecular Disease Mechanisms 124 Hepatic Steatosis

Cytokines and Oxidative Stress & Genes that Genetic Determinants of Hepatic Steatosis Influence NAFLD Predisposition 122 Antwort 121 Antwort

• first hit: accumulation of fat in hepatocytes (lipid partitioning)

◦ pro-inflammatory cytokines (TNF-α) are produced directly by heptaocytes in response to increased supply of free FA and/or by adipose tissue macrophages that increase during obesity

◦ fibrosis is thought to arise as part of normal healing response to inflamma- tion and injury

• subsequent hits:

◦ chronic oxidative stress with production of ROS

◦ secretion of pro-inflammatory cytokines

◦ mitochondrial dysfunction

◦ liver injury, hepatic apoptosis and fibrosis

124 Antwort 123 Antwort

• hepatic lipid export - MTTP, ApoB • Adiponectin: inversely associated with obesity, BMI, metabolic syndrome, visceral adiposity, NAFLD • hepatic lipid uptake - APOCIII • IL-6: implicated in insulin resistance, NASH • hepatic lipid synthesis - DGAT2, SLC25A13 • TNF-α: elevated levels with insulin resistance, metabolic syndrome • insulin resistance - AKT2, ADIPOQ, IRS1, PPARs • CRP: may be marker for hepatic steatosis - but not for severity of NAFLD • hepatic TAG hydrolysis - ATGL, LIPA ◦ insulin signaling and regulation of fat metabolism • FA oxidation disorders - MCAD, LCAD, VLCAD ◦ oxidative stress • lipodystrophies - LMNA; PPARg ◦ response to endotoxins

◦ release of cytokines

◦ severity of fibrosis Molecular Disease Mechanisms 125 Hepatic Steatosis Molecular Disease Mechanisms 126 Hepatic Steatosis

PNPLA3 & HFE Gene Biochemical, Anthropometric and Clinical Features of NAFLD

Molecular Disease Mechanisms 127 Hepatic Steatosis Molecular Disease Mechanisms 128 Hepatic Steatosis

Diagnosis of Liver Disease Dietary Factors in Liver Disease 126 Antwort 125 Antwort

• elevated serum TAGs - > 150mg/dL • patatin-like phospholipase domain-containing protein 3, Adiponutrin

• elevated ALT, but no universal standard what is high • function not entirely clear, triacylgycerolipase that mediates triacylglycerol hydrolysis fastingglucose∗fastinginsulin • insulin resistance: 405 > 3 • SNP that regulates variety of mechanisms involved with development of NAFLD • overweight - BMI > 95th percentile ◦ human hemochromatosis protein • abdominal obesity - weight circumference > 90th percentile ◦ involved with insulin sensitivity, oxidative stress • Acanthosis nigricans

• hepatomegaly - enlarged liver

128 Antwort 127 Antwort

• overconsumption of fructose & soft drinks • liver biopsy - invasive, risky, expensive

• lower consumption of fiber • diagnostic imaging:

• overconsumption of meat/saturated fat/cholesterol ◦ CT: exposure to ionizing radiation

• lower consumption of fish, Ω−3 FA, and lower consumption of some vitamins ◦ MRI: no exposure to ionizing radiation but expensive (E) ◦ ultrasound: accessible, no ionizing radiation exposure • sucrose → increases hepatic TG synthesis ◦ low sensitivity: mild-moderate steatosis • fructose → increases DNL, plasma TG & insulin resistance in animal models ◦ limited beam penetration in obese individuals Molecular Disease Mechanisms 129 Hepatic Steatosis Molecular Disease Mechanisms 130 Hypertension

Treatment of Liver Disease Blood Pressure

Molecular Disease Mechanisms 131 Hypertension Molecular Disease Mechanisms 132 Hypertension

Types of and Risk Factors for Hypertension Renin-Angiotensin System 130 Antwort 129 Antwort

• determined by cardiac output multiplied by peripheral resistance • lifestyle interventions

• major factors maintaining blood pressure: sympathetic nervous system and ◦ physical activity → improves insulin resistance, reduces adipose tissue kidneys ◦ weight loss - 7-10% reductin in total body weight may reduce hepatic fat • optimal: systolic < 120 mmHG, diastolic < 80 accumulation in obese adults and adolescents

systolic blood pressure diastolic blood pressure • bariatric surgery - stomach reduction normal < 120 < 80 • pharmacological agents pre-hypertension 120-139 80-89 hypertension - stage 1 140-159 90-99 ◦ metformin - metabolic effects, momentarily not recommended for NAFLD hypertension - stage 2 ≥ 160 ≥ 100 treatment

◦ vitamin E, Ω − 3 fats

132 Antwort 131 Antwort

• Renin (= angiotensinogenase) is a proteolytic enzyme • primary hypertension: present in ≈ 90% of patients, cause unknown

• produced by juxtaglomerular cells of kidney • secondary hypertension: ≈ 10% resulting from other disease

• secreted in response to ↓ arterial blood pressure, ↓ Na+ in macula densa, ↑ • either caused by an increase in systemic vascular resistance (SVR) or an increase in cardiac output (CO) SNS activity ◦ SVR determined by the resistance of vessels • Renin cleaves Angiotensinogen to Angiotensin I (decapeptide) → ACE con- verts AT-I to AT-II (octapeptide) → AT-II gets degraded to AT-III by pep- ◦ CO determined by heart rate and stroke volume tidase • age above 55 in men and 65 in women

• AT-II has very short half life (1 min) • family history

• AT-II and AT-III stimulate Aldosterone secretion from adrenal cortex • smoking

• obesity, DM and dyslipidemia

• microalbuminuria Molecular Disease Mechanisms 133 Hypertension Molecular Disease Mechanisms 134 Hypertension

Angiotensin II Blood Pressure Regulation

Molecular Disease Mechanisms 135 Hypertension Molecular Disease Mechanisms 136 Hypertension

Antihypertensive Drugs Metabolic Syndrome - Symptoms 134 Antwort 133 Antwort

• blood pressure = cardiac output (CO) × resistance to passage of blood • vasoconstrictor - particularly arteriolar through pre-capillary arterioles (PVR) • direct action and release of adrenaline/noradrenaline • CO and PVR are maintained min to min by arterioles, post-capillary venules, ◦ promotes movement of fluid from vascular to extravascular and heart ◦ more potent vasopressor than noradrenaline - NA - promotes Na+ and water reabsorption • kidney controls volume of intravascular fluid ◦↑ myocardial force of contraction - Ca2+ influx - and ↑ heart rate by sympathetic activity • baroreflex, humoral mechanism, and renin-angiotensin-aldosterone system → cardiac output is reduced and cardiac work is increased regulate above 4 sites • vasoconstriction of renal arterioles

• local agents like NO • decreases NO release

• decreases fibrinolysis in blood

• induces drinking behavior and ADH release by acting in CNS

• mitogenic effect → cell proliferation

136 Antwort 135 Antwort

• glucose intolerance • diuretics: act on kidneys to increase excretion of Na and water from blood → ↓ blood pressure • insulin resistance • ACE inhibitors: inhibit synthesis of AT-II → ↓ peripheral resistance and blood volume • inflammation • angiotensin II blockers: block binding of AT-II to its receptors

• obesity • centrally acting: on α2A receptors → ↓ sympathetic outflow → ↓ blood pressure

