Tuberculosis

1
Tuberculosis

• Juan Pablo Olano M.D.
• Associate Professor
• Department of PathologyUTMB 2007

2
History

• Archeological evidence of TB in mummies and stone
  age skeletons
• Hippocrates Pthisis
• Responsible for thousands to millions of deaths
  during middle ages, renaissance and into the XXI
  century.
• Tubercles, miliary forms described
• 1882 Kochs description of mode of transmission.
• 1918 BCG (bacille Calmette-Guerin) vaccine.
• 1940s-1970s. Therapy Streptomycin, INH,
  rifampin
• 1950s Atypical mycobacteria.
• 1990s Genome sequencing

3
Epidemiology

• Two billion infected
• Eight million new cases per year
• 1.5-3.0 million deaths
• US
• 1953 84,000 cases
• 1983 24,000 cases (10.2 x 100,000)
• 1990 New York 48 x 100,000.

4
Microbiology

• Gram positive rod with waxy wall (long chain
  fatty acids).
• Acid fast
• Complex growth requirements
• Aerobic, non-spore forming, non-motile,
  facultative intracellular
• 0.2-0.5 by 2-4 ?m
• Other members of MTB complex M. microti, M.
  bovis, M. africanum

5
Genome

• Size 4.4 Mbp
• Protein coding genes 3,959 (90.8)
• Pseudogenes 6
• Known function 2,441
• Conserved hypotheticals 912
• Unknown genes 606
• Fatty acid metabolism 250 genes

6
Cell wall of MTB
7
Pathogenesis

• Port of entry
• Adhesion
• Internalization
• Intracellular survival/killing
• Pathology (tissue damage)
• Immune response

8
Portal of entry

• Inhalation
• Contact
• Duration
• Inoculum
• Gi tract, skin (rare)

9
Adhesion

• Complement receptors CR1, CR3 (cholesterol-depend
  ent) and CR4
• Opsonization (C3b via C2a activation or
  alternative pathway activation).
• Non-opsonin driven internalization (CR3 and CR4)
• Mannose residues and lectins
• Scavenger receptor(CD36)
• Enhanced internalization Collectins (surfactant
  protein A), mannose binding lectins (MBL).
• Heparin binding adhesins and fibronectin binding
  proteins.
• Target cells Macrophages
• Other cells PMNs

10
Adhesion and internalization
11
Internalization and intracelullar survival

• Relative importance of each of the adhesion
  mechanisms remains to be determined in vivo.
• Membrane-coated vesicles
• Hydrogen pump ATP-ase inhibition
• Phagolysosomal fusion
• Mycobacterial sulfatides
• Ammonia production
• Urease and glutamine synthetase
• GTPases of Ras family
• TACO (tryptophan aspartate-containing coat)
• Phagosome escape

12
Internalization and intracellular survival

• Endosomal traficking
• PI3 Kinases recruited to phagosome via Rab5
• Generation of PI3P
• Binding to proteins with FYVE motifs
• EEA1 and Hrs Myobacteria deplete these two from
  endosomal membranes
• Inhibition of calcium signaling in early
  endosomes via depletion of sphingosine kinase

13
Endosomal traficking

• Myobacterial factors
• Acid phosphatase (SapM)
• Protein kinase G
• Glycosylated PI lipoarabinomannan (ManLAM)
• PIP3 interference
• Calcium signaling interference

14
Internalization and intracelullar survival

• ROS Controversial
• In vitro Resistant to killing (sulfatides and
  LAM, phenolic glycolipid I)
• In vivo NADPH oxidase deficient mice vs. chronic
  granulomatous disease in humans
• RNI Supported by in vitro experiments. In vivo
  role remains to be determined. Probably
  important. Activated Vit D seems to play a role
• Apoptosis vs. necrosis of macrophages infected
  with MTB.

15
Pathology

• Primary tuberculosis
• Gohns complex
• Dissemination
• Reactivation
• AIDS, neoplasia, immunosupression,
  malnourishment, silicosis
• Common lesion Granuloma

16
Tuberculosis stages
17
Tuberculosis stages
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Lung Granulomas
19
Granuloma
20
Granuloma
21
Primary tuberculosis
22
Miliary tuberculosis
23
Millet seeds
24
Miliary tuberculosis
25
Immune response

• CD4 responses
• MHC class II context
• Gamma interferon
• CD8 responses
• MHC driven
• Th1 and Th2 responses (IL12 and IL4) in humans
  remains uncertain. Theoretically Th1 responses
  are beneficial
• Toll-like receptors
• Essential for innate immune responses. MTB is no
  exception
• Cytokines and lysis of infected cells.
• IL-12 production
• MHC I and II responses
• IFN-?
• TNF-a

