Intragastric and Intranasal Administration of Bifidobacterium Animalis to Reduce Airway Inflammation in Asthma.

Research proposal: Intragastric and intranasal administration of Bifidobacterium animalis to reduce airway inflammation in asthma.
Project submitted to : madam Raheela Rahmat

Submitted by: Shazia Naseer
Father name: Naseer Ahmed
Seat number:
Enrolment Number:

Class: B.S.C (H) 3rd year
Crouse #: 516
Department: Biotechnology
Date of submission: nov5th, 2012

Submitted by: Shazia Naseer
Father name: Naseer Ahmed
Seat number:
Enrolment Number:

Class: B.S.C (H) 3rd year
Crouse #: 516
Department: Biotechnology
Date of submission: nov5th, 2012

Performa for the submission of research proposal: Name and address of the: Department of Biotechnology, university of
Institution: Karachi.
Title of propose project: Intragastric and intranasal administration of Bifidobacterium animalis to reduce airway Inflammation in asthma.
Field of study: Medical microbiology
Nature of the project: Applied
Principle Investigator: Madam Raheela Rahmat
Research officer: Shazia Naseer Ahmed
Proposed duration: 5 months
Total fund requested:

Endorsement Principal Investigator Institutional Head Signature: Name: Madam Raheela Rahmat Dr. Mustafa Kamal Designation: Lecturer Associate Professor & Head of department
SHAZIA NASEER AHMED

ADDRESS: street # 43, house # 1002 block “C”, Jinnah road, sher shah colony Karachi
TEL # 03463593671
E.MAIL: sadiansr.sky@gmail.com
OBJECT:
Looking for internship in a hospital to get experience and training of lab working.

ACADEMIC QUALIFICATION:

MASTERS: doing M.SC in Biotechnology from University of Karachi. B.SC: (science) from Jinnah University for women North Nazimabad. INTERMEDIATE : (science) from Alma Mater Degree College Bihar colony MATRICULATION : (science) from AL –Badar Secondary School Baldia Town

COMUTER SKILLS:
MS-OFFICE 2003 and 2007
INTERNET

WORK EXPERIENCE:
Telemarketing executive at ufone franchise sher shah
6months experience of teaching biology personal: Marital status : Single
N.I.C No : 42401-9571808-8
Date of birth : 17 -03-1987 interpersonal skills : can easily get along with all types of people.

SUMMARY

Introduction
Asthma is a chronic hyperreactivity of the bronchi which occurs during inflammation happens while the attack of allergens. In allergic form of asthma airway of mucous lining becomes sensitized. This sensitization leads to appear symptoms includes wheezing, coughing, chest tightness and shortness of breath in common.
All developing countries like Pakistan where air pollution and weather disturbance is getting worse day bay day Asthma has become often occurring decease in adults and in children .
Two major factors of Asthma:
In general asthma has two forms [1] * Genetic factor : this genetic factor called “Atopy” . in this case baby hereditary infected though allergic asthma . but hypersensitivity reaction and shortness of breath always develop only when patient is exposed to allergen. * Environmental factors: in this form of asthma hypersensitivity reaction and symptoms appears when human airway passage come in contact with the some particles from which body or person is sensitive to these particles . High levels of endotoxin exposure may contribute to asthma risk. respiratory infections are has higher risk of developing asthma especially in young children [1] These particles includes which can become factors , allergens, cigarratte smoke, cockroaches, dust mites, mold, pets and animals, pollen, Classification of Asthma : Asthma attacks are different from person to person some patients have
Twise a week and not more then twice a month but severity of these attacks is varies. Where as some times patience has night time severity and attacks occurse daily in worst conditions . sometimes these attacks continual daytime symptoms and frequent night time symptoms .
If in these cases treatments is not giving to patient on time on the bases of severity asthma is classification on following.

Asthma in pregnancy :
Having asthma in pregnancy can gives drastic result for mother and fetus. In this due to asthmatic decease mother leads to shortness of breath and as a result of this during pregnancy less oxygen delivered to blood. This condition can cause very dangerous effects on the baby development. Many complications happens for instance low birth weight of baby, increase in blood pressure and premature birth.[4][5] * The low oxygen transfer can affect the lungs during pregnancy . * Respiratory infections and asthma during fetus developing months cause of wheezing in infant, and the chance of having heredity factors like atopy become higher in these infants [5]
Asthma attacks :
Include series of reactions like shortness of breath, wheezing, coughing, over inflammation of the chest, in sever condition patient skin and lips turn into blue color. [7] Apart from these common signs bronchospasm and high mucus production is produced. when person is on attack chest pain and flow of blood in body runs faster and blood pressure gets higher which is dangerous and if treatment does not reach to the patient and long time of asthma attack can cause death eventually, due to oxygen deficiency. [8] when person ignores early signs of attack and continuous exposure to allergen pollen, dust mits and smoke in this attack has possibility to develop asthmatics [10]