• dyslipidemia with elevated triglycerides • α and/or β-adrenergic blockers: block of α/β adrenergic receptors in SMC → vasodi- latation • hypertension • calcium channel blockers (CCB): block Ca2+ influx in SMC → relaxation of SMC → • osteoporosis ↓ blood pressure • K+ channel activators: leaking of K+ → hyper polarization of SMC → relaxation • low HDL-cholesterol, high LDL-cholesterol • vasodilators: arteriolar/arterio-venular • related disorders - PCOS and NASH Molecular Disease Mechanisms 137 Hypertension Molecular Disease Mechanisms 138 Cardiovascular Disease

Hyertension Pathways Cardiovascular Disease

Molecular Disease Mechanisms 139 Cardiovascular Disease Molecular Disease Mechanisms 140 Cardiovascular Disease

Consequences of Atherosclerosis & Types of Stroke Myocardial Infarction 138 Antwort 137 Antwort

• typically affects ability of heart to pump blood

• OR: ability of blood vessels to deliver blood

◦ arteries bring oxygenated blood to where it is required

◦ coronary arteries provide heart with blood

• main forms:

◦ atherosclerosis: progressive narrowing of arteries typically caused by fatty deposits

◦ coronary artery disease (CAD)/coronary heart diseae (CHD): blockage of the coronary artery

◦ heart failure

◦ hypertension (high blood pressure)

◦ cerebrovascular diseae

140 Antwort 139 Antwort

• ischemic stroke: (80%) caused by lack of blood flow to brain, typically due • reduced elasticity of arteries → more strain on heart and less able to respond to thrombus + atherosclerosis to different oxygen demands

◦ thrombotic stroke: thrombus starts in artery near brain • can cause an aneurism - dilation of artery - which can rupture and leak blood into surroundings → haemorrhage ◦ embolic stroke: thrombus develops somwhere in body and travels to brain • reduces blood flow or completely block if a thrombus gets loged • transient ischemic attack - TIA: caused by temporary disruption of blood flow to brain, ”mini-stroke”, warning sign! myocardial infarction - MI heart attack, occurs when there is absence of blood flow to heart, most often caused by CAD • hemorrhagic stroke: (20%) caused by uncontrolled bleeding of brain, dis- rupts normal blood flow, kills brain cells, can be caused by weakness of artery wall

◦ aneurysm: weakned vessel full of blood

◦ arteriovenous malformation (AVM): malformed blood vessels in brain that make artery weak, typically prsent at birth Molecular Disease Mechanisms 141 Cardiovascular Disease Molecular Disease Mechanisms 142 Cardiovascular Disease

Lipoproteins Framingham Study

Molecular Disease Mechanisms 143 Cardiovascular Disease Molecular Disease Mechanisms 144 Cardiovascular Disease

Major Changeable CVD Risk Factors Major Changeable CVD Risk Factors (I/II) (II/II) 142 Antwort 141 Antwort

• identification of risk factors for CVD Lipoprotein Apolipoproteins Function HDL ApoA1 reverse cholesterol transport • major changable LDL ApoB100 cholesterol transfer VLDL ApoB100 TAG transporter ◦ hypertension, high blood cholesterol, smoking, physical inactvity, obesity, diabetes Chylomicrons ApoB48 TAG transporter Lp(a) Apo(a) ??? (indicator for CVD risk) • mino changeable

• stress, low Ω-3 FA, high alcohol consumption

• non-changable

• age, male gender, heredity, ethnicity

144 Antwort 143 Antwort

• smoking • hypertension

◦ nicotine can cause lesions in artery walls ◦ can cause damage to blood vessels, puts extra strain on heart

◦ CO causes lesions in artery walls and decreases blood’s ability to transport ◦ cause unknown, but high body fat, high salt intake, lack of exercise are risk oxygen factors

• physical inactivity • high serum cholesterol

◦ exercise can lower blood pressure ◦ typically caused by eating too much saturated fat

◦ increase HDL and lower LDL and VLDL ◦ can deposit in artery walls

◦ reduce str4ess ◦ LDL/VLDL: ”bad” choesterol, forms plaques

◦ maintain body weight and control T2D ◦ HDL: ”good” cholesterol. lowers ox-LDL deposition in artery walls

• obesity: can lead to hypertension, low HDL, T2D • Diabetes Mellitus: impaired ability of blood to store glucose Molecular Disease Mechanisms 145 Cardiovascular Disease Molecular Disease Mechanisms 146 Cardiovascular Disease

Other Risk Factors for CVD Treatment of CVD

Molecular Disease Mechanisms 147 Cardiovascular Disease Molecular Disease Mechanisms 148 Cardiovascular Disease

Plaque Formation CVD Pathways 146 Antwort 145 Antwort

• heart transplants • stress: increased blood pressure, blood clotting, cholesterol levels

• artificial hearts: now used as bridge during surgery, possibly permanent de- • low Ω3-fatty acid intake: inverse correlation with CVD vices in future • alcohol: low daily intake associated with lower CVD risk, high intake can • implanted pace maker damage heart muscle

• coronary artery bypass: replacing blocked/narrow coronary arteries with • gender: males higher risk (biological or cutural?) healthy segment of other arteries • ethnicity: higher risk in african americans, latinos, aboriginals and south • angioplasty: enlarging artery by using ballon-type instrument asians

148 Antwort 147 Antwort Molecular Disease Mechanisms 149 Cancer Incidences Molecular Disease Mechanisms 150 Cancer Incidences

Stage of Cancer & Types of Cancer & Symptoms of Metastasis Most Common Cancers

Molecular Disease Mechanisms 151 Cancer Incidences Molecular Disease Mechanisms 152 Cancer Risk Factors

Factor Contributing to Stomach Cancer Mortality IARC Classification of Carcinogens Decline 150 Antwort 149 Antwort

• carcinoma: epithelial tissues in internal and external lining of body I Early Stage: cancer has spread to other tissues in small area

◦ squamous cell carcinoma: develop in squamous epithelium of organs (skin, II Localized: Tumor is between 20-50mm and some lymph nodes are involved bladder, esophagus, and lung) or a tumor larger than 50mm with no lymph nodes involved

◦ adenocarcinoma: develop in organ or gland (e.g. breast cancer) III Regional Spread: Tumor is larger than 50mm, with more lymph nodes involved across wide region • sarcoma: connective tissue - e.g. bone, tendons, cartilage, muscle, and fat IV Distant Spread: cancer has spread to other parts of the body • leukemia: blood cancers, originate in bone marrow • enlarged lymph nodes • lymphoma: lymphatic system cancers • enlarged liver or enlarged spleen • most common: lung, colon, breast, prostate, stomach, liver • pain or fracture of affected bones

• neurological symptoms

152 Antwort 151 Antwort

• Group 1: agent is carcinogenic to humans • decrease of exposure to Heliobacter pylori - H-pylori - infections

• Group 2A: agent is probably carcinogenic to humans • dietary and other exogenous factors

• Group 2B: agent is possibly carcinogenic to humans • better food preservation and refrigeration → replacing salting, pickling, and smoking • Group 3: agent is not classifiable as to its carcinogenicity to humans • availability of fresh food and vegetables • Group 4: agent is probably not carcinogenic to humans • improved detection and therapy of stomach cancer and H. pylori infections Molecular Disease Mechanisms 153 Cancer Risk Factors Molecular Disease Mechanisms 154 Cancer Risk Factors

Asbestos Other Chemical/Environmental Carcinogens

Molecular Disease Mechanisms 155 Cancer Risk Factors Molecular Disease Mechanisms 156 Cellular Cancer Characteristics

Alcohol & Diethylstilbestrol (DES) Hallmarks of Cancer & Therapeutic Targets 154 Antwort 153 Antwort

