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  1. “In vivo” Techniques:

 1.1. Pulmonary fibrosis animal model.[1-5]

1.1.1. Rat/ mouse with intra-tracheal bleomycin

1.2. Neutrophilic inflammation animal model.[6]

1.2.1. Rat/ mouse with intra-tracheal LPS.

1.2.2. Rat/ mouse with intra-tracheal bleomycin.

1.2.3. Rat/ mouse with inhaled cigarette smoke.

1.3. Pulmonary Hypertension animal model.

1.3.1. Rat/ mouse with monocrotaline.

1.3.2. Rat/ mouse with intratraqueal bleomycin

1.4. Asthma experimental model. [7, 8]

1.4.1. Rat/ mouse con ovoalbúmina (OVA).

1.5. COPD model.

1.5.1. Rat/ mouse with inhaled tobacco smoke with nose-only system (TSE; Inhalation exposure system).

1.5.2. Rat/ mouse with intratracheal elastase.

1.6. Intravital microscopy: study of leukocyte-endothelium interaction. [9-12]

1.6.1. Rat mesentery: stimuly: LPS, citokines, chemokines.

1.6.2. Mouse cremaster: stimuly: LPS, citokines, chemokines, tobacco smoke extract  and OVA antigenic “challenge”.

1.7. Molecular images technique (OncovisionAlbira Equipment for animal follow up, and diverse applications in different animal)

1.7.1. micro-CT: evaluation of pulmonary damage at real time.

1.7.2. micro-CT/PET: evaluation of inflammation and pulmonary damage at real time

1.7.3. micro-CT/SPECT; evaluation of pulmonary and heart circulation- Evaluation of pulmonary damage at real time.

1.8. Pulmonary function parameters measurement in rat and mouse conscious and unconscious (BUXCORsystem: Animal Plethysmograph to measure resistance and compliance in dynamic or static animals and perform dose-response studies).

  1. “In vitro” Techniques:

2.1. Biochemistry:

2.1.1. Determine intracellular calcium by fluorescence microscopy at real time. Pharmacological modulation of homeostasis of intracelular calcium.[13, 14]

2.1.2. Determine inflammatory molecules (citokines, chemokines, metalloproteinases, growth factors etc.)

2.1.1 ELISA [15,16]

2.1.2 Luminex (Technology for measures multiple analytes simultaneously in a single reaction vessel)

2.1.3. Determine by HPLC with fluorescence detector, UV-visible, refraction index: drugs, carbohydrates and taurine in different biologic fluids. [17]

2.1.3.1. Determine by HPLC intestinal permeability, intestinal atrophy and malnutrition by detecting carbohydrates, manitol/lactulose and xylose in urine.

2.1.3.2. Farmacokinetics in human and animal model by HPLC/mass spectrometry.

2.1.3.3. Determine diverse substances in biological fluids in human and animal samples.

2.1.4. Determine reactive oxygen species [16, 18, 19]:

  • H202by ELISA
  • Intracelular reactive oxygen species by DCFDA by microscopic fluorescence at real time.

2.1.5 Determine protein expression.

2.1.4.1 Tranditional Western blott [15]

2.1.4.1 Zeptosens platform: protein microarrays [20]

2.2. Molecular:

2.2.1. Real Time RT-PCR [15]

2.2.2. Real Time RT-PCR based in nanofluids (Fluidigm)

2.2.3. Microarrays based in PCR [21]

2.2.4. Nucleic acid sequencing

2.2.5. Farmacogenetic analysi

2.3. Histology and Immunohistochemistry

2.4. Pharmacomechanics: [22]

  (Human and animal bronchi, pulmonary arteria and pulmonary vein)

 2.4.1. Immersion organ bath studies onset offset off potency

2.4.2. Organ bath EFS

2.4.3. Myograph [23]

2.5. Culture cells:

2.5.1. Cell line culture

2.5.2. Primary culture of human cells

(Diagnose: Healthy,COPD,IPF, PH, asthma, etc.)

2.5.2.1 Obtaining primary keratinocytes.

2.5.2.2 Obtaining human bronchial epithelial cells.[14,24, 25]

2.5.2.3 Obtaining cilliated human bronchial epithelial cells in air-liquid interphase[14, 24, 25]

2.5.2.4 Obtaining human bronquial smooth muscle cells and human arterial smooth muscle cells.

2.5.2.5 Obtaining fibroblasts from human bronchi.[15]

2.5.2.6 Obtaining pulmonary artery endothelial cells.[16]

2.5.2.7. Blood cells (Neutrophils, Eosinophils isolated by MACS, Mononuclears)[19]

2.5.3. Cellular transfection with Respiratory Sincitial Virus (RSV) [26]

2.5.4. Genetic silencing [15]

2.6. “Flow Chamber”:

2.6.1. Study of leukocyte-endothelium interaction in primary cultures of human endothelial cells (HUVECs, HUAECs and pulmonary artery) [9, 12, 27]