Acute form: Acute attacks in asthma happence in those who have sever asthma types death rate is also high in acute attacks because peak expiratory flow gets 75 percent which is higher as compare to mild asthma attack in which it gets till 50 percent. [9]
Pulse rate also reaches to its peak point which is noted at 120/mint and this condition os frightening lead to life threatening state. [9]
Types of Asthma : * Allergic Asthma: Is the most common type of asthma both in adult and children. All basic symptoms chronic cough, chest tightness, peak flow of runny nose watery eyes occurs. Immune system always try to protect the body from allergens. For this immune system secrets IgE . if body or mucus lining re-exposed to an allergen or re-attachment to IgE with allergens release chemicals and this results acute inflammation. An other factor is eosinophils release after binding allergen by this IgE reaches to higher and condition become sevevr [11]
Exercise induced Asthma : an other form os exercise induced asthma in this continuously work out ot vigorous aerobic activity or continuously exercise, where oxygen supply to the lungs is essential . this condition patiens airway become narrow and blood pressure increased and muscle in the line of lung passage injurs due to dehydration. [12]
Occupational Asthma :
This will happens occupationally during work places or in schools, university. At present over 400 work place substance have been identified as having asthmaganic triggers. this is most common at industries side where automobile work is done or where dust is also induced occupational asthma [14][15]

Cough variant Asthma :
15 percent to 20 percent of chidren under 11 years of age have this form of asthma in which chronic caugh is a signal to asthma diagnosis. These children should not only be evaluate for the presence of chronic cough but inhalation of foreging objects mite dust,pollen smoke also cause coughing [16]
Nocturnal Asthma :
Around 75 percent people with asthma have symptoms that disrupt both the length and depth of their nighttime sleep. 40 percent of children experienced night time asthma in which patient stay awake at night and sleep may cause frighting symptoms and they feel intrathoracic pressure during sleep and inflammation get worst in nocturnal asthma . [17]
Inflammation become worse due to increase in secreting eosinophils and lymphocytes [17]
Immunohistopathology of Asthma :
Inflammatory changer occurs throughout the central and peripheral airways. Airway showed infiltration by neutrophils and eosinophils, mast cell . transcription factors include in which proteins bind to the promoter region of gene. This initiate cytokines inflammatory response in asthma. [1]
Some cell of immune resoponse provides mechanism for adhesion of inflammatory cells. These cell are includes intercellular-leukocyle molecule-1, vascular cell molecule-1,endothelial leukocyte adhesion molecule [19]
Inflammation in asthma :
Inflammation of asthma is link with IgE antibody. Immunological hall masks of respiratory allergy are mainly is due to IgE and chemokines.lymphocytes eosinophils into the lungs [20] [1]
Basophils secrete intraleukin-4 and -13 to express CD4 ligand . the inerleukin-4 and interleukin-13 share alpha chain to deliverd signal on CD4 on Bcell binds to its ligand on Tcell it deliver second signal start inflammation [21] Th1 and Th2 cells: the balance and an imbalance between Th1 and Th2 celles play major roll on maintaining asthma attacks . the region of asthma severity this concept relies on the “hygiene hypothesis” which gives the information about avoidance of microbial exposure during time to time easily develop allericdiseases. [22]
TypeIII and TypeIV hypersensitivity in asthma:
In TypeIII hypersensitivity immune complex has different size of macrophages. antigen and antibody compleses that are not bind to cell Surface. Macroghages clesr out large complexes but small complexes which are not clear out though macrophages, insert themselves in small blood vessels. These small complexes become pathogenic for allergic asthma [14] [15] Type IV hypersensitivity is delayed because appearance get in to eyes in two to three days. Macrophage secrets IL-12. This stimulates CD4 and cells secrete cytokines [14] [15]

OBJECTIVES

METHODOLOGY

List of Instruments from Biotechnology Department

1. Weighing machine

2. Centrifuge machine.

3. Compound microscope.

4. Incubator.

5. Weighing machine

BUDGET
Estimated cost of total budget in Rs: YEAR | RECURRING | NON RECURRING | TOTAL (IN RS.) | 1st | 439141 | 215252 | 654393 |

Expenditure of salaries and allowances: S.NO | POST & PAY SCALE | NO.OF POSTS | 1st YEAR | 2nd YEAR | TOTAL (IN RS.) | 1 | Project investigator(PI) | 1 | 30000 | 30000 | 60000 | 2 | Research officer @10000/month | 1 | 120000 | 120000 | 240000 | 3 | Lab attendant @4000/month | 1 | 48000 | 48000 | 96000 | 5 | Contingency | | 75000 | 75000 | 150000 | | Total | | 273000 | 273000 | 546000 |

Expenditure of equipment and supplies in RS: S.NO | ITEMS | 1ST YEAR | 2ND YEAR | TOTAL (IN RS.) | 1 | Equipments | 215252 | 321687 | 536938 | 2 | Chemicals | 95749 | 309899 | 405648 | 3 | Glass ware | 10730 | - | 10730 | 4 | Plastic ware | 6668 | - | 6668 | 5 | Others | 52994 | 35995 | 88989 | | | | | | | Total | 381393 | 667581 | 1048973 |