• pesticides • group of oxidized silicate minerals that occur as fibers

◦ leukemia, lymphoma, multiple myeloma, and soft tissue sarcoma • resistant to heat, fire, chemical, and does not conduct electricity → many industrial uses → nowadays replaced by cellulose fiber ◦ cancers of skin, lip, stomach, brain, and prostate • causes lung cancer and mesothelioma (rare cancer of mesothelia: thin mem- • formaldehyde branes lining chest and abdomen)

◦ emitted from pressed wood building materials • carcinogenesis influenced by fiber size and physical properties

◦ (myeloid) leukemia, possibly nascal cancers • interferes with cell division

• chemical dyes, especially hair dyes before 1980

◦ leukemias and lymphomas

156 Antwort 155 Antwort

• sustaining proliferative signaling → EGFR inhibitors • association with esophageal, head and neck, liver, breast, and colorectal can- cer, squamous cell carcinoma • evading growth suppressors → cyclin-dependent kinase inhibitors • reaction of ethanol to acetaldehyde generates ROS • avoiding immune destruction → immune activating anti-CTLA4 MAb

• enabling replicative immortality → telomerase inhibitors • inhibiton of nutrient absorption (vit B, vit C, folate)

• tumor-promoting inflammation → selective anti-inflammatory drugs • increase in blood estrogen (linked to breast cancer)

• activating invasion & metastasis → inhibitors of HGF/c-Met  synthetic estrogen prescribed to pregnant women in 40s-70s to prevent preg- • inducing angiogenesis → inhibitors of VEGF signaling nancy complications

• genome instability & mutation → PARP inhibitors  endocrine-disrupting chemical

• resisting cell death → pro-apoptotic BH3 mimetics  prenatal exposure linked to clear cell adenocarcinoma: cervix and vagina

• deregulating cellular energetics → aerobic glycolysis inhibitors  ”DES daugthers” 40x risk of cancer Molecular Disease Mechanisms 157 Cellular Cancer Characteristics Molecular Disease Mechanisms 158 Cellular Cancer Characteristics

Hayflick Limit & Evading Growth Suppressors Sustained Proliferative Signaling

Molecular Disease Mechanisms 159 Cellular Cancer Characteristics Molecular Disease Mechanisms 160 Cellular Cancer Characteristics

Inducing Angiogenesis, Activating Invasion and Metastasis & Resisting Cell Death & Enabling Replicative Immortality Avoiding Immune Destruction 158 Antwort 157 Antwort

• generally involves tumor suppressor proteins: modulation of cell growth through • Hayflick Limit number of cell divisions until senescence (in humans 40-60) negative regulation of cell cycle or by promoting apoptosis • self production of growth factors (e.g. HGF) • RB: retinoblastoma - inactivated by CDK phosphorylation • self production of cognate receptors (e.g. Tyrosine kinases)

• p53: ”guardian of the genome” • somatic mutations that activate signaling circuits (e.g. PI3-kinase)

• loss of contact inhibition: • defects in negative-feedback loops (e.g. Ras)

◦ Merlin is a membrane scaffolding protein • Ras proteins essential components of signaling networks controlling proliferation, differen- tiation, and survival ◦ mediates contact inhibition by coupling cell surface adhesion molecules • mutations of H-ras, N-ras, or K-ras genes frequently found in human tumors (≈30%)

◦ loss of Merlin function in various cancers • oncogenic mutations concentrated within 2 hotspots of primary nucleotide sequence of all ras family members

160 Antwort 159 Antwort

• growth of blood vessels from pre-existing vasculature • alterations in cell-to-cell or cell-to-ECM adhesion molecules (e.g. loss of E- cadherin) • cancer cells need high amounts of nutrients and oxygen and fast removal of CO2 and waste • traits of invasion and metastasis:

 Bcl-2 protein family are inhibitors of apoptosis - via binding to pro-apoptotic ◦ loss of adheren junctions proteins like Bax and Bak - in outer mitochondrial membrane ◦ expression of matrix degrading enzymes  Bax and Bak disrupt mitochondrial membrane to release Cytochrome C to ◦ increased motility activate proteases for apoptosis ◦ resistance to apoptosis • immune system provides surveillance to destroy abnormal/cancer cells • telomerase lengthen ends of telomeres → high levels in cancer cells, relatively • some cancer cells avoid immune detection → evade eradication low levels in normal cells (get shorter by 50-200 bp per replication) Molecular Disease Mechanisms 161 Cellular Cancer Characteristics Molecular Disease Mechanisms 162 Genotoxic Carcinogenesis

Deregulating Cellular Energetics Differences Genotoxic/ Non-Genotoxic Carcinogens

Molecular Disease Mechanisms 163 Genotoxic Carcinogenesis Molecular Disease Mechanisms 164 Genotoxic Carcinogenesis

Examples of Genotoxic Carcinogens N -Acetyl Transferase NAT 162 Antwort 161 Antwort

• direct reaction with DNA to alter its structure • cancer cells reprogram their energy production by limiting metabolism largely to glycolysis and lactic acid fermentatin → Warburg effect • direct carcinogens: no metabolic activation (alkylating agents) • counterintuitive: ATP production 18x less efficient by glycolysis → hypothe- • indirect carcinogens: require metabolic activation (aromatic amines, PAHs) sis: increased glycolysis produces molecular intermediates useful for growth - e.g. nucleosides and amino acids  don’t directly cause mutation

 increase cell proliferation

 decrease apoptosis

 induction of metabolic enzymes

 e.g. endocrine disruptors - bisphenol-A (BPA) & estradiol

164 Antwort 163 Antwort

• polymorphisms in NAT can lead to bladder, liver, lung, and colorectal cancer, • UV radiation myeloma and lymphoma • ROS • association between NAT enzyme activities and cancer risk • polycyclic aromatic hydrocarbons (PAHs) - benzopyrene

• NATs involved in metabolic activation and detoxification of arylamines • myotoxins/aflatoxins

• located in cytosol, prevalent in liver of mammals • acrylamide

• cofactor: Ac-CoA → catalyzes acetylations • alkenes and haloalkanes

• two human variants: NAT1 and NAT2 • aromatic amines • furans • competing pathways: detoxification → N-acetylation or activation → damage • polycyclic aromatic amines • polymorphisms divide humans into rapid, intermediate, and slow acetylators • N-nitrosamines • slow acetylators exhibit increased risk in developing bladder cancer Molecular Disease Mechanisms 165 Genotoxic Carcinogenesis Molecular Disease Mechanisms 166 Genotoxic Carcinogenesis

NAT Mechanism Glutathione S-Transferase GST

Molecular Disease Mechanisms 167 Genotoxic Carcinogenesis Molecular Disease Mechanisms 168 Genotoxic Carcinogenesis

DNA Adducts Sites of Oxidative Damage to Nucleotides & Resulting Adducts 166 Antwort 165 Antwort

• large multigene family of enzymes involved in detoxification of potentially • nitrenium potent DNA alkylating agent genotoxic chemicals

• conjugation of the reduced form of glutathione (GSH) to xenobiotic substrates for detoxification

• high concentrations in liver, intestine, kidney

• account for ≈ 10% of cellular proteins

• associated with colorectal cancer

168 Antwort 167 Antwort

• reactions leading to adducts:

◦ oxidation

◦ hydrolysis

◦ photodimerization

◦ alklyation : methlyation adducts/ other bulky alkylation products

• DNA adducts are NOT mutations, instead precursors to mutations when it is the substrate for DNA synthesis

• key basis of genotoxic mechanism of carcinogenesis Molecular Disease Mechanisms 169 Genotoxic Carcinogenesis Molecular Disease Mechanisms 170 Genotoxic Carcinogenesis

