streptococcus pneumoniae isolated from patients infected ... and... · prof. dr. jaafar k.n....

Republic of Iraq Ministry of Higher Education University of Kufa College of Science Department of Biology Microbiology

Bacteriological and Immunological study of

Streptococcus pneumoniae isolated from patients infected

with respiratory tract infection in

AL-Najaf Governorate

A thesis Submitted to the College of Science University of Kufa in Partial Fulfillment

of the Requirements for the Degree of Doctorate of Philosophy in Biology/Microbiology

By

Ahmed Abd Aljabbar Jaloob Aljanaby BSc. College of science-Babylon University 1994

MSc. Microbiology College of science-Kufa University 2004

February 2010 Rabee Alawal 1431

جمهورية العراق

وزارة التعليم العالي والبحث العلمي جامعة الكوفة

كلية العلوم قسم علوم الحياة األحياء ألمجهريه

و مناعية لبكتريا ولوجيةيبكتر دراسة Streptococcus pneumoniae

في التنفسية القناة لمصابين بالتهابالمرضى ا المعزولة من النجف محافظة

إلىمقدمة أطروحة

فلسفة في الدكتوراهوهي جزء من متطلبات نيل درجة الكوفةجامعة – العلوم كلية

ريهألمجه األحياء علوم الحياة /

من قبل

حمد عبد الجبار جلوب الجنابيأ

۱۹۹٤ جامعه بابل كليه العلوم / بكالوريوس علوم حياه /

۲۰۰٤جامعه الكوفة كليه العلوم / / مجهريه أحياءماجستير

شباط ۱٤۳۱ ربيع األول۲۰۱۰

ه ا الرحمن الرحيه

فهو ذي خلقني ال والذي )۷۸( يهدين

هو يطعمني ذا وإ )۷۹( ويقين

مرضت فهو يشفين )۸۰(

ظيم الع لي الع هللا صدق الشعراء

dedication

To

my beautiful country IRAQ

To my mother and my father

my wife

and

my daughters

Noor and Shahad

Ahmed Al-janaby

Examination Committee: We, the examining committee, certify that we have read this thesis and have

examined the student (Ahmed Abd Aljabbar Jaloob Aljanaby), in its content, and that

in our opinion it is adequate as thesis for the degree of Doctorate of Philosophy in

Biology / Microbiology.

Signature: Prof. Dr. Ghazi Mousa Al-khatib Chief of Al-Muthani University

(Chairman)

Signature: Signature: Prof. Dr. Khairy Abdulla Dawood Assist. Prof. Dr. Jaafar K.N. AL-Mousawi College of Veterinary Medicine College of Medicine Al- Qadissya University Kufa University (Member) (Member)

Signature: Signature: Assist. Prof. Dr. Mohamad A.K.AL-Saadi Assist. Prof. Dr. Mahdi Husan Muhal College of Medicine College of Science Babylon University Kufa University (Member) (Member)

Signature: Signature: Prof. Dr. Ibrahim M.S. Shnawa Prof. Dr. Abd Al-majeed A. Al-saadi College of Science College of Science Babylon University Kufa University (Supervisor) (Supervisor) Approved for the college committee of graduate studies. Signature: Prof. Dr. Abd Al-majeed A. Al-sadi Dean college of science University of Kufa

CERTIFICATION

We certify that this thesis entitled (Bacteriological and Immunological

study of Streptococcus pneumoniae isolated from patients infected with

respiratory tract infection in AL-Najaf Governorate)was prepared under our

supervision at the Department of Biology, College of Science, Kufa

University, in partial requirements for the Degree of Philosophy Doctorate of

Science in Microbiology and this work has never been published anywhere.

Signature: Signature:

Prof. Dr. Ibrahim M.S. Shnawa Prof. Dr. Abd Almajeed A. Alsadi

Department of Biology Department of Biology

College of Science College of Science

Babylon University Kufa University

In the view of the available recommendation, I forward this thesis for

debate by the Examination Committee.

Signature:

Prof. Dr. Sami Abd Alrudi Ali

Head of Department of Biology College of Science Kufa University.

I

Acknowledgement

First of all, I would like to thank God the mighty, for helping me in

completing this work. Sincerely I feel deep gratitude to my supervisors Prof. Dr. Ibrahim M.S.

Shnawa and Prof. Dr. Abd Almajeed A. Alsadi for their constant supervision,

infinite tolerance, assistance and guidance without them, I would certainly not

have embarked upon the work at all.