APPENDIX A: equipment S.NO | EQUIPMENTS | TYPE | COST (IN RS.) | | | 277316 | | 1 | Green house | At 30oC,16 h photoperiod | 200000 | 2 | Mass flow controller | 0-250ml | 32550 | 3 | Sulfur analyzer | Model H19024 | 44766 | 4 | Growth chamber | Hotech instruments | 185562 | | Total | | 462878 | | Total + 16 % GST | | 536938 |

APPENDIX B1: Chemicals SN.O | CHEMICALS | CATALOG NO. | QUANTITY | COST IN RS. | 1 | Sodium hypochlorite sol | 23930-5 | 500ml | 4510 | | MS-media | | - | | 2 | CaCl2.2H2O | 35080 | 500g | 3800 | 3 | CoCl2.2H2O | C2911 | 100g | 4119.2 | 4 | MgSO4.7H2O | Ms921 | 500g | 6482.8 |

APPENDIX B2: Chemicals SN.O | CHEMICALS | CATALOG NO. | QUANTITY | COST IN RS. | 1 | β-mercaoto ethanol | BPE-176100 | 100g | 1417.8 | 2 | PVP | P6755 | 100g | 2447.5 | 3 | Chloroform | C5672 | 52ml | 1402.5 | 4 | Iso amyl alcohol | 13643 | 100ml | 1430 | 5 | Ammonium acetate | k/3400/53 | 500g | 2196.2 | 6 | Iso propanol | 405-7 | 480ml | 3465 | 7 | Ethanol 70% | G-9632 | 100ml | 1932 | 8 | TE buffer | T9650 | 1L | 2245.95 | 9 | Comassie blue | G-250 | 25g | 1946 | 10 | Phosphporic acid | p-6560 | 100g | 1436.4 | 11 | 1 M NaOH | S5881 | 500g | 1149.5 | 12 | Liquid nitrogen | d/2970/90 | 53L | 199136 | 13 | Potassium phosphate | P2828-3 | 25g | 1249.6 | 14 | 2mM EDTA | E2826-2 | 500g | 2249.9 | 15 | Nitroblue tetrazolium | N5514 | 10tablets | 2614.4 | 16 | Riboflavin | R0508 | 50g | 2888 | 17 | H2O2 | U1753 | 100g | 4947.6 | 18 | Potassium ferriamide | P8131 | 100g | 1155.2 | 19 | Ferric chloride | F7134 | 100g | 1732.8 | 20 | Xanthine | X0626 | 5g | 3032.4 | 21 | Xanthine oxidase | X2252 | 25units | 4415.6 | 22 | Tris | T6687 | 100g | 1634 | 23 | Ascorbate | A0157 | 100g | 1915 | 24 | NADPH | N5130 | 25g | 5304 | 25 | SO2 gas | 29569-8 | 454g | 17305 | 26 | Glycerol | g-8898 | 500g | 5421 |

Appendix C: glassware SN.O | ITEM | TYPE | QUANTITY | COST IN RS. | 1 | Glass rod | | 5 | 75 | 2 | Beaker | 100ml | 20 | 1960 | 3 | Beaker | 250ml | 10 | 1250 | 4 | Beaker | 500ml | 10 | 1490 | 5 | Test tube | 12*100mm | 50 | 250 | 6 | Burette | 100ml | 5 | 300 | 7 | Flask | 250ml | 20 | 2900 | 8 | Petri dishes | 6” | 50 | 450 | 9 | Pots | 20 cm diameter | 20 | 100 | 10 | Pipettes | 10ml | 5 | 475 | | | | | | | Total +16%GST | | | 10730 |

APPENDIX D: plastic wares SN.O | ITEM | TYPE | QUANTITY | COST IN RS. | 1 | Tips | 1000µl | 1000 | 500 | 2 | Tips | 200µl | 1000 | 500 | 3 | Tips | 10 | 1000 | 1500 | 4 | Eppendorf | 1.5 | 1000 | 800 | 5 | Pipette runner | | 4 | 250 | 6 | Test tube stand | | 5 | 1000 | 7 | Gloves | | 1 packet | 276 | 8 | Squeezer | | 3 | 172 | | | | | | | Total +16% GST | | | 6668 |

APPENDIX E1: ANY OTHER SN.O | ITEM | TYPE | COST IN RS. | 1 | Agro bacterium | LBA4404 | 15000 | 2 | pB1121 | | 10685 | 3 | E.coli SOD gene | | 10000 | 4 | E.coli CAT gene | | 10000 | | | | | | Total+16% GST | | 52994 |

APPENDIX E2: ANY OTHER SN.O | ITEM | TYPE | QUANTITY | COST IN RS. | 1 | PCR kit | - | - | 25000 | 2 | Kodak biomax film | Z35188.1 | 25 | 5540 | 3 | Filter paper | Grade1 | 1 pack | 489 | | | | | | | Total+16% GST | | | 35995 |