c
 



 
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SUBBMITTED IN PARTIAL FULFILLMENT OF THE ACADEMIC REQUIREMENTS

FOR

THE AWARD OF THE DEGREE OF BACHELOR OF TECHNOLOGY

IN

BIOTECHNOLOGY

BY

JAWARHARLAL NEHRU TECHNOLOGICAL UNIVERSITY

HYDERABAD



c

  !

c 

"c
c
 

Dr. Vijaya Lakshmi Valluri Dr. Laxman Rao

Group Leader, Associate Professor 

Immunology & Molecular Biology Division, Dept. of biotechnology

BPHRC, LEPRA India, Hyderabad SNIST.

Department of Biotechnology

Sreenidhi Institute of Science and Technology

Yamnampet, Ghatkesar, Hyderabad 501301

 
c 

I hereby declare that the work presented in this dissertation entitled ³ROLE OF
IL-10 (-1082 G/A) POLYMORPHISM IN TB INFECTED INDIVIDUALS´ is
carried out by me under the supervision of Dr.VijayaLakshmiValluri, Group
Leader, Immunology & Molecular Biology Division, BPHRC, LEPRA India,
Hyderabad. I further declare that this dissertation as not been submi tted to any
institution for other degree or diploma. All the assistance and help received
during the course of investigation has been duly acknowledged.

MITHUN.M
 # $


I present a deep sense of gratefulness towards, %&'()(
(*+,-&(../%&0

Group Leader, Immunology & Molecular Biology Division, BPHRC, LEPRA
India for allowing me to carry out my project.

I would like to express gratitude to, ,(%1( -(1(-, for taking time out from
her busy schedule to serve as my supe rvisor whose expertise, understanding,
and practice, added considerably to our graduate experience. I honor her vast
knowledge and skill in many areas and her assistance in writing report. She was
more of a mentor and friend, than a guide.

I would also like to thank,  %
(2-(3c(4 for taking time out from her busy
schedule to help me as an internal guide at the crucial time when I needed.

A special thanks to the Head of Department of Biotechnology, c #c

,&5((&0 whose motivation and encouragement h elped me to strive in spite of
innumerable hardships. It was through his, persistence, understanding and
kindness that I inculcated a spirit of research and development.

I would also like to thank my friends and family for the support they provided
me throughout the project.


 
  

?  c  

1.1 HISTORY
1.2 PREVALENCE
1.3 MYCOBACTERIUM TUBERCLOSIS
1.4 SYMPTOMS
1.5 DIAGNOSIS OF TB
1.5.1 MANTOUX TUBERCULIN SKIN TEST
1.5.2 THE SPUTUM TEST
1.5.3 CHEST X-RAY
1.6 TREATMENT

2.  #
2.1 IL-10
2.2 POLYMORPHISM

3. c
 
3.1 INCLUSION CRITERIA
3.2 EXCLUSION CRITERIA
3.3 DNA ISOLATION BY FLEXIGENE KIT METHOD
3.4 IL-10 (-1082) A-G POLYMORPHISM USING ARMS PCR
3.5 AGAROSE GEL ELECTROPHORESIS

4. 



5. c


6.  

7.  
 


 c  

&+64%)

Tuberculosis is estimated to have existed 15,000 and 20,000 years. The first evidence of the
infection in humans was found in a cemetery near Heidelberg, in the Neolithic bone remains
that show evidence of the type of angulations, often seen with spinal tuberculosis. Some
authors call tuberculosis the first disease known to mankind. It has often been a difficult
disease to diagnose and has been confused with many other diseases. The actual name
"Tuberculosis" was introduced during the first half of the nineteenth century and it refers to
the diseased condition caused by infectious agents known as tuberculosis bacteria or tubercle
bacilli. It is believed that at some point in its evolution, some species of the bacteria was able
to invade animal hosts. This possibly took place with the first species Mycobacterium bovis,
which is considered by most to be the oldest of the species that make up the Mycobacterium
tuberculosis complex.