Frequent Sites of Alkylation in DNA Aflatoxin

Molecular Disease Mechanisms 171 Genotoxic Carcinogenesis Molecular Disease Mechanisms 172 Genotoxic Carcinogenesis

Acrylamide Polycyclic Aromatic Hydrocarbons - PAH & Oxidation-Induced Mutagenesis 170 Antwort 169 Antwort

• produced by fungi (Aspergillus sp.) found in soil

• dietary exposure: contamination of grains, seeds, and nuts in high tempera- ture and high humidity storage

• liver carcinogen - amongst most potent natural liver toxicants

• contributor to high incidences of liver cancer in China and West Africa

• metabolic activation → alkylation of DNA → two competing damage out- comes: depurination (strongly promoted by alkylation)/ adduct hydrolysis

172 Antwort 171 Antwort

• among others: napthalaene, chrysene, pyrene, coronene, ovalene • is metabolically activated by P450 to form glycidamide (DNA reactive epox- ide) • lead to alkylation of guanine • detoxification by GSH reaction • which leads to either DNA repair or translesion synthesis → transversion from G to T • DNA reacts at multiple positions and at both sides of epoxide

• biomontoring: hemoglobin adducts are markers of acrylamide exposure over preceding few months

• levels increase with dietary intake  polymerase-mediated DNA replication of 8-oxoG often incorporates an A • smokers have 3-4x higher adduct levels than non-smokers  mismatch is not proofread efficiently

 poor proofreading can be attributed to geometry of 8-oxo-G:A → similar to natural G-C • younger children may have slightly higher levels due to increased intake of base pair acrylamide-containing foods relative to body size

 causes G-T transversion Molecular Disease Mechanisms 173 Genotoxic Carcinogenesis Molecular Disease Mechanisms 174 Genotoxic Carcinogenesis

Role of DNA Polymerases in Mutagenesis Determinants of Replication Fidelity and Mutation Rates

Molecular Disease Mechanisms 175 Genotoxic Carcinogenesis Molecular Disease Mechanisms 176 Genotoxic Carcinogenesis

DNA Repair Pathways Nucleotide Excision Repair NER 174 Antwort 173 Antwort

• biochemical factors of polymerases • Y-family polymerases catalyze translesion DNA synthesis

◦ expression levels • error rates: η>β>α>γ>δ>ε

◦ regulation • Polymerase Switching Models ◦ kinetic behavior/accuracy ◦ 2 relevant mechanisms for TLS, depending on lesion structure and active • chemical factors enzymes

◦ structure/size of modification ◦ one TLS polymerase responsible for insertion and extension

◦ Watson-Crick H-bonding ◦ OR: two TLS polymerases responsible for insertion and extension ◦ Base Stacking Interactions with neighboring bases as well as aromatic residues on the B Family Poly- Y Family Poly- enzyme Characteristics merases (δ, ε) merases (η, ι, κ) ◦ A-Rule: DNA polymerases insert A opposite abasic sites Error Rate low (< 10−10/bp) high (10−2 to 10−4 • elimination of damage by repair Fidelity high low Proofreading 3’ exonuclease none Processivity high low

176 Antwort 175 Antwort

• substrates: bulky-helix distorting DNA damage • nucleotide excision repair

◦ thymine dimers and 6,4-photoproducts → UV exposure • base excision repair

◦ chemotherapy-induced DNA crosslinks (e.g. Cisplatin) • mismatch repair

• steps: recognition → assembly of multi-protein complex for remodeling and • homologous recombination cutting → removal of short (12bp) single-stranded DNA segment containing lesion → DNA synthesis → ligation • non-homologous end-joining • dealkylation (direct reversion) Molecular Disease Mechanisms 177 Genotoxic Carcinogenesis Molecular Disease Mechanisms 178 Genotoxic Carcinogenesis

Base Excision Repair BER Substrates BER

Molecular Disease Mechanisms 179 Genotoxic Carcinogenesis Molecular Disease Mechanisms 180 Genotoxic Carcinogenesis

Importance of BER in Protection Against Colon Mismatch Repair Carcinogenesis MMR 178 Antwort 177 Antwort

• small, non-helix-distorting base adducts

• initiation: DNA glycosylases

◦ recognize damage

◦ remove altered bases

◦ form AP sites

• AP sites cleaved by AP endonuclease

• new DNA synthesized by polymerase

◦ in short-patch single nucleotide is replaced

◦ in long-patch 2-10 nucleotides are synthesized and flap endonuclease trims displaced strand

◦ strand sealed by ligase

180 Antwort 179 Antwort

• repairs misincorporated bases during DNA replication • chronic inflammation increases cancer risk

• MutS recognizes and binds mismatch • ROS induce DNA damage recognized by alkyladenine DNA glycosylase (Aag)

• MutL makes incision, DNA contains nicks not yet sealed by DNA ligase • Aag recognition of adduct initiates BER

• patch is removed (RPA - replication protein A, EXO1 - exonuclease 1) • Aag prevents colonic epithelial damage

• DNA synthesis (polymerase δ) and ligation (ligase) • reduces severity of chemical-induced colon tumorigenesis

• hereditary nonpolyposis colorectal cancers attributed to mutations in the • Aag-/- mice accumulate DNA adducts following stimulation of colonic inflam- genes encoding MSH2 and MLH1 → tumor suppressor genes mation Molecular Disease Mechanisms 181 Genotoxic Carcinogenesis Molecular Disease Mechanisms 182 Genotoxic Carcinogenesis

Homologous Recombination Non-Homologous End-Joining NHEJ

Molecular Disease Mechanisms 183 Genotoxic Carcinogenesis Molecular Disease Mechanisms 184 Receptor-Mediated Carcinogenesis

Direct Reversion/Dealkylation Repair Aryl Hydrocarbon Receptors AHR (I/II) 182 Antwort 181 Antwort

• recognition: Ku70/Ku80 encircles duplex DNA at DSB • after double-strand break

• Ku70/Ku80 stabilizes/structurally aligns the two DNA ends and recruits • sections of DNA around 5’ ends of break are cut away in a process called resection DNA-Protein Kinase (PK) • in the strand invasion step that follows, an overhanging 3’ end of the broken DNA molecule then ”invades” a similar or identical DNA molecule that is not broken • DNA-PK phosphorylates and activates NHEJ effector complex (ligase IV/XRC44/XLF) that ligates broken DNA • after strand invasion: DSBR (double-strand break repair) OR SDSA (synthesis-dependent strand annealing)

184 Antwort 183 Antwort

• activated ligand-receptor complex migrates from cytoplasm to nucleus → transcription fac- • via o-6 methylguanine methyltransferase (MGMT) tor • MGMT is consumed, can no longer be used • activates the expression of multiple phase I and II xenobiotic chemical metabolizing enzyme genes (eg. CYP1A1 gene) • non-enzymatic process • ARNT (Ah receptor nuclear translocator) binds ligated AhR and transports it to nucleus

• ligands are generally planar aromatic hydrocarbon compounds:

◦ dioxin

◦ plant flavenoids

◦ polyphenolic compounds

◦ PAHs

◦ endogenous ligands of AhR include tryptophan-derived metabolites Molecular Disease Mechanisms 185 Receptor-Mediated Carcinogenesis Molecular Disease Mechanisms 186 Receptor-Mediated Carcinogenesis

Aryl Hydrocarbon Receptors Dioxins AHR (II/II)

Molecular Disease Mechanisms 187 Receptor-Mediated Carcinogenesis Molecular Disease Mechanisms 188 Hormone-Mediated Carcinogenesis

Belgian Dioxin Scandal 1999 Breast Cancer Risk Factors & Geneeral Hormone Function 186 Antwort 185 Antwort

