effect of mesenchymal stem cells on mycobacterium tuberculosis growth in vitro study n.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
EFFECT OF MESENCHYMAL STEM CELLS ON MYCOBACTERIUM TUBERCULOSIS GROWTH (IN VITRO STUDY) PowerPoint Presentation
Download Presentation
EFFECT OF MESENCHYMAL STEM CELLS ON MYCOBACTERIUM TUBERCULOSIS GROWTH (IN VITRO STUDY)

Loading in 2 Seconds...

play fullscreen
1 / 46

EFFECT OF MESENCHYMAL STEM CELLS ON MYCOBACTERIUM TUBERCULOSIS GROWTH (IN VITRO STUDY) - PowerPoint PPT Presentation


  • 175 Views
  • Uploaded on

EFFECT OF MESENCHYMAL STEM CELLS ON MYCOBACTERIUM TUBERCULOSIS GROWTH (IN VITRO STUDY). Andhika Yudistira, MD. INTRODUCTION. Tuberculosis as infection remains one of major health problems in Indonesia. Indonesia in 2009 : 100 / 100.000 populations in 2009 (5th Rank worldwide)

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'EFFECT OF MESENCHYMAL STEM CELLS ON MYCOBACTERIUM TUBERCULOSIS GROWTH (IN VITRO STUDY)' - aliya


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
effect of mesenchymal stem cells on mycobacterium tuberculosis growth in vitro study

EFFECT OF MESENCHYMAL STEM CELLS ON MYCOBACTERIUM TUBERCULOSIS GROWTH (IN VITRO STUDY)

Andhika Yudistira, MD

introduction
INTRODUCTION
  • Tuberculosis as infection remains one of major health problems in Indonesia.
  • Indonesia in 2009 : 100 / 100.000 populations in 2009 (5th Rank worldwide)
  • Indonesia in 2011 : 0.4 – 0.5 millions cases (4th rank worldwide)

*World Health Organization. Global Tuberculosis Report 2012. Diunduh dari: www.who.int/tb/publications/global_report/gtbr12_main.pdf

*Perkumpulan Pemberantasan Tuberkulosis Indonesia. TBC di Indonesia Peringkat ke-4. 2012. www.ppti.info/2012/09/tbc-di-indonesia-peringkat-ke-5l

slide3

Musculoskeletal tuberculosis accounts for 10% – 15% of all TB notifications in the non-industrialized world.

  • Spine is the most common site (50%), followed by pelvis (12%), hip and femur (10%), knee and tibia (10%), ribs (7%), and mulltiple sites (3%).

50%

10%

Talbot J C, Bismil Q, Saralaya D, Newton DAG, Frizzel RM, Shaw DL. Musculoskeletal Tuberculosis in Bradford – A 6 Year Review. Ann R Coll Surg Engl. 2007. May; 89(4): 405-409.

3%

10%

pathophysiology
Pathophysiology

Watts, H.G., Lifeso, R.M. (1996). Current Concepts Review-Tuberculosis of bones and Joints. JBJA Journal of Bone and Joint Surgery - American 1995 - 1998 February 1996, Volume 78-A: 2.

Spiegel D A, Singh G K, Banskota, A K. Tuberculosis of the Musculoskeletal System. 2005. Techniques in Orthopaedics. Lippincott Williams & Wilkins,, Inc. Philadelphia. 20(2):167-178.

slide6

Mycobacterium tuberculosis has no exotoxins or destructing proteins

  • Macrophage dependent  Extended dormancy
  • MTB role in bone pathology : induce osteolytic activity through chaperonin 10 protein.
  • Chaperonin 10 as a bone resorption stimulator, and prevent osteoblast bone-forming cell pathway