2.6.2. Studies of complete blood, or leukocytes subtypes isolated form healthy individual sor with a pulmonary disease.

2.7. Flow citometry:

 2.7.1. Studies on expression of adhesion molecules in complete blood of different leukocytes subtypes [9].

2.7.2 Apoptosis studies in isolated leukocytes subtypes.[28,29]

2.7.3. Cellular population caracterization, surface molecule expression.

2.8 Dermal Permeability studies “Franz Cells”:

2.8.1 Skin permeability studies (human abdominal skin, scalp and nails) of different pharmaceutical preparations.

2.9. Studies of pharmacologic cell toxicity and chemical/organic products toxicity according to ISO 10993-5:2009

Bibliography:

  1. 1. Cortijo J, Iranzo A, Milara X, Mata M, Cerda-Nicolas M, Ruiz-Sauri A, Tenor H, Hatzelmann A, Morcillo EJ. Roflumilast, a phosphodiesterase 4 inhibitor, alleviates bleomycin-induced lung injury. Br J Pharmacol 2009: 156(3): 534-544.
  2. Barrio J, Cortijo J, Milara J, Mata M, Guijarro R, Blasco P, Morcillo EJ. In vitro tracheal hyperresponsiveness to muscarinic receptor stimulation by carbachol in a rat model of bleomycin-induced pulmonary fibrosis. Auton Autacoid Pharmacol 2006: 26(3): 327-333.
  3. Mata M, Ruiz A, Cerda M, Martinez-Losa M, Cortijo J, Santangelo F, Serrano-Mollar A, Llombart-Bosch A, Morcillo EJ. Oral N-acetylcysteine reduces bleomycin-induced lung damage and mucin Muc5ac expression in rats. Eur Respir J 2003: 22(6): 900-905.
  4. Serrano-Mollar A, Closa D, Prats N, Blesa S, Martinez-Losa M, Cortijo J, Estrela JM, Morcillo EJ, Bulbena O. In vivo antioxidant treatment protects against bleomycin-induced lung damage in rats. Br J Pharmacol 2003: 138(6): 1037-1048.
  5. Cortijo J, Cerda-Nicolas M, Serrano A, Bioque G, Estrela JM, Santangelo F, Esteras A, Llombart-Bosch A, Morcillo EJ. Attenuation by oral N-acetylcysteine of bleomycin-induced lung injury in rats. Eur Respir J 2001: 17(6): 1228-1235.
  6. Sanz MJ, Nabah YN, Cerda-Nicolas M, O’Connor JE, Issekutz AC, Cortijo J, Morcillo EJ. Erythromycin exerts in vivo anti-inflammatory activity downregulating cell adhesion molecule expression. Br J Pharmacol 2005: 144(2): 190-201.
  7. Cortijo J, Sanz MJ, Iranzo A, Montesinos JL, Nabah YN, Alfon J, Gomez LA, Merlos M, Morcillo EJ. A small molecule, orally active, alpha4beta1/alpha4beta7 dual antagonist reduces leukocyte infiltration and airway hyper-responsiveness in an experimental model of allergic asthma in Brown Norway rats. Br J Pharmacol 2006: 147(6): 661-670.
  8. Blesa S, Cortijo J, Martinez-Losa M, Mata M, Seda E, Santangelo F, Morcillo EJ. Effectiveness of oral N -acetylcysteine in a rat experimental model of asthma. Pharmacol Res 2002: 45(2): 135-140.
  9. Sanz MJ, Cortijo J, Taha MA, Cerda-Nicolas M, Schatton E, Burgbacher B, Klar J, Tenor H, Schudt C, Issekutz AC, Hatzelmann A, Morcillo EJ. Roflumilast inhibits leukocyte-endothelial cell interactions, expression of adhesion molecules and microvascular permeability. Br J Pharmacol 2007: 152(4): 481-492.
  10. Abu Nabah YN, Losada M, Estelles R, Mateo T, Company C, Piqueras L, Lopez-Gines C, Sarau H, Cortijo J, Morcillo EJ, Jose PJ, Sanz MJ. CXCR2 blockade impairs angiotensin II-induced CC chemokine synthesis and mononuclear leukocyte infiltration. Arterioscler Thromb Vasc Biol 2007: 27(11): 2370-2376.
  11. Buenestado A, Cortijo J, Sanz MJ, Naim-Abu-Nabah Y, Martinez-Losa M, Mata M, Issekutz AC, Marti-Bonmati E, Morcillo EJ. Olive oil-based lipid emulsion’s neutral effects on neutrophil functions and leukocyte-endothelial cell interactions. JPEN J Parenter Enteral Nutr 2006: 30(4): 286-296.
  12. Mateo T, Abu Nabah YN, Abu Taha M, Mata M, Cerda-Nicolas M, Proudfoot AE, Stahl RA, Issekutz AC, Cortijo J, Morcillo EJ, Jose PJ, Sanz MJ. Angiotensin II-induced mononuclear leukocyte interactions with arteriolar and venular endothelium are mediated by the release of different CC chemokines. J Immunol 2006: 176(9): 5577-5586.