• bind to AhR • inhibits apoptosis by

• a family of compounds with diverse chlorine substitutions at the benzene rings of PCDDs - ◦ induction of TGFα → activation of NF-κB polychlorinated dibenzo-p-dioxin) - and PCDFs - polychlorinated dibenzofurans ◦ induction of mdm2 → inhibition of p53

• sustained hyperactivation of AhR due to long half live in humans (dioxin ≈ 10 years, high binding affinity, antagonism

• induces expression of enzymes that bioactivate other carcinogens • TCDD: industry byproduct, used as pesticides since before WWII • induce growth factors • can be detected anywhere in the environment, most human exposure from food → accumulates in fat • promote inflammation signals • very high lipophilicity and stability - half live ≈ 7 years • overexpression of AHR and its target growth factors characterized in cancer • classified as known human carcinogen cells

• classification based on mechanistic considerations focusing on Ah receptor

188 Antwort 187 Antwort

• prolonged exposure to high levels of estrogens • farms in Belgium ordered to destroy livestock given feed contaminated with dioxin • in obese women, adipose tissue major source of estorgens • bans on Belgian agriculture exports of eggs, chickens, pork, and beef • hormone replacement therapy (HRT) raises likelihood of breast cancer com- pared to no treatment • contamination thought to have come from tanks used to hold animal fats for producing animal feeds • high levels of blood estrogen in post-menopausal women • tanks previously used to hold industrial oil containing dioxins • increased rate in women taking estrogens • tanks not sufficiently cleaned so animal fats became contaminated with dioxin-  control of enzymatic reactions bearing oil residues

 control of movement of ions or molecules across membranes • tainted animal feeds were supplied to hundreds of Belgian farms, and were exported to France, Holland, and Germany  control of gene expression and protein synthesis • food crisis precipitated a massive political crisis inside Belgium  act at very low concentrations: nano to picomolar range (109 to 1012) Molecular Disease Mechanisms 189 Hormone-Mediated Carcinogenesis Molecular Disease Mechanisms 190 Hormone-Mediated Carcinogenesis

Mechanism of Endocrine Modulation by Estrogen Features of ER Disrupting Compounds Agonists

Molecular Disease Mechanisms 191 Hormone-Mediated Carcinogenesis Molecular Disease Mechanisms 192 Hormone-Mediated Carcinogenesis

Endocrine Disrupting Compounds Tamoxifen 190 Antwort 189 Antwort

• compete with endogenous estrogen for ER binding sites • two mechanisms:

• shared E2 structural features determined by ER binding pocket ◦ genomic: intracellular estrogen receptors

• similar ligand binding pockets, but subtle difference in amino acids lining the ◦ non-genomic: membrane estrogen receptors (mER) → activation of kinase pocket contribute to ligand selectivity signaling cascades

• naturally binding ligand: 17β-estradiol (E2)

• PCBs: once widely used as dielectric and coolant fluid in electrical appa- ratus, synthetic hormone mimic, high fat solubility and stability can cause bioaccumlation

192 Antwort 191 Antwort

• used to prevent breast cancer in women and treat breast cancer in women • industrial phenolics and men ◦ bisphenol A • competitive inhibitor of estrogen that blocks the action of estrogens in several ◦ alkyl phenols tissues, notably the breast • phytoestrogens (dietary estrogens) - e.g. genistein • therapeutic compound acting as a ligand for estrogen receptors (ER), but with distinctly different spectrum of activities from natural hormone 17-βestradiol • organochlorine pesticides

• exhibits anti-estrogen activity on mammary tissues in postmenopausal women ◦ methoxychlor ◦ DDT • tamoxifen enters cell, binds to estrogen receptors → when estrogen enters cell, cannot bind to receptors • pharmaceutical agents

◦ tamoxifen

◦ diethylstilbestrol (DES) Molecular Disease Mechanisms 193 Epigenetics of Carcinogenesis Molecular Disease Mechanisms 194 Epigenetics of Carcinogenesis

Basics of Epigenetics Epigenome, Histone Modifications, & Cancer Epigenome

Molecular Disease Mechanisms 195 Epigenetics of Carcinogenesis Molecular Disease Mechanisms 196 Epigenetics of Carcinogenesis

CpG Islands & DNMTs TETs 194 Antwort 193 Antwort epigenome balanced state of chromatin, nucleosome (147 bp of DNA around • heritable changes in gene expression not attributable to changes in sequence histone octamer) positioning and DNA methylation • mediate reprogramming during development and maintenance of cell identity • repressive vs. enhancing modifications for histones and DNA → methylation during the life of an organism and acetylation of DNA and histones • histones: 16 modifications, DNA 4 • histone methylation can lead to either transcriptional repression or activation • common epigenetic changes: ◦ H3K4 Me: activation ◦ DNA methylation ◦ H3K9 Me: silencing  genome wide hypomethylation → transcriptional activation of oncogenes

 cancer epigenome characterized by global losses in DNA methylation  promoter-specific hypermethylation → silencing of tumor suppressor genes

 certain genes have abnormal gains of DNA methylation and repressive histone ◦ histone modification modifications in promoter region CpG islands ◦ ncRNA expression

196 Antwort 195 Antwort

• regulate gene transcription by controlling 5-hmC, 5-formylC and 5-carboxyC • DNA region of > 500 bp with a high CpG density, found upstream of many mammalian levels → modulate chromatin structure genes

• TET2 mutated in many cancers, TET1 and 3 mutations are rare • DNA methylation - especially around promoter regions - often inhibits transcription •≈ 29000 regions in human genome • TET2 mutations (→ non-functional enzyme) lead to methylation imbalance • potential biomarker for cancer • drug development with TET as target difficult → inactivated in cancer • de novo methylation: DNMT3a and DNMT3b

• maintenance metyhlation: DNMT1

• demethylation can occur in absence of DNMT1

◦ passive demethylation: continued rounds of DNA replication /active demethylation: with- out DNA replication

• decitabine: hypomethylating agent for treatment of acute myeloid leukemia (AML) → in- hibits DNMT methylation Molecular Disease Mechanisms 197 Viral Carcinogenesis Molecular Disease Mechanisms 198 Viral Carcinogenesis

Virus Basics RSV Host Infection

Molecular Disease Mechanisms 199 Viral Carcinogenesis Molecular Disease Mechanisms 200 Viral Carcinogenesis

RSV - Rous Sarcoma Virus v-src 198 Antwort 197 Antwort

• consist of RNA or DNA packaged in a protein capsid

• can multiply only inside host cell

• outcome of viral infection:

◦ lysis of infected cells with release of viral particles

◦ integration of nucleic acid sequence into host chromosome

• Rous Peyton showed role of a Rous Sarcoma Virus - RSV - in cancer trans- mission in chicken

200 Antwort 199 Antwort

• first discovered oncogene • RNA genome

• transduced allele of a cellular gene - c-src • four genes:

• lacks regulatory C-domain → C-terminal deletion, which leads to constitutive ◦ gag - capsid protein activation ◦ pol - reverse transcriptase • effects of src on tumor behaviour: ◦ env - envelope gene ◦ increased cell motility ◦ src - tyrosine receptor kinase: attaches phosphate groups to tyrosine in host ◦ ECM degradation cell proteins

◦ adherens junction degradation

◦ stimulated angiogenesis Molecular Disease Mechanisms 201 Viral Carcinogenesis Molecular Disease Mechanisms 202 Viral Carcinogenesis

Carcinogenic Viruses for Humans Burkitt’s Lymphoma

Molecular Disease Mechanisms 203 Viral Carcinogenesis Molecular Disease Mechanisms 204 Radiation-Induced Carcinogenesis