Meghji, S. White, P A. Nair, S P. Reddi, K. Heron, K. Henderson, B. Zalliani, A. Fossati, G. Mascagni, P. Hunt, J F. Roberts, M M, Coates, A. Mycobacterium tuberculosis Chaperonin 10 Stimulates Bone Resorption : A Potential Contributory Factor in Pott's Disease. 1997. J Exp Med, Rockefeller University Press. Volume 186 No. 8.

slide7

Orthopaedic Management in Musculoskeletal TB

  • Establish Diagnosis
  • “Treating rather than Testing”
  • Chemotherapy – Anti Tuberculous Drugs
  • Surgical Debridemant
  • Joint Arthrodesis
  • Correction Deformity
  • Improve Function

Spiegel D A, Singh G K, Banskota, A K. Tuberculosis of the Musculoskeletal System. 2005. Techniques in Orthopaedics. Lippincott Williams & Wilkins,, Inc. Philadelphia. 20(2):167-178.

mesenchymal stem cells
Mesenchymal Stem Cells
  • Discovered in 1968, by A.J. Friedenstein : “ MSCs are a subset of non-haematopoetic pluripotent cells found in adult bone marrow and are capable of differentiating into adipocytes, fibroblasts and even myoblasts”
  • Stem cell nature of these cells : ability to self renewal and differentiation into various mesenchymal elements. (bone, fat, cartilage, and muscle)
  • Over the years, MSCs are increasingly used in clinical practice for cell therapy.
  • There is growing understanding among scientific community that many of infectious diseases may be cured or controlled using stem cells.

Augello A, Kurth TB, De Bari C. Mesenchymal Stem Cells : A Perspective from In Vitro Cultures to In Vivo Migration and Niches. 2010. European Cells and Materials Vol.20 : 121-133.

Karl H. Kraus, Carl Kirker-Head, Vet MB. Mesenchymal Stem Cells And Bone Regeneration, Veterinary Surgery 35:232-242, 2006, The American College of Veterinary Surgeons.

mesenchymal stem cell has immunomodulator properties
Mesenchymal Stem Cell has Immunomodulator Properties
  • MSCs are rare population of non-haematopoetic stromal cells, present in bone marrow and connective tissues.
  • MSCs as multipotent cells: bone, cartilage, adipose tissue, muscles.
  • MSC have potent immunosuppresive and immunomodulator capacity.
  • It was reported that MSCs posess antingen-presenting properties
  • Under inflammatory conditions, MSCs can act as proffesional antigen-presenting cells

Chelluri, I. K. Prasad, C t. Vennila, A G. Gokhale, V. Adavi, R. Preliminary Report on Immunomodulation of Mesenchymal Stem Cells in M.tb Infection, 2010. The Internet Journal of Infectious Diseases. 2010 Vol 8 No.l.

Shilpa R, Pawan Sharmaa, Sarman Singhb, Luc Van Kaerc, and Gobardhan Das. Mycobacterium tuberculosis evades host immunity by recruiting mesenchymcl stem cells, www.pnas.as.org/cgi/doi/l 0.1073/pnas. 1007967107.

Hoogduijn M J, Popp F, Verbeek R, Masoodi M, Nicolaou A, Baan C, Dahlke M. The Immunomadulatory Properties of Mesenchymal Stem Cells and Their Use for Immunotherapy. 2010. International Immunopharmacology. Doi : 10.1016/j.intimp.2010.06.019.

effects of mscs on immunocytes
Effects of MSCs on Immunocytes

MSC modulates immune response by interaction with :

  • T cells
  • B cells
  • Dendritic cells
  • Treg cells
  • NK cells
  • Γδ cells

Shi M, Liu Z-W. Wang FS. Immunomodulatory Properties and Therapeutic Application of Mesenchymal Stem Cells. Clinical and Experimental Immunology. 2011. British Society for Immunology, Clinical and Experimental Immunology, 164: 1-8.

slide11

MSCs possess immunosuppressive : inhibit T-cells, B-cells, NK cells proliferation and functions.