Thanks to the University of kufa, College of Science and department of

Biology for providing the necessary facilities during this study.

Thanks to the AL-Sadder Teaching Hospital in AL-Najaf City for providing

the necessary facilities during this study.

I would like to express my thanks to Assist prof. Dr. Assad Aljanaby

Department of histopathology in AL-Sadder Teaching Hospital in AL-Najaf City

for assist diagnosis the histopathology sections.

I like to extend special thanks to Assist prof. Dr. Ali AlMohana University

of Kufa College of Medicine for providing the most necessary chemicals

materials during this study.

My special thanks to all bacteriologist in laboratory of microbiology in

AL-Sadder Teaching Hospital in AL-Najaf City for their assistance in my study

especially Mr.Forat , Mr.Raffed, Mr .Ihssan, Mr.Raad, Mr.Ahmed, Mss.Ishtar

and Mss.Yossra.

II

Abstract: A total number of 410 out patients attending the chest unit of AL-Sadder

Teaching Hospital in AL-Najaf Governorate who were suspected of having

respiratory tract infection from first February 2008 to the end January 2009

were included in this study with age range between 18-60 years old (males

and females).

The sputum with Gram stain revealed the presence of lymphocytes

were 190 specimens from a total of 410 specimens (46.341%) were positive,

while the monocyte cells were present in 99 specimens (24.146%),the 3rd

group of specimens is that with dominant neutrophils with 90 positive

specimens (21.951%), the number of specimens positive for acid fast bacilli

was 31 specimens (7.560%). From the 90 specimens showing neutrophil cell

domination, and the positive results for culture , microscopic and

biochemical's characteristics for Streptococcus pneumoniae was 22 specimens

only (24.444%), while 68 specimens (75.555%) were negative results for

colonies by cultivation , microscopic and biochemical characterization for

S.pneumoniae .

The results demonstrate that the most serotype distribution in AL-Najaf

Governorate was serotype 6 while the serotype 1 was the most virulent isolate

in Mice and Rabbits, thus it was chosen in this study because it is the most

virulent one , and isolate 1 from serotype 6 because it is the most dominant

serotype.

Three immunization protocols were used in this study to enhance

specific immunoglobulin titer in rabbits. The results demonstrates that the

protocol 2 (rabbits intramuscular 1mg capsule mix with 1ml lanolin for 15

days) was the best method for enhancing systemic specific immunoglobulin

of the rabbits, while protocol number 3 (Intranasal 1×108 cfu/ml of heat killed

III

S.pneumoniae for 30 days) was the best method for enhancing mucosal

specific immunoglobulin of the rabbits, and the results indicate that capsular

polysaccharides serotype 1 gave the highest response than serotype 6, and the

statistical analysis proved that specific immunoglobulin titer in systemic was

highest than mucosal washing in both for serotype 6 and 1 was significant at

p value = 0.0007,on the other hand there was significant increase in specific

immunoglobulin titer in serotype 1 systemic than serotype 6 systemic was

significant at p value =0.0339, also the results indicates that the immunized

rabbits with intramuscular 1mg capsule serotype 1 mix with 1ml lanolin for 15

days provided 80% protection and with serotype 6 provided 60% protection in

laboratory rabbits post 36 days of immunization and infection with live

S.pneumoniae (immune protection), and serotype 1 provided specific

immunoglobulin titer in systemic higher than serotype 6 systemic , p= 0.0425

and the specific immunoglobulin titer in systemic was highest than mucosal

washing for both serotype 6 and 1 and was significant at p value = 0.0200

and 0.0032 respectively.