HPV - Human Papillomavirus Radiation Basics 202 Antwort 201 Antwort

• starts in immune B-cells • EBV - Epstain-Barr

• fastest growing human tumor • Hepatitis B & C

• endemic in equatorial Africa • Kaposi’s sarcoma herpes virus

• caused by Epstein-Barr (EBV) → first discovered human oncogenic virus • HIV-1 - human immunodeficiency virus type 1

• EBV leads to reciprocal chromosomal translocation of chromosome 8 and 14 • HTLV-1 - human T-cell lymphotropic virus type 1

• activates Myc oncogene by juxtapositioning it to an Ig gene locus → tran- • HPV - human papilloma virus scriptionally active

• Myc Protein accumulates more than in normal B cells

204 Antwort 203 Antwort

• non-ionizing radiation: cannot break chemical bonds but can vibrate atoms • DNA virus form papillomavirus family - e.g. radiowaves, microwaves, infrared, visible light • group of > 200 related viruses - ≈ 40 spread sexually • ionizing radiation: breaks chemical bonds - e.g. α & β particles, γ rays • causes several types of cancer - ≈ 5 % worldwide:

• α particles: two protons and two neutrons, block with paper ◦ cervical cancer: virtually all cases caused by HPV, only two types responsibel: HPV 16 & 18 • β particles: high energy electron, block with skin/body tissue ◦ anal cancer: caused by HPV 16 • γ radiation: high energy photon, block with lead ◦ oropharyngeal cancers: middle part of throat, including soft palate, base of tongue, ton- • radiosensitivity of tissue correlates with proliferation rate: lymphatic tissue sils, linked to HPV 16 (lymphocyte) > white blood cells, immature erythrocytes > gastro-intestinal ◦ rare cancers: vaginal cancers, vulvar cancers, penile cancers, most caused by HPV 16 lining > gonadic cells > skin > blood vessels, body cavity lining > glands, liver > connective tissue > muscle > nerves • leads to an overexpression of E6 and E7 → oncoproteins that inibit Rb and p53 tumor suppression • miners of radioactive ore have high incidences of lung cancer → radon and • vaccination available: Gardasil, Gardasil 9, Cervarix thoron gas from rocks and soil Molecular Disease Mechanisms 205 Radiation-Induced Carcinogenesis Molecular Disease Mechanisms 206 Radiation-Induced Carcinogenesis

Direct and Indirect DNA Damage & Dicentric Chromosomes Assay & Ultraviolet Radiation Xeroderma Pigmentosum - XP

Molecular Disease Mechanisms 207 Cancer and Inflammation Molecular Disease Mechanisms 208 Cancer and Inflammation

Immunity and Cancer Molecular Mechanism of Inflammation-Induced Cancer 206 Antwort 205 Antwort

• chromosomes with two centromers

• considered a biomarker of radiation

• widely used for radation dose estimation following accidents and nuclear ex- posures

Xeroderma Pigmentosum

• increase in skin cancer risk 1000-2000x • sun exposure causally linked to basal cell carcinoma, squamous cell carcinoma and malignant melanoma • defects in DNA repair (TT dimers) • UVA (320 to 480nm) - 95% of sun, production of ROS • defective XP proteins in NER • UVB (280 to 320nm) - most correlated with cutaneous cancer ◦ 7 variants (XPVA most common type) • UVC (200 to 280nm) - most dangerous (not in sunlight, e.g in welding ◦ another variant - XPV - defective in TLS by DNA polymerase η torches)

208 Antwort 207 Antwort

• inflammation and innate immunity have a vital and complex role in driving tumorigenesis

• strong epidemiological and preclinical data support anti-inflammatory ap- proach to prevention and treatment of cancer

• several therapeutic agents targeting inflammatory cytokines, transcription factors and immune cells are being developed

• combining treatments targeting adaptive and innate immune systems in tu- mor microenvironment might be advantageous Molecular Disease Mechanisms 209 Cancer and Inflammation Molecular Disease Mechanisms 210 Cancer and Inflammation

Cytokines & Cytokine Receptors JAK-STAT3 in Cancer

Molecular Disease Mechanisms 211 Cancer and Inflammation Molecular Disease Mechanisms 212 Cancer and Inflammation

NF-κB microRNA regulating JAK-STAT3 & IL-1β & IL-18 210 Antwort 209 Antwort

• broad category of small proteins (≈ 5-20 kDa) which are important in cell signaling

• include chemokines, interferons (IF), interleukins (IL), lymphokines, and tumor necrosis factors (TNF)

• cytokine receptor families

◦ JAK/STAT

◦ PI3K/AKT

◦ MAPK

◦ NF-κB

TNFα IL17 IL6 IL10 IL12 IL23 TGFβ TRAIL tumor pro- + + + + + + moting tumor pro- + + + ≈ + + tective

212 Antwort 211 Antwort

• miR-337-3p & miR-135a: inhibit JAK/STAT leading to apoptosis • triggered by inflammation

• let-7: pleotropic effects: chemoresistance or sensitivity, metastasis, malignant transformation

• miR-17-Sp & miR-20a: tumor protective

• miR-24, miR-629, miR-9: tumor promoting effects

IL-1β & IL-18)

• inflammatory cytokines

• immune surveillance: IL-18 induces tumor surveillance in colon cancer

• tumor progression: IL-1β induces tumor growth in breast cancer Molecular Disease Mechanisms 213 Cancer and Inflammation Molecular Disease Mechanisms 214 Cancer and Inflammation

Bortezomib Chimeric Antigen Receptor T-Cell Therapy CAR-T

Molecular Disease Mechanisms 215 Cancer and Inflammation Molecular Disease Mechanisms 216 Introduction to Aging

CAR-T 2.0 Hallmarks of Aging & Interventions 214 Antwort 213 Antwort

• engineering patients’ immune cells to treat their cancer • first proteasome inhibitor anticancer drug

• challenges: • reversible inhibitor of proteasome

◦ dosing: small dose may not obtain ideal curative effect, large dose increases cytokine • boronic acid group can bind and complex active site of threonine hydroxyl release syndrome and tumor lysis syndrome (β5-subunit) and block chymotrypsin-like activity of the proteasome ◦ toxicity: cytokine release syndrome - a severe, body-wide immune reaction • treatment of relapsed and refractory multiple myeloma (hard cancer to treat) ◦ treatment: only approved for small subset of cancers • suppression of NF-κB signaling pathway → downregulation of anti-apoptotic  Kymriah: people up to 25 with acute lymphoblastic leukemia of B-cell origin who are target genes resistant to treatment or have relapsed twice (≈ 100 cases/yr

 Yescarta: large B-cell lymphoma in adults after two other treatments have failed (≈ • NF-κB involved in B-cell maturation → Bortezomib treats B cell malignancies 7.500 people/yr)

◦ price: Kymriah 475.000$, Yescarta 373.000$ one time cost

216 Antwort 215 Antwort

• Primary Hallmarks: molecular damage • brain swelling commonly occurs with CAR-T cell therapy (> 12 patients have died so far) ◦ genomic instability - elimination of damaged cells, telomere attrition - telomerase reacti- vation & epigenetic alterations - epigenetic drugs • making a kill switch ◦ loss of proteostasis - activation of chaperones and proteolytic systems ◦ two engineered proteins located inside CAR-T cell that dimerize when ex- • Antagonistic Hallmarks: consequences on cellular level posed to small-molecule called rimiducid