  • Modulate activities of DCs, induce Treg cells.
  • These properties is dependent on cell – cell contact or IDO, PGE2, NO, IFN-γ, TGF-β secretions
  • Inflammatory : MScs prevent naive CD4 cells into TH17
slide12

Raghuvanshi : “MSCs are recruited to the site of MTB infection”

  • MSCs contains the pathogenic microorganisms but doesn’t eliminate them.
  • Inhibit T-cell atcivation by producing NO, TGF-β, or IDO
  • NO controls the growth of MTB organisms and confines them within granuloma-like structures
  • Persistent MTB infections !
slide13

It is believed that MSCs can act as Antigen Presenting Cells under inflammatory conditions

  • MSCs secreted IL6, IL8, chmokines CCl2, TIMP2
  • Pro inflammatory : attracts Monocytes, DCs, T cells and NK cells  antigen eradications actively or through T cells and NK cells, and NO.
  • Anti inflammatory : Chemotaxis targets neutrophils, monocytes.
  • MSCs have dual immunomodulatory capacity, depend on IFN-γ and TNF-α.
slide15

MSCs role in immunomodulation is still questionable

  • Mensyuknil (2011) studied effect of MTB debris on MSCs growth (in vitro).  NO effect on MSCs growth by MTB debris
  • Interaction between living Mycobacterium tuberculosis and Mesenchymal Stem Cells in one single medium is required to evaluate.

Mensyuknil, U. Rahyussalim Pengaruh Debris dan Supernataan Kuman Mycobacterium Tuberculosis yang Diambil Dari Lesi Spondylitis Terhadap Pertumbuhan Sel Punca (Studi in vitro). 2011. Program Studi Orthopaedi dan Traumatologi, Fakultas Kedokteran Ur.iversitas Indonesia, RS Cipto Mangunkusumo, Jakarta.

research questions
Research Questions
  • Is there any effect on Mycobacterium tuberculosis growth by performing co-culture Mycobacterium tuberculosis and Mesenchymal Stem Cells ?
  • By performing co-culture  is there any effect on Mesenchymal Stem Cells growth ?
research hypothesis
Research Hypothesis
  • MSCs have immunoregulator properties and have direct eradication effect on Mycobacterium tuberculosis
  • Effect MSCs on eradication of Mycobacterium tuberculosis or MTB growth are seen in Acid fast staining, bacterial culture, and PCR.
slide18
Aim
  • To evaluate interaction between living Mycobacterium tuberculosis and Mesenchymal Stem Cells in co-culture growth
theoretical framework
Theoretical Framework

MTB

MSCs

MSCs recruited by MTB

(APC)

Macrophage coated MTB

MHC I _CD8+

T eff

MHC II – CD4+

Th

Adaptive immune

MHC I  Teff

MHC II  B cells

Activate NK - DCs

IFN-γ & TNF-α

Pro infllamatory

mediators

Th1  Makrofag

Th2  B cells

NK cells

NO

Immune Exhaustion

Granuloma

IDO, NO, PGE2

HGF, TGF-β

IFNγ

Granuloma degradation

Replications & Activations

Immunosuppresive

Effects

Inhibit MTB replications

MTB eradications

(-)

Tissue Necrosis

conceptual framework
Conceptual Framework

MSC

MTB

Co-Culture

MTB & MSC

  • AFB
  • Culture
  • PCR
  • AFB
  • Culture
  • PCR
  • AFB
  • Culture
  • PCR
research method
Research Method
  • Exclusion
  • Contamination (+)
  • RPMI (-)
study protocol
Study Protocol

MTB culture in LJ

MSC culture in RPMI

5000 cells/cm2

1 McF =

108 cfu/ml

104 cfu/ml

5000 cells/cm2

MTB in RPMI

2,5cc MTB + 2,5 cc RPMI

Co-Culture MSC+MTB in RPMI

2,5cc MTB + 2,5 cc MSC

MSC in RPMI

2,5cc MSC + 2,5 cc RPMI

Incubation in 5% CO2, Temp 37°C

  • AFB
  • Culture
  • PCR
  • MSC counting
  • AFB
  • Culture
  • PCR
  • MSC counting
  • AFB
  • Culture
  • PCR
  • MSC counting