The levels of cytokines were detected by three protocols, the results

indicate that the protocol 3 gave the highest level in systemic and mucosal

preparation of all cytokine levels ,then protocol 2 and 1 respectively, and the

results indicate through protocol 2 for serotype 1 systemic there was no

significant difference between TNFβ / TNFα , TNFβ / IL-6 , TNFα / IL-6 and

IL-6 / IL-10, while it was significant between TNFβ / IL-10 (p=0.0342) ,

TNFα / IL-10 (p= 0.0018 ) and the results indicate for serotype 1 mucosal

there was no significant difference between TNFβ / TNFα and IL-6/ IL-10,

while there was significant difference between TNFβ / IL-6(p= 0.0004) ,

TNFβ / IL-10(p=<0.0001) , TNFα / IL-6(p=0.0060) and TNFα / IL-

10(p=0.0037),on the other hand the results indicate for serotype 6 systemic

IV

there was no significant difference between TNFβ / TNFα , TNFβ / IL-6 ,

TNFβ / IL-10 and IL-6 / IL-10,while there was significant difference between

TNFα/IL-6(p=0.0019) and TNFα /IL-10(p=0.0305),also the results indicate

that for serotype 6 mucosal preparation there was no significant difference

between TNFβ /IL-6 , TNFβ /IL-10 and IL-6/ IL-10,however there was

significant difference between TNFα / TNFβ (p=0.0027 ), TNFα / IL-

6(p=0.0007 ) and TNFα / IL-10(p=0.0001 ).

The results demonstrate that all cytokine levels post 36 day of

immunization and challenge (immune protections) increased significantly than

in controls and than lanolin, and the most significant increase was in mucosal

than systemic it was the highest level of all cytokines. On the other hand the

results indicate that there was no significant increase for all cytokines except

TNFβ and IL-10 (p=0.0447) in serotype 1 systemic, and there was a significant

increase in TNFα and IL-6 , IL-10 (p= <0.0001 , 0.0004) respectively, also in

IL-6 and IL-10(p=0.0038) serotype 1 mucosal ,on the other hand the results

indicate that there was no significant increase for all cytokines levels for

serotype 6 systemic and was significant for TNFα and IL-10(p=0.0173), and

for IL-6 and IL-10(p=0.016) for serotype 6 mucosal.

V

Contents Number Title Page Acknowledgement I Abstract II Contents V

1 Introduction 1-1 Overview 1 1-2 Aims of the study 3

2 Review of Literatures 2-1 Pneumonia 4 2-2 Etiology of pneumonia 4 2-2-1 Bacterial pneumonia 4 2-2-2 Atypical bacterial pneumonia 6 2-3 Streptococcus pneumoniae 7 2-3-1 History of Streptococcus pneumoniae 7 2-3-2 Biology of Streptococcus pneumoniae 8 2-3-3 Pathogenesis of Streptococcus pneumoniae 10 2-3-3-1 Infection route 10 2-3-4 Pathogenicity of Streptococcus pneumoniae 14 2-3-5 Immunology of streptococcus pneumoniae 14 2-3-5-1 Host defense mechanism 14 2-3-5-2 Innate immunity 17 2-3-5-3 Capsule polysacchrides of S.pneumoniae immunogens and

immunity: 18

2-4 Role of specific antibodies 19 2-5 Streptococcus pneumoniae immunogens evaluations for

vaccination 20

2-5-1 Role of adjuvant 22

2-6 Role of cytokines 24 2-6-1 Tumor necrosis factor alpha (TNF-α) 24 2-6-2 Tumor necrosis factor beta (TNF-β) 25

2-6-3 Interleukin-6(IL-6) 26

2-6-4 Interleukin-10(IL-10) 27 3 Materials and Methods

3-1 Materials 30 3-1-1 Kits 30 3-1-2 Patients 30

3-1-3 Rabbits 30 3-1-4 Mice 30 3-1-5 Solutions 31 3-1-5-1 Normal saline 31

VI

3-1-5-2 Formal saline 31 3-1-5-3 Tris buffer 31 3-1-5-4 Polyethylenglycol solution 31 3-1-5-5 Sodium deoxycholate solution 31 3-1-5-6 Phenol red inulin broth 31 3-1-5-7 Iodine solution 32 3-1-5-8 α- naphthol reagent 32 3-1-6 Culture media 32 3-1-6-1 General culture media 32 3-1-6-2 Gentamicin blood agar 32 3-1-6-3 Skim milk , tryptone, glucose and glycerol medium(STGG) 32 3-1-7 Stains 32 3-1-7-1 Gram stain 32 3-1-7-2 Ziehl-Neelsen stain 33 3-1-7-3 Nigrosin stain 33 3-1-8 Lanolin 33 3-2 Methods 33 3-2-1 Sputum specimens 33 3-2-2 Sputum Ziehl-Neelsen stain method 33 3-2-3 Sputum gram stain method 34 3-2-4 Sputum culture method 34 3-2-5 Streptococcus pneumoniae identification methods 35 3-2-5-1 Gram stain 35 3-2-5-2 Optochin test 35 3-2-5-3 Bile solubility test 35 3-2-5-4 Capsule staining method 35 3-2-5-5 Inulin fermentation method 35 3-2-5-6 Mouse virulence test 36 3-2-5-7 Streptococcus pneumoniae biochemical test kit 36 3-2-5-8 Serotype identification test by slide agglutination method 36 3-2-5-9 Bacterial storage 36 3-2-6 Pneumonia model in rabbits caused by S.pneumoniae 38 3-2-7 Antigens preparation 38 3-2-7-1 Capsule polysaccharide isolation method 38 3-2-7-1-1 Capsule polysaccharide detection methods 39 3-2-7-2 Heat- inactivated Streptococcus pneumoniae 40 3-2-8 Immunization protocols 41 3-2-8-1 Immune protocol 41 3-2-8-2 Immunization protocol and immune protection 42 3-2-8-2-1 immunization protocol 42 3-2-8-2-2 immune protection 43 3-2-9 Samples 44 3-2-9-1 Blood sample 44 3-2-9-2 Mucosal sample 44

