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Pathological study on the therapeutic effect of MSC on exper

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mohamed refat

on 18 January 2015

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Transcript of Pathological study on the therapeutic effect of MSC on exper

Experimental Design


Prof. Dr. Mohamed M. Lotfy Prof. Dr. Kawkab Abd El Aziz

Pathological study on the therapeutic effect of MSC on experimentally infected mice with H9N2 Avian influenza virus
Introduction
Avian Influenza virus H9N2
Avian influenza (AI) or “bird flu”
is a highly infectious disease of birds.
AI viruses are negative single-stranded
enveloped RNA viruses that belong to
the influenza A genus of the
Orthomyxoviridae family
Zonootic Importance :

Ann Acad Med Singapore 2008;37:504-9
For a strain of influenza virus to cause a human pandemic, it must be (i) novel to the human immune system
(ii) virulent in the human host
(iii)transmissible from person to person
It has been suggested that it is not impossible that H9N2 influenza virus shows a low level of human-to-human transmission
Maines et al.,2006
Butt et al., 2005; Guo et al., 1999; Peiris et al., 1999
Transmission of Avian Infleunza in murine model
The BALB/c mouse is one of the most important laboratory animals for studies relating to transmissible mechanism in experimental infections with avian and human influenza virus and development of therapy methods
Hatta, M., et al.Growth of H5N1 influenza A viruses in the upper respiratory tracts of mice. PLoS Pathog 3 (10):1374-1379
Infection of host cells is mediated by specific interactions between the viral hemagglutinin (HA) and cell oligosaccharides containing sialic acid (SA) residues
The majority of avian influenza viruses bind to receptors with sialic acids containing an α-2, 3 linkage to the galactose (SAα-2, 3-gal), while human viruses prefer receptors that contain an α-2, 6 linkage (SAα-2, 6-gal)
Suzuki, Y.,2005.Sialobiology of influenza—molecular mechanism of host range variation of influenza viruses (Review). Biol Pharm Bull 28:399–408
expression of alpha- 2,3 and alpha-2,6 sialic acid-linked receptors has been defined in the tissues of BALB/c mice
Biotinylated Maackia
amurensis
lectin II (MAAII),
specific for
α-2,3-linked sialic
acid to detect AIV-Rs
a) nasal cavity
b) trachea
c) lung
Mice  are  not  a  natural  host  for  influenza. 
However,  some  studies  suggest  that  H9N2 viruses replicate in the murine respiratory tract 
without  prior  adaptation, 
and  may  even  prove  lethal .
.Guo  YJ,  Krauss  S,  Senne  DA,  et  al.  Characterization  of  the  pathogenicity  of  members  of  the  newly  established  H9N2  influenza virus lineages in Asia. Virology, 2000; 267(2), 279‐88
Vet Res Commun (2009) 33:895–903 DOI 10.1007/s11259-009-9307-3
Bone Marrow Derived Mesenchymal stem cells
The biology of stem cells have role in regeneration are currently the subject of intensive and extensive research in many laboratories around the world because of the promise of stem cells as therapeutic agents to regenerate tissue damaged by disease or injury.
MSC have the potential to differentiate into various connective tissue lineages which include adipose tissue, marrow stroma, cartilage, tendon and bone
While the vast majority of influenza A virus infections resolve without complications, approximately 3–5 million affected individuals worldwide develop severe and potentially fatal disease annually .
Severe influenza can activate deleterious innate
immune responses and cause acute lung injury (ALI)/acute
respiratory distress syndrome (ARDS), which directly contribute to influenza-associated morbidity and mortality
ALI/ARDS is characterized by
increased permeability of the microvascular endothelium and disruption of the alveolar-capillary membrane barrier,
leading to pulmonary edema
accompanied by neutrophil, macrophage, and erythrocyte infiltration
Labeling of stem cells with PKH26 dye
Avian influenza virus H9N2 will obtained from animal research institute and prepare for desired dose for intranasl adminstration
staphylococcus aureus will be obtained from Microbiology department and prepare
Sampling
Mice will be observed during experiment period to record the signs and deaths occurred at the times of scarification. mice will be sacrificed on
1day ,
3 day ,
7 day post infection
from each group 5 mice will sacrificed in each sample.
Sample
Tissue sample
1-upper respiratory ( nasal chonchae , nasal cavity)
2-trachea 3- lung 4-heart 5-liver 6-kidney 7-spleen 8-brain
Fluid sample
Blood
BAL
Tissue Sample