M.bovis eventually passed to humans at a time coinciding with the domestication of animals.
Human bones from the Neolithic show a presence of the bacteria although the exact
magnitude (incidence and prevalence) is not known before the nineteenth century. Over time,
the various cultures of the world gave the illness different names: yaksma (India), phthisis
(Greek), consumptione (Latin) and chaky oncay (Incan), Scrofula, tabes, bronchitis, and
inflammation of the lungs, hectic fever, gastric fever, and lupus. It was also known as the
great white plague or "consumption´ .In 1720, the English physician Benjamin Marten was
the first to conjecture, in his publication, A New Theory of Consumption, that TB could be
caused by "wonderfully minute living creatures", which, in the body could generate the
lesions and symptoms of the disease. He stated, moreover, "It may be therefore very likely
that by an habitual lying in the same bed with a consumptive patient, constantly eating and
drinking with him, or by very frequently conversing so nearly as to draw in part of the breath
he emits from the Lungs. The first real breakthrough in understanding tuberculosis came
when a German bacteriologist named Robert Koch isolated the infectious agent known as
tuberculosis bacteria or tubercle bacilli in 1882. He was later awarded the Nobel Prize for
physiology or medicine in 1905.

%75(.7387

According to the World Health Organization (WHO), nearly 2 billion people, one-third of the
world's population, have TB. In 1998, a total of 18,371 active TB cases, in all 50 states and
the District of Columbia, were reported to the Centers for Disease Control and Prevention
(CDC). It is estimated that 1.8 million people died of tuberculosis in 2008. There were an
estimated 9.4 million new cases of tuberculosis in 2008 of which the majority were in Asia
and Africa. It is thought that the rates of new tuberculosis infections and deaths per capita
have probably been falling globally for several years now. However, the total number of new
tuberculosis cases is still slowly rising due to population growth. Tuberculosis is not only a
problem in low and middle income countries. There were 14,097 new cases reported in the
 U.S. in 2005. In 2005, 8,113 new cases of tuberculosis were reported in England, Wales and
Northern Ireland. Although the UK's national rate is very low in comparison with most of the
world, London has become one of the world's tuberculosis hotspots. In parts of London,
tuberculosis rates are ten times the national rate - higher than in some countries of the former
Soviet Union.

)849(867%&/-6/97%8/.4+&+

M. tuberculosis is an obligate pathogen and can infect a wide variety of animals. Humans are
the principal hosts. Mycobacterium tuberculosis (M.TB.) are characterized by slender,
straight or slightly curved bacillus, they are non-motile and non-encapsulated and do not form
spores. M.TB is not classified as either Gram-positive or Gram-negative because it does not
have the chemical characteristics of either, although the bacteria do contain peptidoglycan
(murein) in their cell wall. If a Gram stain is performed on M.TB, it stains very weakly
Gram-positive or not at all (referred to as "ghosts"). It is slow growing, dividing every 18-24
hr, resistant to drying and chemical disinfectants but sensitive to heat (Pasteurization) and UV
light.

These organisms are:

i. Obligate aerobes growing most successfully in tissues with high oxygen content, such as
the lungs.

ii. Facultative intracellular pathogens usually infecting mononuclear phagocytes

(macrophages).

iii. Slow-growing with a generation time of 12 to 18 hours.

iv. Hydrophobic with high lipid content in the cell wall. Because the cells are hydrophobic and
tend to clump together, they are impermeable to the usual stains, e.g. Gram's stain.

v. Known as "acid-fast bacilli" because of their lipid-rich cell walls, which are relatively
impermeable to various basic dyes unless the dyes are combined with phenol. Once stained,
the cells resist decolourization with acidified organic solvents and are therefore called
"acid-fast".