VII

3-2-10 Blood culture 44 3-2-11 Histological investigation 45 3-2-12 Separation of serum immunoglobulin 45 3-2-13 Separation of mucosal immunoglobulin 45 3-2-14 Interfacial capillary tube test for systemic and mucosal for

specific immunoglobulin titer.(INFCT) 46

3-2-14-1 Interfacial capillary tube precipitation test for systemic specific immunoglobulin titer

46

3-2-14-2 Interfacial capillary tube precipitation test for mucosal specific immunoglobulin titer

47

3-2-15 Cytokines detection 47 3-2-15-1 Interlukin-6 (IL-6) enzyme immunoassay 48 3-2-15-2 Interlukin-10 (IL-10) enzyme immunoassay 49 3-2-15-3 Tumor Necrosis Factor alpha(TNFα) enzyme immunoassay 50 3-2-15-3 Tumor Necrosis Factor beta(TNFβ) enzyme immunoassay 51 3-2-15-5 Calculation of results 52 3-2-16 Statistical analysis 56

4 Results Page 4-1 Sputum gram stain 57 4-2 Streptococcus pneumoniae identification 57 4-3 plate culture , microscopic and biochemical characteristics of

S.pneumoniae sputum 57

4-4 Serotype identification for Streptococcus pneumoniae isolated from 22 patients infected with pneumonia

59

4-5 Mice virulence test 59 4-6 Pneumoniae model in rabbits caused by S.pneumoniae 62 4-7 Capsule polysaccharide isolation 65 4-7-1 Total protein concentration calculation 65 4-8 Heat - inactivated Streptococcus pneumoniae 66 4-9 Systemic and mucosal of specific immunoglobulin titer in

rabbits post three different immunization protocols 66

4-10 Specific immunoglobulin titer and immune protection 67 4-10-1 Specific immunoglobulin titer 67 4-10-2 Immune protection 68 4-11 Cytokines detections 71 4-11-1 Systemic and mucosal of cytokines levels in rabbits post three

different immunization protocols 71

4-11-2 Systemic and mucosal of cytokines levels in rabbits post intramuscular capsule 1mg mix with lanolin 1ml for15 days

73

4-11-3 TNFβ levels in rabbits post intramuscular capsule 1mg mix with lanolin 1ml for15 days

75

4-11-4 TNFα levels in rabbits post intramuscular capsule 1mg mix with lanolin 1ml for15 days

76

4-11-5 IL-6 levels in rabbits post intramuscular capsule 1mg mix with lanolin 1ml for15 days

77

VIII

4-11-6 IL-10 levels in rabbits post intramuscular capsule 1mg mix with lanolin 1ml for15 days

78

4-11-7 Cytokines levels in rabbits post 36 days of immunization dose and challenge (immune protection)

79

5 Discussion Page 5-1 Streptococcus pneumoniae isolation and identification 82 5-1-1 Sputum gram stain 82 5-1-2 plate culture , microscopic and biochemical characteristics of

S.pneumoniae . 83

5-2 Serotype identification for Streptococcus pneumoniae isolated from 22 patients suffering from pneumonia

84

5-3 Streptococcus pneumoniae virulence 85 5-4 Specific immunoglobulin titer enhancing by three immunization

protocols 88

5-5 Immune response to heat killed S.pneumoniae 92 5-6 Capsule polysaccharide of S.pneumoniae and immune protection 95 5-7 Capsule polysaccharide and heat killed S.pneumoniae activated

cytokines production 97

5-8 Cytokines levels post 36 days of immunization dose and challenge (immune protection)