◦ deregulated nutrient sensing - dietary restriction: IIS and mTOR inhibition, AMPK and ◦ rimiducid activates caspase-9, which kick-starts process of CAR-T-cell sui- sirtuin activation cide ◦ mitochondrial dysfunction - mitohormetics, mitophagy & cellular senescence - clearance of senescent cells

• Integrative Hallmarks: culprits of phenotype

◦ stem cell exhaustion - stem-cell based therapies

◦ altered intercell communication - anti-inflam drugs & blood-borne rejuvenation factors Molecular Disease Mechanisms 217 Introduction to Aging Molecular Disease Mechanisms 218 Introduction to Aging

Definition & Concepts of Aging Model Organism for Aging Research

Molecular Disease Mechanisms 219 Introduction to Aging Molecular Disease Mechanisms 220 Introduction to Aging

Telomere Attrition & Epigenetic Alterations Loss of Proteostasis & Cellular Senescence 218 Antwort 217 Antwort

mouse C. elegans Aging the progressive loss of physiological integrity, leading to impaired func- 2.5-3.5 tion and increased vulnerability to death Lifespan 3-4 weeks years ≈ 30% (or 65% disease related • can be measured and thus studied Gene Homology to Humans ≈ 90% genes) Costs (maintaining/housing) expensive cheap • is plastic - genetic (inheritity of lifespan 15-30%), enviromental and stochastic Ethical issues lots none factors Scalability for high- through- weak high • is universal put • by understanding the underlying mechanisms of aging, we can identify novel • automated lifespan analysis strategies to promote healthy aging • discovery of TOR signaling importance in aging → 6-weeks mTOR inhibitor • insulin/IGF-1 signaling implicated in GWAS lead to a 20% enhanced response to influenza vaccines in elderly • Metformin (drug for Type II diabetes) decreases mortality in diabetic and non-diabetic patients

220 Antwort 219 Antwort

Loss of Proteostasis Telomere Attrition

• leads to aggregation of misfolded proteins • most mammalian somatic cell do not express telomerase → progressive and culmulative loss of telomere-protective sequence from chromosome ends Cellular Senescence • telomere exhaustion explains limited proliferate capacity of some types of in-vitro-cultured cells → Hayflick limit or replicative senescence • can be defined as a stable arrest of cell cycle coupled to sterotyped phenotypic changes • premature aging of telomerase-deficient mice can be reverted when telomerase is genetically reactivated • caused by: telomere shortening, non-telomeric DNA damage. activation of ARF4a/INK lokus (correlates with chronological age) Epigenetic Alterations  transcriptional noise • senescence is anti-cancer and (in young) anti-aging or pro-aging (in old)  RNA processing aberrations

 impaired DNA repair

 chromosomal instability Molecular Disease Mechanisms 221 Introduction to Aging Molecular Disease Mechanisms 222 Introduction to Aging

Deregulated Nutrient Sensing & Mitochondrial Stem Cell Exhaustion & Altered Intercellular Dysfunction Communication

Molecular Disease Mechanisms 223 Aging and Metabolism Molecular Disease Mechanisms 224 Aging and Metabolism

Calorie Restriction and Aging Benefits of Calorie Restriciton 222 Antwort 221 Antwort

Stem Cell Exhaustion

• decline in regenerative potential of tissues

Altered Intercellular Communication

• neuroendocrine dysfunction

• inflammaging

• immunosenescence

• bystander effects

224 Antwort 223 Antwort

• lower plasma glucose and insulin/IGF-1 levels calorie restriction limited food intake without causing nutritional deficiencies

• protection against autoimmune disease • 1934: McCay shows feeding rats calorie-reduced, micronutrient enriched diet • elevated levels of apoptosis in tumors leads to longer lifespans

• reduced angiogenesis • 1983: reduced food uptake due to eat-2 mutation increases lifespan in C. elegans • postponed or attenuated onset of cancer, immunosenescence, and inflammation • until recently only intervention known to result in a consistent positive cross • delayed onset of other age-related disease species effect on lifespan • possible explanations: • also tested in Rhesus monkeys ◦ reduced oxidative damage

◦ downregulation of insulin/GH/IGF-1 axis

◦ low-level stress Molecular Disease Mechanisms 225 Aging and Metabolism Molecular Disease Mechanisms 226 Aging and Metabolism

Rate-of-Living Hypothesis & AMP-Dependent Kinase Free Radical Theory of Aging AMP-K

Molecular Disease Mechanisms 227 Aging and Metabolism Molecular Disease Mechanisms 228 Aging and Metabolism

Metformin Insulin/IGF-1-Signaling 226 Antwort 225 Antwort

• key sensor of cellular energy status • large animals live longer and have lower metabolic rates

• work and hypoxia lead to decrease of ATP and increase of AMP → activates • hypothesis: maximum life span is inversely proportional to basal metabolic AMPK → phosphorylates enzymes needed for metabolism rate

• increases catabolic pathways: glycolysis, βoxidation (only in liver) • many inconsistencies → mole rat

• decreases anabolic pathways: gluconeogenesis, protein synthesis, lipogenesis, • Free Radical Theory: nutrient metabolism requires mitochondrial oxida- cholesterol synthesis tive phosphorylation → generates ROS → cause cellular damage

- - • ROS: H2O2, •OH , NO3 , HOCl

• antioxidants block effects of calorie restriction → increase overall mortality and cancer growth

• ROS in small dose beneficial → hormetic response

228 Antwort 227 Antwort

• in C. elegans: functioning daf-16 required for extended lifespan • used for treatment of T2D, prediabetes, PCOS (polycystic ovary syndrome), gestational diabetes • daf-16 is a FOXO-family transcription factor • found to reduce blood sugar • FOXO targets: cell-cycle inhibiton, oxidative-stress resistance, apoptosis, metabolism • mechanism unclear: complex I inhibition, AMPK activation

• prevents weight gain and cancer

• adverse effects: gastrointestinal irritation, lactic acidosis Molecular Disease Mechanisms 229 Aging and Metabolism Molecular Disease Mechanisms 230 Aging and Metabolism

mTOR Pathway Rapamycin

Molecular Disease Mechanisms 231 Aging and Metabolism Molecular Disease Mechanisms 232 Cognitive Aging

Sirtuin & Resveratrol Definitions of Learning, Memory and Retrieval Mechanism & Why Do We Have Memory? 230 Antwort 229 Antwort

• produced by Streptomyces hygroscopicus from Easter Islands as secondary • promotes cell growth metabolite • regulates lifespan across different species • potent antitumor & immunosuppressive properties • mTORC1

• yest genetic screen revealt Target Of Rapamycin as TOR1 and TOR2 ◦ potently regulated by rapamycin

• inhibits mTor - a serine threonine kinase - in mammalian cells ◦ regulates translation, cell growth regulation

• found as two complexes with distinct function and different sensitivities to ◦ main substrates S6 kinase & 4E-BP1 rapamycin • mTORC2

• prolongs lifespan but does not prevent signs and symptoms of aging ◦ acutely resistant towards rapamycin

◦ regulates Actin reorganization, survival

◦ main substrates: AKT, SGK1, PKCa

232 Antwort 231 Antwort learning process by which an organism acquires knowledge about its environ- Sirtuin ment • deacetylates or mono-ADP-ribosylate proteins memory storage or retention of knowledge • needs NAD+ as substrate retrieval mechanism search and readout of stored information • increases lifespan • to retain useful skills, knowledge and expertise • activated by calorie restriction • to recognize familiar people and places Resveratrol • to go beyond conditioning in learning from experience → lessons from one’s • like calorie restriction, activates SIRT1 past and experience of others • highly contained in red wine

• also increases lifespan Molecular Disease Mechanisms 233 Cognitive Aging Molecular Disease Mechanisms 234 Cognitive Aging