Tuberculosis (TB) is a chronic bacterial infection caused by Mycobacterium tuberculosis. It

is spread through the air and usually infects the lungs, although other organs and parts of the
body can be involved as well. Most people who are infected with tuberculosis harbor the
tuberculosis bacterium without any tuberculosis symptoms. This is known as latent
tuberculosis. If the body's resistance is low because of aging, malnutrition, infections such as
HIV, or other reasons, the bacteria may break out of hiding and cause active tuberculosis. TB
bacteria become active if the immune system can't stop them from growing. The active
bacteria begin to multiply in the body and cause active tuberculosis. The bacteria attack the
body and destroy tissue. If this occurs in the lungs, the bacteria can actually create a hole in
the lung. Some people develop active tuberculosis soon after becoming infected, before their
immune system can fight the TB bacteria. Other people may develop active tuberculosis later,
 when their immune system becomes weak for another reason. Babies and young children
often have weak immune systems. People infected with HIV, the virus that causes AIDS, also
have very weak immune systems. Other people can have weak immune systems, too,
especially people with any of these conditions:

hSubstance abuse

hDiabetes mellitus

hSilicosis

hCancer of the head or neck

hLeukemia or Hodgkin's disease

hSevere kidney disease

hLow body weight

hCertain medical treatments (organ transplants)

:)-;64-+

hCommon cough with a progressive increase in production of mucus

hCoughing up blood.

hFever, particularly with rise of temperature in the evening

hLoss of appetite

hWeight loss

hNight sweats

hTiredness

hChest pain

hShortness of breath.
    

  "c
#

The most commonly used diagnostic tool for TB is a simple skin test. A small amount of a
substance called PPD tuberculin is injected just below the skin of inside forearm. Within 48 to 72
hours, a health care professional will check the arm for swelling at the injection site, indicating a
reaction to the injected material. A hard, raised red bump (induration) means the patient is likely to
have TB infection. The size of the bump determines whether the test results are significant. The
results of this test must be interpreted carefully. The person's medical risk factors determine at
which increment (5 mm, 10 mm, or 15 mm) of induration the result is considered positive. A
positive result indicates TB exposure.

 

Sputum is material coughed up from the lungs. Sputum is a thick fluid produced in the lungs and in
the airways leading to the lungs. A sputum culture is a test to detect and identify bacteria or fungi
(plural of fungus) that are infecting the lungs or breathing passages. To perform a sputum test, the
patient coughs deeply to expel sputum into a sterile cup. Three sputum samples are collected on
two days, on the first day and one on early morning and one spot on the second day. The sputum is
then analyzed in a laboratory. The lab technician smears a sample of the sputum on a microscope
slide and then stains it with a special stain that causes TB bacteria to show up under fluorescent
light. Substances that promote the growth of bacteria or fungi are added.

This will indicate the presence or absence of TB in the sputum sample. If no bacteria or fungi
grow, the culture is negative. If organisms that can cause infection (pathogenic organisms) grow,
the culture is positive. Patients with two positive smear results are smear positive cases and are
diagnosed by the physician as having tb. They are further classified as new or older cases based on
their treatment history, and appropriate therapy is prescribed. Patients with only one smear positive
result of smear examination will be referred to the nearest X-ray facility. Patients who have one
smear positive and a chest X-ray compatible with tb as diagnosed by an MO are considered to be
suffering from TB and are registered as smear-positive cases. Patients in whom all 3 samples are
smear negative are prescribed symptomatic treatment or broad spectrum antibiotics for 1-2 weeks.
These patients are prescribed with general antibiotics such as co-trimoxazole which do not have
any anti-tuberculosis activity.

 ,7+6"%()

A posterior-anterior (PA) chest X-ray is the standard view used. In active pulmonary TB,
infiltrates, consolidations or cavities are often seen in the upper lungs with or without mediastinal
or hilar lymphadenopathy. However, lesions may appear anywhere in the lungs. Old healed
tuberculosis usually presents as pulmonary nodules in the hilar area or upper lobes, with or without
fibrotic scars and volume loss. Bronchiectasis and pleural scarring may be present. Nodules and
fibrotic scars may contain slowly multiplying tubercle bacilli with the potential for future
progression to active tuberculosis.
<%7(6-736

