THE MAIN EMPHASIS IN PRESENT POWER POINT IS GIVEN TO THE MECHANISMS OF CELL DEATH ESPECIALLY NECROSIS AND APOPTOSIS.

2.  Introduction
 Cell injury
 Necrosis
 Apoptosis
 Apoptotic pathways
 Different agents involving apoptosis
 Molecular assays of apoptosis
 Crosstalk and Conclusion
 Over-expression & under-expression

3. Normal cell: homeostasis
 Maintenance of the integrity of cell membrane
 Aerobic respiration
 Synthesis of functional and structural proteins
 Preservation of genetic apparatus of the cell

4.  Irreversible form of cell injury
in the form of:
 Necrosis
 Apoptosis,
 Autophagy,
 some other alternative ways -
 Necroptosis,
 Anoikis,
 Entosis,
 Cornification.
Robbins Basic pathology

5. Robbins Basic pathology

6. Cell injury during ischaemia/hypoxia
Mechanisms –
Depletion of ATP
Mitochondrial Damage
Loss of Calcium Homeostasis
Accumulation of Oxygen-Derived
free radical
Defects in Membrane PermeabilityRobbins Basic pathology

7. Necrosis

8. NECROSIS TYPES
CO &CN poisoning (Hypoxia)
Thiamine def.
Vit- E Def
Mouldy corn poisoning
Coagulation Necrosis
Liquefactive necrosis
Ischemia & infarction
F. Necrophorum (toxins)
Vit- E def.
Hg salt ,thallium, phenol
poisoning
Robbins Basic pathology

9. Robbins Basic pathology
Caseous Necrosis
TB

10. Nutritional fat necrosis
Pancreatic fat necrosis:
Traumatic fat necrosis:
Robbins Basic pathology

11. 1. Dry gangrene
2. Moist gangrene
3. Gas gangrene
Gangrenous necrosis
• Necrotic tissue invaded by saprophytic bacteria(putrefactive)
Fibrinoid necrosis
Ag-Ab complexes and fibrin accumulate in
arteries or other vessels.
Robbins Basic pathology

12. APOPTOSIS
The name was first introduced by John Kerr in 1972. ( Lockshin., 1964)
Review articles Apoptosis—an introduction Alfons Lawen
It is a form of cell death designed to eliminate unwanted host cells
through activation of a coordinated, internally programmed series of
events effected by dedicated set of gene products.
Apoptosis is needed for proper development
Apoptosis is needed in pathological process
Need of apoptosis

13. David and Emanuel, 2006

14. Elmore,S.,2007

15. Fas-mediated signaling TNF Receptor-mediated signaling
Important caspases:
 Initiator— 2,8,9,10
 Effectors- 3,6,7
 Inflammatory 1,4,5
 Others- 11,12,13,14
Important ligands
and receptors
 FasL :FasR
 TNFa :TNFR
 Apo 3L :DR3
 Apo 2L :DR4
 Apo2L :DR5
Pathologic Basis of Veterinary Diseases,4th
Edition , M. Donald et.al.

16. Intrinsic pathway(Mitochondrial events)
Other proteins:
 Smac / DIABCO
 Htr /Omi
 AIF, Endonuclease-G
 CAD
 p53, ATM, and PARP-PUMA,BIM (undergo p53
mediated apoptosis)
The Bcl-2 family
Antiapoptotic
Bcl-2
Bcl-xL
Bcl-w
Mcl-1
Survivin
IAP
Proapoptotic:
Bax
Bik
Bak
Bad
Bcl-xS
(Elmore,S.,2007)
Pathologic Basis of Veterinary Diseases,4th Edition ,
M. Donald et.al.

17. Phagocytosis
Cell surface markers
 Phosphatidylserine
 Thrombospondin-1
 Calreticulin
 Annexin 5
 Annexin 1
Caspase 3 activates
 Endonucleases
 Proteases
 Protein Cross-linking
EXECUTION
PATHWAY
Elmore,S.,2007

18. S.NO BACTERIA APOPTOSIS MECHANISM
1. Clostridium dificile Enterotoxin A, B leads to cyt.c release
2. Pseudomonas inhibit cyt.c release
3. Chlamydia Inhibit cyt.c release
4. Staphylococcus aureus Cyt.c release
5. S.pneumoniae cyt.c release
6. B. anthracis Zn metalloprotease inhibit MAPK pathway
7. Helicobacter pylori action of VacA on the mitochondria
8. Neisseria NFkB activation of the apoptotic mechanism
9. Escherichia coli hemolysin- Hly on mitochondria
10. Listeria , Yersinia Lly-o release cyt.c
11. Shigella, Salmonella Caspase 8 activation
12. Legionella caspase 3 activation
13. Mycobacterium,
Rickettsia and Coxiella
Mcl 2 protein inhibitiit apoptosis,MyD88 mediated (NFkB
activation ) apoptosis
L. Marcelo et.al .,2006
Main mechanisms of bacteria-induced cell apoptosis

19. Gonzalez,A.M.F.et.al.,2013

20. S.NO. Parasite Predilection Mechanism
1. Plasmodium falciparum Endothelium Inos leads to caspase 8,9 activation
hepatocytes ROS mediated (cyt.c)
2. Toxoplasma gondaii cerebellum Fas/FasL mediated
eye do
3. Entamoeba histolytica intestine FasR mediated
4. Cryptosoridium parvum intestine Fas/FasL mediated
5. Trichinella spiralis muscles Pro-inflammatory cytokines mediated
6. T.cruzi ROS mediated(cyt.c)
7. Leishmania donovanii Inhibit MQ apoptosis through GM-CSF
production
8. HAT Brain Fas mediated
Main mechanisms of Parasites induced cell apoptosis
B.Anne-Lise et.al. ,2010