Day 3

Day 7

Day 9

Day 3

Day 7

Day 9

Day 3

Day 7

Day 9

co culture
Co-Culture

Transferring MTB from 3 days optimization RPMI (108 cfu/ml)  centrifuge  (104cfu/ml)

  • 2.5 cc of RPMI
  • 2.5cc of MTB in RPMI
  • 2.5cc of MSC in RPMI
slide25

25cc Tc-Flask

  • 3 flasks of culture of MSC
  • 2 flasks of culture of MTB
  • 3 flasks of co culture

Incubation in CO2 incubator

5%, Temp 37°C

evaluation each group
Evaluation Each Group

Each flask is being observed under inverted microscope

Flasks from 3 groups are taken out from the incubator

slide27

MTB Evaluation

  • MTB preparation
  • MTB samples for PCR
  • MTB Culture in LJ
  • MTB AFB
  • 1 cc for PCR exam
  • 2 cc for AFB and Bacterial culture in LJ medium
msc counting
MSC counting
  • Add trypsin 2cc  incubate 37°C for 7 min
  • Add inactivating medium 4cc
  • Collect to 15cc tube  centrifuge 1200rpm for 4 min
  • Debris are taken  haematocytometer
  • Microscope counting
results day 3
ResultsDay 3

Flasks under Microscope

MSC culture

MTB culture

MSC-MTB co culture

AFB Ziehl Neelsen

MSC-MTB co culture

MTB culture

slide31

LJ medium for culture

MTB culture

MSC-MTB co culture

PCR Realtime

Cyclus Threshold

results day 7
ResultsDay 7

Flasks under Microscope

MSC culture

MTB culture

MSC-MTB co culture

AFB Ziehl Neelsen

MSC-MTB co culture

MTB culture

slide33

LJ medium for culture

PCR Realtime

Cyclus Threshold

MSC-MTB co culture

MTB culture

results day 9
Results Day 9

Flasks under Microscope

MSC culture

MTB culture

MSC-MTB co culture

AFB Ziehl Neelsen

MSC-MTB co culture

MTB culture

slide35

LJ medium for culture

PCR Realtime

Cyclus Threshold

MSC-MTB co culture

MTB culture

pcr cyclus threshold
PCR - Cyclus Threshold

P 0.04

P 0.07

P 0.07

*Kruskal Wallis test

p 0.08

p 0.77

p 0.56

*Mann-Whitney test

msc cell counting
MSC : Cell Counting

Cells : viable cells x 2 x 104 x volume (cc)

cell counting msc
Cell Counting MSC

P 0.05

P 0.05

P 0.04

*Kruskal Wallis test

P 0.037

P 0.046

P 0.046

*Mann-Whitney test

discussion
DISCUSSION

Why RPMI ?

  • DMEM (for MSCs) and MGIT/LJ (for MTB) are best for each MSC and MTB. But MSCs can’t be cultured in MGIT/LJ as well as MTB in DMEM
  • Roswell Park Memorial Institute Medium is a kind of medium in culturing cells and tissues.
  • RPMI-1640 as a medium to culture leucocytes, bone marrow, and hybridoma.
  • RPMI can be used in culturing Mycobacterium tuberculosis*
  • RPMI can be used in culturing MSCs, though DMEM is still superior compared to RPMI**

*Zhang M, Gong J, Lin Y, Barnes P F. Growth of Virulent and Avirulent Mycobacterium tuberculosis Strains in Human Macrophages. 1998. Journal of Infection and Immunity. V.66(2) : 794-799.