26
Immune response (cont.)

• CD4 responses
• MHC class II Macrophages
• IFN-?
• Interferon and NOS-2 independent mechanisms are
  at play.
• Activation and maturation of APC via CD40-CD40L
• Apoptotic cell death of targets controversial
• Downregulation of MHC class II molecules

27
Immune response (cont.)

• CD8 responses Lysis of affected cells and IFN-?
  production.
• MHC class I presentation of mycobacterial
  antigens is in its infancy.
• MHC class I restricted
• 65, 38, 19 kDa proteins.
• Via pores in endosomal vesicles.

28
Immune response (cont.)

• Non-restricted CD8 cells
• Via CD1 molecules
• Lipids and glycolipids
• CD4, CD8 or CD4 and CD8 neg
• Mycolic acid, LAM PIM
• Different CD1 sample different compartments
• Dendritic cells and macrophages

29
Immune response (cont.)

• Cytokines
• TNF alpha
• Granuloma formation, macrophages activation,
  tissue pathology and systemic manifestations.
• IL-1beta
• Expressed in excess at the site of disease.
• Animal models support role in containing
  infection
• IL-6
• Controversial role. Can be both pro- and
  anti-inflammatory.

30
Immune response (cont.)

• Cytokines (cont.)
• IL-12
• Crucial role. Induces IFN gamma production and
  early immune events.
• Human and mice deficiencies support role.
• IL-15 and IL-18
• IL-18 synergistic with IL-12. Also induces other
  chemokines, transcription factors and other
  inflammatory cytokines. IL-15 stimulates T-cell
  and NK cell proliferation
• Lymphotoxin alpha 3 Granuloma stability

31
Immune response (cont.)

• Cytokines (cont.)
• IFN gamma
• Role well defined
• Antigen dependent (CD4 cells) and independent
  (macrophages, gamma-delta T cells and NK cells
  possibly).
• Activation of macrophages and their intracellular
  killing mechanisms
• Induction of LRG-47 (P47 phosphatases belonging
  to GTPase family)
• Induction of autophagy

32
Immune response (cont.)

• Anti-inflammatory cytokines
• IL-10, TGFbeta and IL-4
• IL-10 Down regulation of TNF, IFN and IL-12.
  IL10 production higher in anergic patients
• TGF beta Synergistic with IL-10. Also inhibits
  proliferation of T- cells. Possible role in
  tissue damage.
• IL-4 IFN and macrophage activation.
• Chemokines
• IL-8, MCP-1 and RANTES

33
Immune response (cont.)

• Toll-like receptors
• Toll/Cactus/Dorsal IL-1 receptor/I-?/NF-?B.
• TLR2 (LAM) and TLR4
• Production of IL-12, NOS-2, TNF-a.
• Other TLR possibly involved.

34
Immunopathology
35
Immune response
36
Virulence factors

• Phenolic glycolipids
• Hemolysin-like substance (listeriolysin)
• Lipoarabinomannan T-cell proliferation and
  interferon inducible genes, ROS scavenger.
• Antigen 85 Induction of antibodies
• Tissue damage Mycolic acids, muramyl dipeptide,
  TNF alpha, lysosomal enzymes, oxygen radicals

37
Host susceptibility

• Natural resistance macrophage associated protein
  (Nramp-1)
• Membrane protein
• Metal ion transporter (Fe)
• Essential for intracellular killing
• Internalized with membrane coated vesicles
• Phagosomal maturation
• Mice and human studies conflicting
• Spectrum of disease HLA

38
Vaccines

• BCG
• Protection Children vs. adults
• Severe forms
• Live vs. killed vaccines
• BCG manipulation.
• BCG as vector for other vaccines

39
Latency and persistence

• Spore-like status sigF gene
• Marginal metabolic activity.
• Other genes acr (alpha crystallin)
• Isocitrate lyase
• Switching to lipid catabolism and nitrogen
  respiration

40
Diagnosis

• PPD
• Smears
• Cultivation
• Standard media LJ
• Radioactive
• Non-radioactive
• Molecular techniques

41
Medications

• Isoniazid, ethambutol, rifampin and ethambutol,
  pyrazinamide
• Cycloserine, streptomycin, kanamycin and
  ethionamide
• Fluoroquinolones
• New targets Isocitrate lyase, antigen 85