A vaccine, called BCG (Bacille Calmette-Guérin), is routinely administered in parts of the world
where tb is common. BCG vaccination results in a positive TB skin test, so that a positive reaction
is no longer indicative of a recent TB infection. Five drugs are most commonly used today to treat
tuberculosis: isoniazid (INH, Laniazid, Nydrazid); rifampin (Rifadin, Rimactane); pyrazinamide
(Tebrazid); streptomycin; and ethambutol (Myambutol). The first three drugs may be given in the
same capsule to minimize the number of pills in the dosage. The standard "short" course treatment
for tuberculosis (TB) is isoniazid, rifampicin, pyrazinamide, and ethambutol for two months, then
isoniazid and rifampicin alone for a further four months. The patient is considered cured at six
months (although there is still a relapse rate of 2 to 3%). For latent tuberculosis, the standard
treatment is six to nine months of isoniazid alone. If the organism is known to be fully sensitive,
then treatment is with isoniazid, rifampicin, and pyrazinamide for two months, followed by
isoniazid and rifampicin for four months.

)64*&37+

Cytokines are a group of soluble proteins that are released by a cell to send messages which are
delivered to the same cell (autocrine), an adjacent cell (paracrine), or a distant cell (endocrine). The
cytokine binds to a specific receptor and causes a change in function or in development
(differentiation) of the target cell. Cytokines are involved in reproduction, growth and
development, normal homeostatic regulation, response to injury and repair, blood clotting, and host
resistance (immunity and tolerance). Unlike cells of the endocrine system, many different types of
cells can produce the same cytokine, and a single cytokine may act on a wide variety of target
cells. Cytokines may produce the same effect on a target, so the loss of one type of cytokine may
have few if any consequences for the organism; this situation is called redundancy. The response of
a target cell may be altered by the context in which it receives a cytokine signal. Cytokines may be
divided into six groups: interleukins, colony-stimulating factors, interferons, tumor necrosis factor,
growth factors, and chemokines.


 

IL10 is a cytokine produced primarily by monocytes and to a lesser extent by lymphocytes. This
cytokine has pleiotropic effects in immunoregulation and inflammation. It down-regulates the
expression of Th1 cytokines, MHC class II Ags, and co-stimulatory molecules on macrophages. It
also enhances B cell survival, proliferation, and antibody production. This cytokine can block NF-
kappa B activity, and is involved in the regulation of the JAK-STAT signaling
pathway.Interleukin-10 is a potent immunomodulatory cytokine which directly or indirectly affect
multiple cell types, including macrophages, monocytes, dendritic cells, CD4 T cells, and CD8 T
cells. The dominant function of IL-10 is to deactivate macrophages, resulting in diminished Th1
cytokine production. IL-10 can be found in the serum and bronchoalveolar lavage fluid of active
TB patients


› ›

  

Polymorphism occurs when two or more clearly different phenotypes exist in the same population
of a species. In order to be classified as such, morphs must occupy the same habitat at the same
time and belong to a panmictic population.

Polymorphism is common in nature. It results from evolutionary process. It is heritable and is

modified by natural selection. In polymorphism an individual genetic make-up allows for different
morphs, and the switch mechanism that determines which morph is shown in environment.

The present study involves certain point mutation in IL-10 gene at -1082 position and is aimed
towards the influence of this polymorphism in case of HIV infection.

c
 

Peripheral Blood was collected from 25 HIV positive patients at ICTC clinic and 25
asymptomatic healthy controls from the employees of Blue Peter Public Health & Research
Center, LEPRA India.


 cc

l 7(.6,)436%4.+ Individuals without clinical symptoms and history of Tuberculosis

and Negative for HIV test.
l /97%8/.4+&+  Patients with Pulmonary TB confirmed by sputum smear (AFB),
X-Ray positivity and extra pulmonary TB confirmed by biopsy and FNAC at DOTS
clinics of BPHRC.

"
 cc
h Pregnant woman were excluded from the study.
h Patients who are weak or unwilling to participate or unable to give informed consent
were excluded from the study.