**Pittenger MF. Mesenchymal Stem Cells from Adult Bone Marrow. 2008. Methods in Molecular Biology, vol.449. Humana Press, Totowa, New Jersey.

discussion1
Discussion

Mesenchymal Stem Cells in Tc-Flask

  • Using capped Tc-Flask  Biosafety
  • MSCs seeding invitro  Various amount
  • Eyckman : compared seeding 5000/cm2 – 25000 /cm2 – 85000/cm2
  • 5000/cm2 results in more efficient in cell attachments compared to the others. *

Eyckmans J, Lin GL, Chen CS. Adhesive and Mechanical Regulation of Mesenchymal Stem Cell Differentiation in Human Bone Marrow and Periosteum-Derived Progenitor Cells. 2012. Biology Open 000, 1-11.

discussion2
Discussion

Effect MSCs on Mycobacterium tuberculosis growth

  • All evaluations for MTB existence/growth in co culture (AFB – Bacterial cuture – PCR) confirmed that MSCs has no eradication effect on MTB.
  • On AFB staining : coculture group showed more densed MTB compared to MTB group especially in day 7 and day 9.
  • On bacterial culture : Both MTB group and coculture group defines the same positive (+) result
  • On PCR assay : p 0.04 / 0.07 / 0.07 (3 groups) and p 0.08 / 0.56 / 0.77 (MTB : Coculture)  showed no difference in MTB existence.
discussion3
Discussion

MSCs Immunomodulator Properties ?

(Previous Studies) :

  • MSCs might support eradication of MTB (microorganism) infection through its immunomodulator properties*
  • MSCs inhibit T cells and provide immune tolerance for Mycobacterium tuberculosis (evade host immunity) **
  • MSCs immunomodulation effect depends on surrounding inflammatory environment.***  In Vitro (-)

*Rahyussalim. Transplantasi Sel Punca Mesenkimal Pada Defek Spondilitis Tuberkulosis : Pengaruh Terhadap Perbaikan Pembentukan Tulang Baru Dan Eradikasi Infeksi Pada Model Kelinci. Fakultas Kedokteran Universitas Indonesia. 2013.

** Shilpa R, Pawan Sharmaa, Sarman Singhb, Luc Van Kaerc, and Gobardhan Das. Mycobacterium tuberculosis evades host immunity by recruiting mesenchymcl stem cells, www.pnas.as.org/cgi/doi/l 0.1073/pnas. 1007967107.

*** Hoogduijn M J, Popp F, Verbeek R, Masoodi M, Nicolaou A, Baan C, Dahlke M. The Immunomadulatory Properties of Mesenchymal Stem Cells and Their Use for Immunotherapy. 2010. International Immunopharmacology. Doi : 10.1016/j.intimp.2010.06.019.

discussion4
Discussion

Coculture Study Mycobacterium tuberculosis – Mesenchymal Stem Cells

  • MSCs viabe cells are hardly seen in the coculture group
  • MSCs are believed to have Antigen Presenting Cellrole in its immunomodulator properties
  • Absence of “real” inflammatory mediators in invitro study.
  • However, MTB are known not to possess/produce exotoxins
  • Is it exotoxins ? / or “Medium competition” ?
  • Novak : “MTB demonstrated polyketide synthesis gense, indicating that MTB produces a related toxin”.
  • Finding an MTB homolog of this toxin will be a focus of future research

Novak K. Tuberculosis Toxic Avengers. 1999. Science; 283:854. http://www.nature.com/nm/focus/tb/research_news.html

conclusions
Conclusions
  • Mesenchymal Stem Cells have no effect in Mycobacterium tuberculosis eradication (In Vitro)
  • Mycobacterium tuberculosis somehow suppress Mesenchymal Stem Cells growth ( In Vitro)

However, effect of Mycobacterium tuberculosis on MSCs growth (In Vitro) creates another question ? Is it just “Medium Competition” or “MTB prouce toxins or virulence factors” ?