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Nanotube Bound Fibroblast Defense and Patient Drug Resistance: NIDHI Criteria R.Sharma*,N.Goel**, S.Kwon***
*Center of Nano and Biotechnology, Florida State University & TCC, Tallahassee, FL 32310, USA **Microbiology Department, Post Graduate Institute of Medical Sciences, Rohtak, Haryana, India ***Department of Biological Engineering, Utah State University, Logan, UT 84322, USA
Anatomy of Human Lung
nasal cavity pharynx
. Q Smooth muscle cells br
If all these weaken or fail. apoptosis and metabolic integrity). abscess. pus. Stage 2.A clinical survey of patients with LRTI active infection for presence of bacteria in lungs and clinical symptoms of drug resistance. PURPOSE: ³NEED OF IMMUNE FIBROBLAST DEFENSE IN HIGH LTRI INFECTION´ (NIDHI) Stage 1. tissue structural damage. . Starts with tumor. If it progresses further. Stage 3. Starts with necrosis. debris (all visible by microscopy)-Drug response and resistance starts.Study Design 1. Alveolar cells keep intact themselves (hypoxia.Physical characterization of cultured alveolar fibroblast cells customized with carbon nanotube coated delivery system.Gluconeogenesis control and inflammatory characteristics of fibroblasts in prototype 3D lung tissue 3. 2. proliferation.
Fibroblasts experience difficulty (not known) to wrestle with bacteria (unclear which type and drug resistance) in LRTI infection.Lung Fibroblasts and Their Fate 1. In LRTI. 2. initially cytokine induced release of nitric oxide 3.What difficulties can be there in transferring engineered tissue in human beings? 2. Alveolar fibroblasts maintain homeostasis in lung 2. Criteria: 1.Who are the patients who can be benefited best by this technique? Need of matched fibroblast characteristics . Fibroblasts circumvent in tissue with dying alveolar cells (not known). Fibroblast enhance vascularization of lung Fate of fibroblasts: 1.
2)The lung fibroblasts were embedded in collagen I in Transwell polyester 3)Above the 10 mm porous Transwell polyester membrane epithelial cells were seeded 4)The epithelial and fibroblast cells communicate 5)These cell system was used for cell-specific protein expression.Fibroblasts in Airway Model Proposed Airway Model: 1)It maintains the normal anatomical arrangement (orientation and dimensions) of epithelial and fibroblast cells. Transelectric Resistance .
Why in-vitro co-culture system? Experimental access to the airways involved in asthma Post-mortem analysis of the smaller airways and direct bronchial biopsy of the upper airways (approximately generations 2-5) Monolayer culture of the key individual cell types These systems are limited by the absence of cell-cell interactions. . which are present in vivo.
In-vitro Co-culture System Airway Lumen mucus CCD18lu (fibroblast) A549 monolayer Macrophage Epithelial cells Fibroblasts collagen gel polycarbonate membrane . Qbr Smooth muscle cells .
Tissue-Engineered Bronchial Mucosa The normal human bronchial epithelial cells are cultured as a monolayer over the thin polyester membrane and the subepithelial human lung fibroblast-embedded collagen gel. . 3 major steps and three weeks in culture.
Physiological Characteristics of Airway Epithelial Cells Cultured airway epithelium expresses three key phenotypic markers .I.
2 0.04 0 12 10 8 6 4 2 0 The Expression of MMP-2 and MMP-9 (Co-culture Effect) 20 18 16 0.32 MMP-2 production in ng/ml no physical denudation 0.08 0.24 0.4 0.16 0.16 0.36 0.The Expression of MMP-2 and MMP-9 in Epithelial Cell Monolayer 20 0.12 0.36 18 MMP-9 production in ng/ml 16 no physical denudation with physical denudation 0.12 0.4 A549 cells alone co-culture MMP-9 production in ng/ml 0.32 MMP-2 production in ng/ml 14 12 10 8 6 4 2 0 0.28 A549 cells alone co-culture 0.2 0.28 14 with physical denudation 0.08 0.24 0.04 0 .
II. Physiological Characteristics of Airway Epithelial Cells µTight Junction¶ .
Transepithelial Electrical Resistance (TER) 1.Kwon et al. MA) attached to a dual ³chopstick´ or transcellular resistance measurement chamber (Millipore. MA).cm2).Different concentrations of CNTs were exposed to the co-culture layers for 6 hours. Electric current could then be passed across the epithelium to measure TER (ohms.Human bronchial epithelial cells were grown at the interface of air and liquid. Bedford. 4.TER of human bronchial epithelial cell with fibroblasts-embedded collagen layers cultured in TranswellTM was monitored using a portable Voltohmmeter (Millipore.Each of the two electrode systems contained Ag/AgCl electrode for measuring voltage and a concentric spiral of silver wire for passing current across the epithelium. 2.2008 IJToxicol) . 5. Bedford. 3. TER values higher than the background fluid resistance means confluent airway epithelium with tight junctions. (S.Culture media was provided from the bottom through the porous membrane.
10-20% of SWCNTs rapidly compromised the barrier function of the epithelium and the TER decreased to 120 ohms. After removing SWCNTs. the TER completely recovered to the control level .The TER of the controls (5% and 20% of Triton X-100 and 0% of SWCNT) were stable around 500 ohms.cm2.cm2 for 48 hours.
