seminario a cerca del articulo descrito

1. Diseño del título
Andrés Canchila
Daniela Castillo Sanchez
Medicine students. 3th semester
Molecular Biology

2. INTRODUCTION

3. THYMIC STROMAL LYMPHOPOIETIN
• Is a protein belonging to the cytokine family.
• It is known to play an important role in the maturation of T
cell populations through activation of antigen presenting
cells.
• TSLP is produced mainly by non-hematopoietic cells such as
fibroblasts, epithelial cells and different types of stromal or
stromal-like cells.

4. ASTHMA
• Asthma is a disease that causes the
airways of the lungs to swell and
narrow. It leads to wheezing,
shortness of breath, chest tightness,
and coughing.
• Asthma is caused by swelling
(inflammation) in the airways. When
an asthma attack occurs, the lining
of the air passages swells and the
muscles surrounding the airways
become tight.

5. RHINOVIRUS
• It is the most common viral infectious agent in humans
and is the predominant cause of the common cold.
Rhinovirus infection proliferates in temperatures between
33–35 °C (91–95 °F), the temperatures found in the
nose.
• The primary route of entry for human rhinoviruses is the
upper respiratory tract (mouth and nose). Rhinovirus A
and B bind to ICAM-1 (Inter-Cellular Adhesion Molecule
1) also known as CD54 (Cluster of Differentiation
54) receptors on respiratory epithelial cells
• As the virus replicates and spreads, infected cells release
distress signals known
as chemokines and cytokines (which in turn activate
inflammatory mediators). Cell lysis occurs at the upper
respiratory epithelium.

6. APOCYNIN
• Also known as acetovanillone
• The function of apocinin base in
prevents the inflamation process as
result of his ability to prevent the
formation of free radicals. Apocynin is
an inhibitor of NADPH Oxidase enzyme
activity preventing the formation of
superoxide from O2 , normaly we use
this superoxide in the immune system
to kill bacteria and fungi, but the
apocynin prevents te formation of
superoxides in human white blood cells
or neurthopilic granulocytes.

7. AIRWAY REMODELING
• Refers to the structural
changes that occur in
both large and small
airways relevant to
miscellaneous diseases
including asthma.

8. RELATION BETWEEN CONCEPTS
• Rhinovirus and Asma are promotors of the airway
remodeling because they increase the expression of TGF-B ,
apocinin prevents de inflamation derivated of the rhinovirus
and asma actions trought the ability to prevent the
formation of free radicals. Also, the TSLP is a citokine that is
significantly elevanted in asthma and allergy diseases

9. GENERAL OBJECTIVE
The objective of this article is to find the influence of the
Human Rhinovirus on the genes involved in the
mechanism of airway remodelling which is in close
relation with asthma, and study the impact of thymic
stromal lymphopoietin (TSLP) and oxidative stress on
airway remodelling in the context of this infection.

10. MÉTODOS

11. CULTIVO CELULAR
• Los fibroblastos humanos
normales (NHLF) fueron
cultivados en el kit de
fibroblastos FMG-2
desarrollado para apoyar el
crecimiento de los
fibroblastos primarios.
Contiene 2% de suero

12. PACIENTES
• 10 voluntarios no
fumadores fueron
involucrados en el estudio.
• 5 hombres y 5 mujeres
• células mononucleares de
sangre periférica fue aislada
por centrifugación con
histopaque 1017

13. PREPARACIÓN DEL VIRUS
Se usó el virus humano RV16, que fue
producido en células suceptibles a
infección (HeLa) hasta los efectos
citopatológicos fueran evidentes
Las cepas de virus, mediante la
infección de capas subconfluentes
de células HeLa
HVR fue expuesto a 58°C 1
hora para inactivarlo de las
células HeLa
Las células objetivo fueron
infectadas con la aplicación
de 50µl de vehículo o rinovirus
Las células fueron incubadas durante
24 horas a 33°C y 5% de Co2

14. PROCEDIMIENTO EXPERIMENTAL
• La infeccion con HRRV
16 fue hecha por 48
horas, luego de esto las
celulas NHLF y PMBC
fueron incubadas con
apocinina o con TSLP.
La muestra control solo
tenia el medio.

15. Extracción de RNA
• El RNA total fue aislado de
una célula usando “RNA cell
Mini Kit with QIAshredder
(Qiagen). El RNA se trató,
purificó y se extrajo en 30 µl
de Rnasa libre de agua y
almacenado a -80°C para el
análisis.
• Se realizó una transcripción
reversa al RNA usando “High
capacity cDNA kit”

16. PCR
• Desnaturalización: En un tubo se agregan primers,
nucleótidos libres, ADN y Taq polimerasa. Se calienta
a 95°C lo que provoca la separación de las dos
cadenas de ADN.
• Alineamiento: Se enfría a 55°C. Los primers se unen
a las regiones complementarias de ADN separadas.
Allí, todo el ADN está en forma de cadena simple,
menos las dos regiones en las cuales los primers se
ligarán a los dos lados de la secuencia de ADN.
• Polimerización: La temperatura se eleva a 72°C. La
Taq polimerasa comienza a sintetizar nuevo ADN,
que ocurre a una tasa de aproximadamente 20
nucleótidos/s y en un minuto es sintetizada una
nueva copia del fragmento que se quiere analizar.

17. Immunoblot
• FUNDAMENTO: Es una
tecnica de 3 pasos. Separando
por tamaño, transferiendo a
un soporte solido y observdo a
través de la proteinas
marcada primaria o
secundariamente con
anticuerpos
• Se hizo para ver la expression
de proteinas de tgf-b y de la
arginasa1 con el hrv el tmls y
la apocinina

18. ELISA
• FUNDAMENTO:Enzime-linked
immunosorbent Assay: Un
antigeno es detectado por un
anticuerpo, dicho anticuerpo está
unido a una enzima marcada que
es capaz de producir un producto
detectable, como un cambio de
color u otro tipo de cambio.
• PARA QUE: Se utilizó para ver la
medida de la proteína TIMP-1 en
el sobrenadante de NHLF y PBMC

19. RESULTADOS

20. FIGURA 1

21. FIGURA 3

22. FIGURA 5

23. FIGURA 7

24. DISCUSSION

25. AUTOR WHAT HE/SHE SAID? YES/ NO
Sokolowska et al., 2005; Tang et
al., 2006
“The mechanisms regulating airway
remodeling changes and the order in
which these changes develop remain
poorly understood”
Tacon et al., 2012
It is thought that HRV infection causes
asthma exacerbations by altering the
epithelial cell biology of the airway in a
manner that promotes airway
inflammation and remodeling by up-
regulating the epithelial production of
mediators involved in the inflammatory
and remodeling processes, therefore
promoting them
Kenyon et al., 2003; Minshall et
al., 1997
In asthmatics, increased TGF-B1
mRNA expression levels are
correlated with the depth of
subepithelial fibrosis
Thomas et al., 2009
TGF-β, as a key regulator of tissue
remodeling, has been shown to
enhance rhinovirus replication and
pro- inflammatory responses in
airway fibroblasts

26. CONCLUSIONS

27. • Asthma can occurs because
of the interaction between
the inflamattion and
remodeling of the airways
• In asthma and allergic
diseases the TSLP is
increased, because in the
epithelial cells, mast cells,
airway smooth muscle cells
and fibroblasts
• TSLP production is
increased in inflamed tissues
because of the action of
viruses and allergens (HRV
or Asthma)
• TGF-B has the capicity of
create fribrosis in the tissue,
this can be mediated by the
action of NADPH oxidase
that create free radicals

30. THANK YOU
GRACIAS