101

Conclusions 103 Recommendations 104 References 105-

136

List of tables NO. Title Page

1 Kits 30 2 Final immunization protocol for 5 replicates 42 3 Immune protection 43 4 TNFβ (systemic) 53 5 TNFβ (mucosal) 53 6 TNFα (systemic) 54 7 TNFα (mucosal) 54 8 IL-6 (systemic) 55 9 IL-6 (mucosal) 55

10 IL-10 (systemic) 56 11 IL-10 (mucosal) 56 12 Direct gram stain and Ziehl-neelsen stain for (410) sputum specimens

from patients infected with respiratory tract infection 57

13 Results of identification for S.pneumoniae 57 14 plate culture , microscopic and biochemical characterization of

S.pneumoniae sputum isolates 58

15 Serotype identification of S.pneumoniae isolated from 22 specimens positive for S.pneumoniae

59

16 Virulence test for S.pneumoniae serotypes in mice by intraperitoneal 60

IX

route with 1×108 cfu/ml 17 Virulence test of S.pneumoniae in rabbits by intranasal route with

1×108 cfu/ml 63

18 Capsule polysaccharide detection parameters 65 19 Heat - inactivated S.pneumoniae parameters 66 20 Comparison between systemic and mucosal of specific immunoglobulin

titer in rabbits post three different immunization protocols for serotype 6 and 1 ( 3 replicates in each serotype and in each protocol).

67

21 Comparison between systemic and mucosal of specific immunoglobulin titer in rabbits post intramuscular capsule 1mg mix with lanolin 1ml for 15 days(for serotype 6 and 1)(5 replicates in each serotype)

68

22 Immune protection to intranasal challenge with S. pneumoniae live culture of(A) homologues and (B) heterologues

69

23 Comparison between systemic and mucosal of specific immunoglobulin titer in rabbits post 36 days of immune protection for serotype 6 and 1.(3 replicates for each serotype)

69

24 TNFβ levels in rabbits post three different immunization protocols 71 25 TNFα levels in rabbits post three different immunization protocols 71 26 IL-6 levels in rabbits post three different immunization protocols 72 27 IL-10 levels in rabbits post three different immunization protocols 72 28 Cytokines levels in rabbits post 36 days of immune protection

compared with control and lanolin 79

29 Cytokines levels in rabbits post 36 days of immune protections 81

List of schemes NO. Title Page

1 Isolation of S.pneumoniae from sputum of patient infected with respiratory tract infection.

37

2 Flow chart for determined of specific immunoglobulin titer in rabbits post three immunization protocols.(3 replicates for each protocol in serotype 1 and 6).

42

3 Flow chart for determined of cytokines level in rabbits post three immunization protocols and post immune protection.(3 replicates for each protocol in serotype 1 and 6)

43

4 Flow chart for the separation of rabbit systemic(A) and mucosal(B) immunoglobulin.( Shnawa and Thwaini, 2000)

46

X

List of figures NO. Title Page

1 Sputum gram stain showing:- A / Neutrophil domination without epithelial cells ( pneumonia infection). B / Domination of epithelial cells without neutrophils ( Saliva not sputum specimens)

58

2 Sputum gram stain(2000x) showing :- A /Sputum from patient with pneumoniae showing (1)Neutrophils and (2)S.pneumoniae cells. B / Sputum from patient with pneumoniae showing neutrophils only without S.pneumoniae cells

58

3 A / Liver of mouse intraperitoneal injection with 0.3ml of normal saline (control). B /Liver of mouse intraperitoneal infected with 0.3ml contained 1×108 cfu/ml of live S.pneumoniae serotype 1 for 12 hours , showing (1) vascular congestion (2) cellular swelling and (3) Lysis of cells (Haematoxylin and Eosin staining).

61

4 A / Lung of mouse intraperitoneal injection with 0.3ml of normal saline (control). B /Lung of mouse intraperitoneal infected with 0.3ml contained 1×108 cfu/ml of live S.pneumoniae serotype 1 for 12 hours , showing inflammatory cells between alveoli (Haematoxylin and Eosin staining).