 
"# 

c  


1. 1.25 ml of ³FG1´ buffer is pipetted into 2.0ml eppendroff tube and 250ul whole blood
is added to it and is mixed by inverting the tube 5 times.

2. The tube containing mixture is centrifuged for 20sec at 10,000 rpm to obtain the
pellet.

3. The supernatant is discarded and the pellet is retained.

4. Required volume of FG2 buffer protease is added. (5ul of protease for 500ul of FG2
buffer).

5. To the pellet 250ul of ³FG2 buffer mix´ is added and the tube is vortexed
immediately till the pellet is completely homogenized.

6. The homogenized mixture is centrifuged briefly for 3-5 sec.

7. After centrifugation it is It is placed in a dry bath and is incubated at 65°c temperature

for 5minutes

8. 250ul of isopropanol is added to the eppendrof tube and is mixed thoroughly by

inversion until the DNA precipitates out and is visible as threads or a clump.

9. The eppendrof tube is centrifuged for 3 min at 10,000 rpm.

10.The supernatant is discarded and the tube is left for 2 minutes on a clean piece of
absorbant paper while pellet remaining in the tube.

11.250ul of 70% ethanol is added to the tube containing pellet and it is vortexed for 5
sec.

12. It is then centrifuged for 3min at 10,000 rpm.

13. The supernatant is discarded and the tube is left for 2 minutes on a clean piece of
absorbant paper while pellet remaining in the tube.

14.The tube is kept for air drying until all the liquid has got evaporated from the pellet.

15.After air dry, 200ul of ³FG3´ buffer is added to the tube and it is vortexed for 5 sec at
low speed,

16.The DNA is dissolved by incubating at 65°c in a heating block or water bath.

17.The DNA is then stored at 4°c in a refrigerator.

 18.Concentration of DNA is measured by using spectrophotometer and it is made up to
50ng/ul.

:
 

 cc

ARMS (Amplification Refractory Mutation System) PCR was performed for the detection
of specific mutation in IL-10 gene. A PCR was set up for detection of polymorphism. A set
of 4 primers or oligonucleotides were added in a single tube to analyze a single polymorphic
site.

(9.74-;4+&6&434=c-&2

(-7 4./-7

Nuclease free water 6.5ul

Buffer 1ul

DNTP¶s 0.25ul

TAQ Polymerase 0.25ul

Primer Beta Actin(+) 0.25ul

Primer Beta Actin(-) 0.25ul

Forward IL-10 (+) primer 0.25ul

Reverse IL-10 (-) primer (A or G 0.25ul

specific)

Template DNA 0.5ul

Q-Solution 0.5ul

 
 /.


c)8.&3>431&6&43+ 

S.NO TEMPERATURE TIME STEP

1 95°C (1 cycle) 60 sec Initial Denaturation

2 95°C 90 sec Cycle Denaturation

3 62°C 90 sec Primer Annealing

4 72°C 60 sec Primer Extension

5 Go to Step 2 30 CYCLES Cycles

6 72°C 10 min Substrate Clearance

7 4°C HOLD Storage

 ?c 
cc 

2gm of Agarose was dissolved in 100ml of 1XTAE buffer by heating in microwave oven.

After ensuring complete digestion, agarose was cooled to 60°C. Ethidium Bromide
(10mg/ml) was added at a concentration of 5ul/100ml of digested agarose to give a final
concentration of 5ug/100ul of agarose and mixed well.

The solution was poured on to gel casting tray with combs avoiding the formation of air
bubbles.

The tray was kept for cooling, allowing the polymerization of gel.

>(%4+77..786%4;,4%7+&+:

1X TAE buffer was filled in the electrophoresis chamber and the solidified agarose on the
electrophoresis plate was fixed in the chamber. The combs were then removed; amplicons
and 100base pair molecular size marker was loaded in the wells. Electrophoresis was carried
out applying a constant voltage of 100 Volts for 45 minutes.