Blood sample
will be collected on anticoagulant
will be submitted for CBC picture
Broncho alveolar lavage
will be collected for detection of INFγ & TNFα
Under supervision of
Submitted By:
Mohamed Refat Mousa
For the degree of
M.V.Sc
Prof. Veterinary pathology
faculty of veterinary medicine
Cairo University
Prof. Veterinary pathology
faculty of veterinary medicine
Cairo University
Aim of Study
1- study zonootic importance of egyption isolate of AI H9N2 by Transmission in murine model
2- study complication of the virus after secondry bacterial infection
3- study therapeutic effect of the MSC on viral infection and its complication as anti inflammatory and immunomodulatory effect
Balb/c mice
Type: Balb/c mice.
Number: 135
Sex: Female
Age: from 3 to 6 weeks
Weight: 20-35 grams
will be obtained from Egyptian company for production of vaccines, sera and drugs ( EGY VAC).
Formalin fixative tissue sample:
1- H& E stainning
2-Immunohistochemistry
Unstained paraffin-embedded sections which will prepared from different collected tissue sample for immunohistochemistry staining
Fluid sample
Material & Method
- physiologically stable and show little to no toxic side-effects on cell systems.
- ideal for in vitro cell labelling and in vivo cell tracking.
- visualized in culture up to 100 days after staining (for non-dividing cells).
- divide equally when the cells divide.
-After staining with PKH dyes, Most commonly, 4-6 divisions can be visualized.

enriched on brain heart infusion broth for 24 hr. at 37 oc
Then grown on blood agar, MANITOL SALT AGAR ,tryptic soya agar for 24 hr. at 37 oc hr 24
Confirmation by biochemical tests:-
• Oxidase (-)
• Catalase (+)
• Coagulase (+ )
• Mannitol fermentation (+)

Calculation of colony forming unit from colony from tryptic soya agar equal to McFarland 0.5
LI yan, CHI ying, BIAN qian,WEN tian,ZHANG wenshuai, Mesenchymal Stem Cells therapy for H9N2 Avian Influenza Viruses Induced Acute Lung Injury in Mice
Fluorescence imaging of frozen sections
used for detection of labelled stem cells in different organs
by fluorescent microscope
Isolation of BM-MSCs
flushing the tibiae and femurs with Dulbecco’ s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine medium .
Nucleated cells were isolated and resuspended in complete culture medium supplemented with 1% penicillin-streptomycin .
Cells incubated at 37° C in 5% humidified Co2 for 12-14 days as primary culture or upon formation of large colonies
pluripotent stromal cells with multilineage potential that can be isolated from
embryonic tissue
adipose tissue
liver
muscle and dental pulp

adult bone marrow
remains the most common source of MSCs for preclinical and clinical studies
have therapeutic modality in various inflammatory disease including ALI
MSCs and inflammatory lung diseases
Why Murine model
-BMD MSCs attenuate leukocytic infiltration in lung and protect against pulmonary edema.
- decrease plasma level of pro-inflammatory cytokines as IL-1B, IFNγ, IL-6 , TNF, MIP-1α.
- decrease Th-1 response and maintain level of Th-2 cytokines.
PANMINERVAR MED 2009;51:5-16
Oxidative stress in ALI
occur by reactive oxygen metabolites produced by activated leukocyte caused tissue injury
MSCs attenuate oxidation of Cys & GSH redox system in vivo
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