21. S.NO. DISEASES MECHANISM
Immunosuppressive:
1. AIDS HIV Tat protein increase FaS mediated
Neuro-degenrative diseases
1. Alzheimer's diseases Beta -plaques, increase FaS &TNFR
2. Parkinson disease Lewy body accumulation in neuron, FAS,TNFR
mediated
3. Huntington diseases Htt protein activate caspase 6,8.
4. Amyotrophic lateral sclerosis Cu, Zn SOD mutation trigger cyt.c release.
Heart &brain diseases
1. Myocardial ischemia increase ORS & BAX, fall in Bcl2
2. Stroke(Brain ischemia) Fas/FasL mediated apoptosis
OVEREXPRESSION OF APOPTOSIS
Favaloro,B.et.al.2012

22. ROLE OF APOPTOSIS IN CANCERS
(Decreased apoptosis)
Bcl2 family proteins alterations
S.N
O
Mechanism Diseases
1. Bcl2 alteration Hodgkin disease, breast
cancer
2. Bax deletion Brain tumors
3. BH3 protein
decrease(Bid)
Chronic myelomonocytes
leukemia
4. P53 gene
mutation
Li Fraumenic syndrome
Apoptosome Defect
1. Apaf 1 inactivation Melanomas,
Glioblastomas
Altered caspase activity
1. C-FLIP Compete FADD
2. IAP activation
Death receptor pathway defect
1. CD95 (FaS)
inactivation
Hepato-ca
esophagea
Favaloro,B.et.al.2012

23. What to do????????
Use of anti-apoptotic therapy:
S.NO Anti-apoptotic agent condition
1. IAP stimulation (XIAP,
Survivin)
Stroke, spinal cord injury
2. Caspase inhibition by Z-
VAD-fmk
Myocardial infarction
3. ICE inhibitor Rheumatoid arthritis
4. PARP inhibitor Reperfusion injury
5. BAX inhibitors Protect cells from injury
Gonzalz,A.M.F.et.al.2013

24. S.NO. alterations Assay
1. Cytoplasmic alterations
 Apoptotic cells  H&E stain, toluidine stain
 Smaller apoptotic bodies  TEM(gold standard)
 Phagocytosis of apoptotic bodies  TEM
2. DNA Fragmentation
 Endonuclease degradation
products
 DNA Laddering technique
 TUNEL method
3. Caspase detection Western blot, Mab, Poly. Ab,
4. Apoptotic & Pro-apoptotic genes PCR micro assay
5. Membrane alteration PS by FITC labeled Annexin 5
6. Mitochondrial assay
 Cyt.c release, Bax, Bcl2 EM, Flouresence
ASSAYS USED?????
Elmore,S.,2007

25. TUNEL
Cells with fragmented nuclei can also be visualized by an
assay that labels DNA ends Terminal deoxynucleotidyl
transferase–mediated dUTP Nick End-Labeling
DNA ladder
When a DNA sample from an apoptotic
cells is electrophoresed, the
fragmentation pattern gives rise to a
“DNA ladder
FACS Analysis
Molecular biology of cells,4th edition
TEM
Single-cell electrophoresis
(Comet assay)

26. Cross-talk between different modes of cell death.
 Necroptosis
(RIP 1 dependent)
 Anoikis(shedding)
 Entosis
(cannibalism, cell eating cell)
 Cornification
(keratinocytes,caspase14
dependent)
Some other alternative ways of cell death
Yaun,J.,2013

27.  Necrosis and apoptosis are main mechanisms of cell death.
 Necrosis is accidental, uncontrolled, un-programmed cell death.
 Apoptosis can be a physiological and pathological process.
 Apoptosis Involve Extrinsic (ligands and receptor mediated),Intrinsic (Cyt.c
mediated) and Cytotoxic T-cells mediated Granzyme pathway.
 p53 gene (TP53) is a tumor suppressor gene which on mutation favors
apoptosis.
 Over-expression and under-expression of apoptosis both are harmful for
normal homeostasis.
 Cell death have clarified many aspects of this fundamental process and
brought to the attention of scientist its role in a large number of different
diseases.

28. Références:
 Hotchkiss,R.S.,Strasser,R.et.al.2009.Mechanisms of cell death. The New England Journal
of Médicine,361:1570-83
 Bienvenu A. L., Rey E.G. and Picot, S. 2010. Apoptosis induced by parasitic diseases.
Parasites & Vectors. 3:106
 Chowdhury, I., Tharakan, B. and Bhat, G. K. 2006. Current concepts in apoptosis: the
physiological suicide program revisited. Cellular & Molecular Biology Letters 11:506 –
525
 Woodruff,T.M., Thundyil., et.al. Pathophysiology, treatment and animals and cellular
models of human ischemic stroke. Molecular Degenration,2011,6:11.
 1Wang, C. and Youle, R. J. 2009. The Role of Mitochondria in Apoptosis. Annu. Rev.
Genet., 43:95–118
 Marcelo, A. and Wanderley, S. D. 2006. Bacteria-induced apoptosis: an approach to
bacterial pathogenesis. Braz. J. Morphol. Sci. 23(1):75-86.
 Elmore, S. 2007. Apoptosis: a review of programmed cell death. Toxicol. Pathol. 2007,
35(4): 495–516.
 Barcinski, M. A. and DosReis, G. A. 1999. Apoptosis in parasites and parasite-induced
apoptosis in the host immune system: a new approach to parasitic diseases. Braz. J. Med.
Biol. Res., 32(4): 395-401.
 Yaun,J. and Kroemer,G. 2013 Alternative cell death mechanisms in development and
beyond.,24:2592-2602.
 Gonzalz,A.M.F.et.al.2013.The modulation of apoptosis by oncogenic viruses. Virology
Journal.10:182

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