IJToxicol) . Afterward. Data were calibrated to the appropriate calibration curve ( Sigma protocols).1 N HCl in anhydrous isopropanol) was added to each well.Kwon et al. After 48 hours. 850 mL of the MTT solubilization solution (10% Triton X-100 in 0. Cytotoxicity of Engineered Fibroblasts Cytotoxicity testing The MTT assay (Sigma) for mitochondrial activity of cells. 150 mL of MTT (5 mg/ml) was added to each well and incubated for 4 hours.III. The resulting formazan crystals was solubilized in acidic isopropanol and quantified by measuring absorbance at 570 nm. Cells were exposed to varying concentrations of CNTs. (S.
one of the two stable oxidized products of NO in the liquid phase.Kwon et al. WI) was used to detect the level of nitrite (NO2¯). Griess Reagent system (Promega Corporation. (S.Inflammatory response (NO) Inflammatory Response by NO production Nitric oxide (NO) production was measured to identify the level of inflammation. IJToxicol) .
MTT activity was decreased as concentration of SWCNTs increased. Each NO production was normalized by total proteins. Cellular metabolic activity was observed for exposure of different following concentrations of SWCNTs to both cell layers.Cytotoxicity & Inflammatory Response Epithelial cells increased NO formation as the concentration of SWCNTs increased (top panel). especially for epithelial cells .
Culture threshold was taken as follows: 105 Colony forming unit (CFU)/mL for ETA. bacteria etc. radiological) First line drug treatment First line drug treatment fails patients progress with disease. biochemistry. drug resistance(second line treatment) second line treatment fails patient progress is poor. 104 CFU/mL For BAL. Stage 2: What are lab parameters ? Lab tests of severe infection and damage by virus. pathological. respiratory block signs. . failure of gas exchange and pulmonary capability Physical symptoms of respiratory arrest. lab respiratory.. microbiological.01mL of sample. The growth on the plates was observed after 24 hours of incubation.Sequential approach of LRTI obstructive disease Stage 1: What are the LTRI symptoms ? Physical signs Laboratory parameters(physiological. Semi-quantitative culture was done and plates were incubated at 37 C. Stage 3: Failure of drug response. renal panel abnormal tests extensive lung machine treatment (if poor progress it suggests the need of fibroblast replacement either in early 3 stage or late 3 stage Drug sensitivity test: 5% sheep blood agar. 103 CFU/mL For PSB. Mac-Conkey Agar plate and chocolateAgar plate using a 4 mm nihrome wire to hold .
7 66.Drug Resistance Pattern (%) of gram-negative bacteria (GNB) isolated from lower respiratory tract against first line and second line antibiotics Drug A.5 96.2 100 100 11.aer (57) ND* ND 61.4 .6 Ac 97.7 Cz 100 Mr 25.pneu (22) 90.4 ND 68.6 95.4 Ao ND TZP ND Nt ND 0f ND Ck ND K.5 12.4 22.7 0 91.bau (38) Co 79.7 100 91.6 Ak 87.9 9.7 49.7 ND ND ND ND ND E.4 68.4 Cac 97.1 100 76.5 100 100 ND ND ND ND ND 100 0 41.coli Ent (17) (12) 100 0 72.2 ND ND ND ND ND C.8 ND ND 94.2 68.9 9.2 Cf 89.1 63.1 70.fr (8) 100 0 75 100 87.1 100 100 ND ND ND ND ND P.5 Do 2.
sepsis. lost pulmonary function and oxygen holding capacity Clinical condition good NO need of fibroblast Antibiotics and Ventilation therapy Deteriorating condition with pathogen or pandrug resistant pathogen Fibroblast transfusion or Lung-heart perfusion . Pandrug resistance Dysponea. mutilobar .Steps to decide need of fibroblasts Check untreatable ICU infections (Acinetobacter baumannii and pseudomonas aeruginosa) Check symptoms and drug resistant to Aminoglycosides or ciprofloxacin plus one of antipseudomonal lactamase stable lactam antibiotic and carbapenems. rapidly progressing lung shadow in imaging. shock (BP<90 /<60 mm Hg).
cytotoxicity. airway block and no response to AED and cardiac resuscitation In lungs. unaffected cytokines. Upon exposure to toxins. hormone dependence Quick Action Transfusion of Transfusion fibroblasts in lungs Recovered Lungs Transfusion and Capacity . alveolar cell regulatory failure: by poor inflammatory response. inflammatory response and alveolar hypoxia Terminal patient in ICU Deteriorated Glucose control BP drop <90/<50 mmHg (-¨ 10/9 mmHg / hr) Sudden oxygen insufficiency (respiratory acidosis/alkalosis) PV. hypoxia Engineered Fibroblast characteristics: High TER. low RBC. Cardiac Output Pulmonary collapse. normal hypoxia and respiratory burst enzymes alveolar enzymes Less oxygen.Match and Need for Fibroblasts in LRTI Engineered Fibroblasts: Resistant to LRTI responsive bacteria Enhanced host independent capacity to reduce alveolar cytotoxicity. LC.
. The engineered fibroblasts have specific characteristics of TER.Conclusion The simple criterion of respiratory track infection patient selection who may need of the engineered fibroblasts without stem cell hazards. cyto-inflammatory response Fibroblasts may be good alternative to regenerate 3D lung architecture in future by transfusion.
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