  13. Milara J, Mata M, Serrano A, Peiro T, Morcillo EJ, Cortijo J. Extracellular calcium-sensing receptor mediates human bronchial epithelial wound repair. Biochem Pharmacol 2010: 80(2): 236-246.
  14. Cortijo J, Milara J, Mata M, Donet E, Gavara N, Peel SE, Hall IP, Morcillo EJ. Nickel induces intracellular calcium mobilization and pathophysiological responses in human cultured airway epithelial cells. Chem Biol Interact 2010: 183(1): 25-33.
  15. Milara J, Serrano A, Peiro T, Gavalda A, Miralpeix M, Morcillo EJ, Cortijo J. Aclidinium inhibits human lung fibroblast to myofibroblast transition. Thorax 2012: 67(3): 229-237.
  16. Milara J, Juan G, Ortiz JL, Guijarro R, Losada M, Serrano A, Morcillo EJ, Cortijo J. Cigarette smoke-induced pulmonary endothelial dysfunction is partially suppressed by sildenafil. Eur J Pharm Sci 2010: 39(5): 363-372.
  17. Prieto L, Badiola C, Cortijo J, Perez-Frances C, Gutierrez V, Lanuza A. Phenol-containing saline solution as a diluent for adenosine 5′-monophosphate in bronchial challenge testing. Chest 2005: 127(1): 125-130.
  18. Milara J, Ortiz JL, Juan G, Guijarro R, Almudever P, Martorell M, Morcillo EJ, Cortijo J. Cigarette smoke exposure up-regulates endothelin receptor B in human pulmonary artery endothelial cells: molecular and functional consequences. Br J Pharmacol 2010: 161(7): 1599-1615.
  19. Milara J, Juan G, Peiro T, Serrano A, Cortijo J. Neutrophil activation in severe, early-onset COPD patients versus healthy non-smoker subjects in vitro: effects of antioxidant therapy. Respiration 2012: 83(2): 147-158.
  20. Milara J, Navarro R, Juan G, Peiro T, Serrano A, Ramon M, Morcillo E, Cortijo J. Sphingosine-1-phosphate is increased in patients with idiopathic pulmonary fibrosis and mediates epithelial to mesenchymal transition. Thorax 2012: 67(2): 147-156.
  21. Mata M, Pallardo F, Morcillo EJ, Cortijo J. Piclamilast inhibits the pro-apoptotic and anti-proliferative responses of A549 cells exposed to H(2)O(2) via mechanisms involving AP-1 activation. Free Radic Res 2012: 46(5): 690-699.
  22. Cortijo J, Sarria B, Mata M, Naline E, Advenier C, Morcillo EJ. Effects of ouabain on human bronchial muscle in vitro. Naunyn Schmiedebergs Arch Pharmacol 2003: 368(5): 393-403.
  23. Moral-Sanz J, Gonzalez T, Menendez C, David M, Moreno L, Macias A, Cortijo J, Valenzuela C, Perez-Vizcaino F, Cogolludo A. Ceramide inhibits Kv currents and contributes to TP-receptor-induced vasoconstriction in rat and human pulmonary arteries. Am J Physiol Cell Physiol 2011: 301(1): C186-194.
  24. Milara J, Armengot M, Banuls P, Tenor H, Beume R, Artigues E, Cortijo J. Roflumilast N-oxide, a PDE4 inhibitor, improves cilia motility and ciliated human bronchial epithelial cells compromised by cigarette smoke in vitro. Br J Pharmacol 2012: 166(8): 2243-2262.
  25. Cortijo J, Mata M, Milara J, Donet E, Gavalda A, Miralpeix M, Morcillo EJ. Aclidinium inhibits cholinergic and tobacco smoke-induced MUC5AC in human airways. Eur Respir J 2011: 37(2): 244-254.
  26. Mata M, Morcillo E, Gimeno C, Cortijo J. N-acetyl-L-cysteine (NAC) inhibit mucin synthesis and pro-inflammatory mediators in alveolar type II epithelial cells infected with influenza virus A and B and with respiratory syncytial virus (RSV). Biochem Pharmacol 2011: 82(5): 548-555.
  27. Sanz MJ, Alvarez A, Piqueras L, Cerda M, Issekutz AC, Lobb RR, Cortijo J, Morcillo EJ. Rolipram inhibits leukocyte-endothelial cell interactions in vivo through P- and E-selectin downregulation. Br J Pharmacol 2002: 135(8): 1872-1881.
  28. Martinez-Losa M, Cortijo J, Juan G, Ramon M, Sanz MJ, Morcillo EJ.Modulatory effects of N-acetyl-L-cysteine on human eosinophil apoptosis. Eur Respir J 2007: 30(3): 436-442.
  29. Martinez-Losa M, Cortijo J, Piqueras L, Sanz MJ, Morcillo EJ. Taurine chloramine inhibits functional responses of human eosinophils in vitro. Clin Exp Allergy 2009: 39(4): 537-546.

 

 

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