Stages of Memory Long-Term Memory

Molecular Disease Mechanisms 235 Cognitive Aging Molecular Disease Mechanisms 236 Cognitive Aging

Neuroplasticity Long-Term Potentiation and Depression LTP & LTD 234 Antwort 233 Antwort

1. incoming sensory information

2. encoding → information is converted to storage

3. storage → information is retained in memory

4. retrieval → information is retrieved from memory

Memory Subcategory Brain Region procedural memory: habits, skills basal ganglia classical conditioning amygdala, cerebellum habituation reflex pathways medial temporal lobe, dien- episodic memory & semantic memory cephalon

236 Antwort 235 Antwort

• LTP: process by which synaptic connections between neurons become stronger with frequent • lifelong ability of brain to recognize neural pathways based on new experiences activation • changing of structure, function and organization of neurons in response to new experiences • may be mechanism underlying learning and memory • axonal sprouting • LTD: process by which synaptic connections between neurons are weakened ◦ healthy axons sprout new nerve endings that connect to other pathways in CNS • may be important to memory formation → perhaps by resetting previous synaptic changes that allow for new memories to be formed via LTP ◦ can be used to strengthen existing conditions

• Case of H.M.: Henry Molaison suffered from intractable epilepsy, underwent bilateral ◦ repair damaged parts of CNS by repairing damaged neural pathways and restoring them removal of medial temporal lobes in 1953 → surgery successful but he developed a severe memory deficit: he could no longer form any new memories, only remembering information • synaptic pruning for seconds to minutes ◦ synapses start to form around 36th week of gestation and get denser for about 2 years → • bilateral removal of hippocampal formation causes severe form of anterograde amnesia converging to a fully connected neural network (no memories formed following trauma) ◦ after it gets as dense as possible (15.000 synapses per neuron = 2x average adult brain) , • retrograde amnesia: events for period of time before trauma are forgotten, memories synapses that do not conduct action potential on a frequent basis start to disappear and from distant past and following trauma intact number of connections start to decline, others are kept and streng Molecular Disease Mechanisms 237 Cognitive Aging Molecular Disease Mechanisms 238 Cognitive Aging

8-Arm Radial Arm Maze Brain Regions for Learning

Molecular Disease Mechanisms 239 Cognitive Aging Molecular Disease Mechanisms 240 Cognitive Aging

Effects of Aging on Brain and Cognition Risk Factors for Aging 238 Antwort 237 Antwort

• hippocampal formation important for short-term storage and retrival of declar- • to study effects of hippocampal lesions on learning and memory ative memory • food pellet placed at end of 4 of 8 arms • prefrontal cortex: working memory, sustained attention and behavioral/cognitve flexibility • animal has to learn which arms are bated (= contain food pellet)

• dorsal hippocampus: responsible for spatial reference and working memory • reference memory: critical information to be remembered stays the same throughout test condition → learn position of arms with pellets • ventral hippocampus: responsible for acquisition and expression of fear and anxiety-related behaviors • working memory: critical information to be remembered depends on sub- ject’s previous actions → after entering bated arm, subject needs to remember that that arm now does not contain a food pellet → information changes with subject’s performance

240 Antwort 239 Antwort

• modifiable: unhealthy diet, physical inactivity, tobacco use, alcohol use, • it becomes harder to ignore irrelevant information with age → ”cocktail party drug use, polypharmacy, stress, sleep phenomenon” → difficult to hold relevant information

• intermediate: raised blood pressure, raised blood glucose, abnormal lipids, • harder to switch between tasks with different requirements overweight/obesity, heart disease, pulmonary disease, mental illness • for mental activities aging is usually noticed in 50s, although decline starts • non-modifiable: age, family history, genetic factors in 30s

• loss of neuronal plasticity

• loss of brain volume with age: white matter > gray matter, especially hip- pocampus and frontal lobes, loss of myelin, synapses, and dendritic arbor, cytoskeletal changes → accumulation of neurofibrillary tangles and deposi- tion of amyloids in brain and blood vessels Molecular Disease Mechanisms 241 Cognitive Aging Molecular Disease Mechanisms 242 Cognitive Aging

Criteria for Evaluating Animal Models Azheimer’s Disease

Molecular Disease Mechanisms 243 Cognitive Aging Molecular Disease Mechanisms 244 Cognitive Aging

Stages of AD Progression of AD 242 Antwort 241 Antwort

• chronic neurodegenerative disease • Construct Validity: mechanism underlying human conditions is similar in animal model • largest cause of dementia in elderly - 60-80% of all cases • Face Validity: phenomenological expressions in human condition and their •≈ 13% of all people older than 65 have AD resemblance to those in animal model • 4th leading cause of mortality • Predictive Validity: model should predict outcomes that are expected in • symptoms: progressive dementia, loss of memory, cognitive decline, impaired judgment, human model, components that exacerbate conditions in humans should have changes in personality effects in the same direction in animal, likewise drugs that alleviate condition • protective factors: physical activity, cognitive and social activity, education, APOE in humans should have same direction of effect in animal model

• risk factors: alcohol misuse, unhealthy diet, smoking, hypertension, obesity, dyslipidemia, • Reliability: consistency that model can be reproduced across laboratories diabetes, vascular insults, neuronal damage, APOE and other genes → reliable and reproducible experimental system essential for scientific study

• genetic risk factor: most important: apoE4, one of three variants, one allele → 3x risk, homozygous → 10x risk

• animal model: Tg mice - Aβ deposition in cortex and hippocampus, memory impairment

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• amyloid is a general term for protein fragments that body produces normally 1. no impairment

• β amyloid is a protein fragment snipped from an amyloid precursor protein 2. very mild cognitive decline: memory lapses, forgetting familiar words or objects (APP), in a healthy brain, protein fragments are broken down and eliminated, 3. mild cognitive decline: family and friends start noticing problems with name-finding, in AD accumulation to hard, insoluble plaques planning or organizing, problems with memory or concentration

• neurofibrillary tangels are insoluble twisted fibers found in brain’s nerve cells, 4. moderate cognitive decline: problems in knowledge of current events, impaired abil- primarily consist of protein called Tau, which forms microtubules ity to perform challenging mental arithmetic, managing finances, memory of personal history, may be moody and withdrawn • microtubules help transport nutrients from one part of nerve cell to another, 5. moderately severe cognitive decline: major gaps in memory and cognitive function, in AD tau protein is abnormal and microtubule structures collapse need assistance with day to day activities, cannot remember address or phone number

• thinning of cortex 6. severe cognitive decline: memory continues to worsen, significant changes in per- sonality, need help with dressing, toileting, may be incontinent • decline of processing speed, working memory, long-term memory 7. very severe cognitve decline: final stage, loss of ability to respond to environment, • older people’s performances in cognitive test are not only poorer but also including speaking and movement control more varied → inconsistent Molecular Disease Mechanisms 245 Cognitive Aging

Hypotheses for AD 245 Antwort

• BAP: accumulation deposit outside of cell of a unsoluble fragment of amy- loid precursor protein or APP - Ab-42 - helps other protein fragments to accumulate → plaques or migration of Ab-42 cause neuronal death

• tau: abnormal phosphorylation of tau proteins make them ”sticky” by caus- ing them to become paired helical filaments - PHFs - → accumulation and formation of neurofibrillary tanges (NFT) → breakup of microtubules → loss of axonal transport → cell deat

• tau hypothesis not in favor anymore

• current theory: protein accumulation → plaques and tangles, inflammation → unregulated activation of glia, lipid distribution → lipid membrane site of APP cleavage