48/-736(6&43(31367%;%76(6&43:

Agarose gel was placed on a UV transilluminator (Bio-Rad) and documented by

photography. The presence or absence of specific PCR products was noted. The relative
lengths of the specific PCR products were helpful in the interpretation of the results.


 &>7.;&86/%7%7;%7+736&3>6,7
 
;4.)-4%;,&+-

Genotypes of the patients according to their IL-10 polymorphisms. Allele of the polymorphic
region of IL-10 (G/A), corresponding to each patient are analyzed using Amplification
Refractory Mutation System-PCR technique described in PROTOCOL. Products amplified
were run on a 2% agarose gel.
 

:



Analysis of case-control data was performed with javastat online statistical tool. Chi-Square
(Ȥ2) test was used for comparing genotype frequencies. A 2 x 2 cross-tabulation method was
used to determine odds ratio (OR) with 95% confidence interval. A µp¶ value of <0.05 was
considered significant.


c c  
  
 



      

 ?  c   ? 5(./7 c   ? 5(./7 c 
5(./7
 c
 14 (56%) - - 10 (40%) - - 1 (4%) - -
@ 

 18 (72%) 0.23 2.02 6 (24%) 0.2258 0.474 1 (4%) 1 1

@  (0.63 - 6.40) (0.14 ± 1.56) (0.09 - 10.12)

*OR-ODD¶S RATIO *CI-CONFIDENCE INTEVAL

c

percentage variation was observed for IL10-1082 position with 40% of control and 24% of
TB patients having heterozygous A/G genotype. While 56% of controls and 72% of TB
patients population had the homozygous A/A genotype. However, they did not reach the
significance level (A/G, p=0.2258, OR=0.474; A/A, p=0.23, OR=2.02)

 

The genetic component that contributes to susceptibility and progression of pulmonary

tuberculosis probably involves an interaction between multiple alleles located on different
genes and chromosomes. Tuberculosis clinical severity is multi-factorial and cytokines play a
pivotal role in the modulation of disease severity.

Below are the results of IL-10 (-1082 G/A) gene polymorphism in Iranian patients with
pulmonary tuberculosis

GENOTYPE PTB (%) NORMAL (%) p-value OD(CI)

A/A 7(17.5%) 18(17.6%) 0.98 0.34(0.99-2.82)
G/A 31(77.5%) 79(77.5%) 0.99 0.39(1.00-2.64)
G/G 2(5%) 5(4.9%) 1.00 0.13(1.02-6.32)
According to their study no significant association was observed between control and TB,
which is similar to my study.


< 
 

No significant association was observed between control and TB with respect to this
polymorphism. Increase in sample number may provide significant values
Reference

1. National Tuberculosis and Respiratory Disease Association. Diagnostic standards and

classification of tuberculosis. New York: National Tuberculosis and Respiratory
Disease Association; 1969.
2. Dhingra VK, Rajpal S, Aggarwal N, Taneja DK (2004) Tuberculosis Trend among
Household Contacts of TB Patients.
3. Guwatudde D, Nakakeeto M, Jones-Lopez EC, Maganda A, Chiunda A, et al. (2003)
Tuberculosis in Household Contacts of Infectious Cases in Kampala, Uganda. Am J
Epidemiol 158: 887±898.
4. Ates O, Musellim B, Ongen G, Topal-Sarikaya A (2007) Interleukin-10 and Tumor
Necrosis Factor-a Gene Polymorphism in Tuberculosis.
5. Ambreen Ansari1, Najeeha Talai, Bushra Jami, Zahra Hasan, Tashmeem Razzaki,
Ghaffar Dawood4, Rabia Hussain. Cytokine Gene Polymorphisms across Tuberculosis
Clinical Spectrum in Pakistani Patients
6. Bloom BR, Small PM, 1998. The evolving relation between humans and
Mycobacterium tuberculosis.
7. Jodene fitness, sian Floyd, david k.warndroff, lifted sichali, simon malema, Amelia c.
crampin, paule.m. fine, and Adrian v.s.hill. Large-scale candidate gene study of
tuberculosis susceptibility in the karonga district of northern Malawi.