61

5 A / Liver of a rabbit intranasal injected with 0.5ml of normal saline (control). B /Liver of a rabbit intranasal infected with 0.5ml contained 1×108 cfu/ml of live S.pneumoniae serotype 1. Four days post inoculation ,showing:-(1) vascular congestion and (2) Cellular swilling and (3) Lysis of cells. (Haematoxylin and Eosin staining).

64

6 A / Lung of a rabbit intranasal injected with 0.5ml of normal saline (control). B /Lung of a rabbit intranasal infected with 0.5ml contained 1×108 cfu/ml of live S.pneumoniae serotype 1.Four days post inoculation showing:- inflammatory cells between and in alveoli.(Haematoxylin and Eosin staining).

64

7 Molisch test (positive). (1) H2SO4 (2) α-naphthol (3) polysaccharides 65 8 A / Liver of a rabbit intranasal with 0.5ml normal saline (control).

B /Liver of a rabbit post 36 day of immune dose and infection(immune protection).No pathological effects.(Haematoxylin and Eosin staining).

70

9 A / Lung of a rabbit intranasal with 0.5ml normal saline (control). B / Lung of a rabbit post 36 day of immune dose and infection (immune protection). No pathological effects.(Haematoxylin and Eosin staining)

70

10 Cytokines levels in rabbits post intramuscular serotype 1 capsule 1mg mix with lanolin 1ml for 15 days.

74

11 Cytokines levels in rabbits post intramuscular serotype 6 capsule 1mg mix with lanolin 1ml for 15 days

74

12 TNFb levels in rabbits post intramuscular capsule 1mg mix with lanolin 1ml for 15 days heat killed S.pneumoniae for 30 days.

75

XI

13 TNFa levels in rabbits post intramuscular capsule 1mg mix with lanolin 1ml for 15 days

76

14 IL-6 levels in rabbits post intramuscular capsule 1mg mix with lanolin 1ml for 15 days

77

15 IL-10 levels in rabbits post intramuscular capsule 1mg mix with lanolin 1ml for 15 days

78

16 Cytokines levels in rabbits post 36 days of immune protection compared with control and lanolin.

80

List of abbreviations:

AOM : Acute otitis media

APCs : Antigen-presenting cells

AS : Alsever solution

BCR : B-cell receptor

CAP : Community-acquired pneumonia

CbpA : Choline binding protein A

CD : Cluster of differentiation

CDL : Cluster of differentiation ligand

CPSS1 : Capsule polysaccharide serotype 1



CRP : C-reactive protein

CSIF : Cytokine synthesis inhibitory factor

ELISA : Enzyme linked immunosorbant assay

FS : Formal saline

G-CSF : Granulocyte colony stimulating factor

GM-CSF : Granulocyte macrophage colony stimulating factor

HPV : Human Para influenza virus

Ig : Immunoglobulin

XII

I.M : Intramuscular

INFCT : Interfacial capillary tube test

IN : Intranasal

IL : Interleukin

INF-γ : Interferon-gamma

LAP : Leucine amino peptidase

MEF : Middle ear fluid

MHC : Major Histocompatibility Complex

MPHA : Modified passive heamagglutination test

MIP : Macrophage inflammatory protein

OD : Optical density

PAF : Platelet activating factor

PAFr : platelet-activating factor receptor

PAMPs : Pathogen associated molecular patterns

PBS : Phosphate buffer solution

PEG : Polyethylene glycol

PHA : Passive heamagglutination test

PRRs : Pattern recognition receptors

Ply : Pneumolysin

PMNs : Polymorphonuclear cells

PspA : Pneumococcal surface protein A

PspC : Pneumococcal surface protein C

PsaA : pneumococcal surface adhesin A

Pht : Pneumococcal histidine triad

PYR : Pyrrolidonyl acylamidase

RSV : Respiratory syncytial virus

rpm : Round per minute

XIII

SARS : Severe acute respiratory syndrome

SE : Standard error

STGG : Skim milk , tryptone, glucose and glycerol medium

TD : T cell -dependent

TI : T cell –independent

TI-1 : T cell-independent type 1

TI-2 : T cell-independent type 2

Th1 : T helper one

Th2 : T helper two

TNF : Tumor necrosis factor

TLR : Toll-like receptor

VH : Variable heavy

VL : Variable light

WHO : World health organization

Chapter one Introduction

۱

1-Introduction: 1-1:Overview:

Streptococcus pneumoniae is an important pathogen that causes both

serious invasive infections, such as septicemia, meningitis and pneumonia, and

mild upper respiratory infections (Henrichsen,1995).It belongs to the normal

nasopharyngeal microbial flora that consists of bacteria with physiologic and

genetic properties suitable for colonization and multiplication under certain

conditions (Carvalho et al.,2003). S.pneumoniae was described for the first

time over 130 years ago (Charalambous et al.,2003). In spite of the

development of new possibilities to examine S. pneumoniae, the traditional

phenotypic definition of S. pneumoniae has not changed.(Barocchi et

al.,2007).S.pneumoniae is a gram-positive, α-hemolytic, bile-soluble and

commonly capsulated streptococcus that is usually identified with a relative

case (Anderton et al.,2007).Identification is based on the bacterial colony

morphology on a blood agar plate, optochin sensitivity, bile solubility and the

presence of a capsule (Doit et al.,2002; Tarja,2006). So far, 90 different

capsular monovalent have been identified , they are grouped into 8 serotypes

based on antigenic specificities similarities, In addition, un encapsulated

isolates are rather common in the nasopharynx (Bentley et al .,2006).

S.pneumoniae annually causes 10 million deaths worldwide including

the deaths of 1 million children of low development countries(WHO,1998).

Type specific immunity, based on the capsular polysaccharides (PS),is well

established (Robbins et al.,1983) . But includes fewer serotypes than the PS

vaccine and omits several that are prevalent worldwide (Dagan et

al.,1992).Other drawbacks of the conjugate vaccine exhibit a limited effect on

pneumonia (Black et al ,2000; Eskola et al.,2001).High costs, and the potential

for serotype replacement, which has already been suggested in recent clinical


۲

trials (Lipsitch,1999;Eskola et al.,2001).

Several investigators have identified protective antigens common to

pneumococci of many or all serotypes, several such species antigens in

purified or vectored form have shown protection in animal models, but it is

uncertain whether, when, and at what cost any of these will be developed as an

effective vaccine for humans (Briles et al.,2000;Ogunniyi et

al.,2000).Particularly in low-income countries, as an alternative presentation

of species antigens, immunization might elicit mucosal immunity and, with

suitable adjuvant, systemic immunity as well (Wizemann et al.,2001).The

majority of invasive S.pneumoniae infections are caused by only a small range

of serotypes with the most prevalent strains varying among age groups and

geographical regions (Henriques et al., 2000).

Different capsular types confer different levels of virulence and strains

with equally thick capsules but different capsular serotypes can vary

considerably in virulence ,however, this indicates that not only the capsular

type but also the genetic background is important for the virulence potential of

pneumococcal strains (Sandgren et al., 2004).

Capsular polysaccharides are immunogenic and give rise to serotype

specific protective antibodies (Brueggemann et al.,2003; Sjostrom et al.,

2006).

1-2:Aims of the study:-

To investigate the potential virulence and immunogenicity of S.pneumoniae

human clinical isolates in rabbits. For application of this aims the following

steps were attempted:-

1-Testing the virulence of all bacterial serotypes in mice and rabbits.

2-Selecting the most virulent and most dominant serotype in Al-Najaf

Government.


۳

3- Assay the ability of S.pneumoniae capsule to induce a humoral or cellular

immunity post vaccination by capsular antigen of the most virulent and most

dominant serotype bacteria. Then assaying the vaccination process by :-

i- Rabbit immunization with capsular antigen of bacteria by using several

protocols.

ii- Selecting the best immunization protocol that provides the highest antibody

level systemically and locally.

iii- Testing the ability of vaccine post challenging rabbits by live

S.pneumoniae post immunization, and estimate the percentage of immune

protection.

iv- Estimation of the Tumor Necrotic Factor beta(TNFβ) , Tumor Necrotic

Factor alpha(TNFα) , Interleukin 6(IL-6) and Interleukin 10(IL-10), in

systemic and mucosal surface of immunized rabbits.

Chapter two Review of Literatures

٤

2- Review of Literatures: 2-1:Pneumonia:

Pneumonia is an inflammation of the lung tissue, frequently, it is

described as lung parenchyma/alveolar inflammation and abnormal alveolar

filling with fluid (consolidation and exudation)(WHO,2007).Pneumonia can

result from a variety of causes, including infection with bacteria, viruses,

fungi, or parasites, and chemical or physical injury to the lungs, it cause may

also be officially described as idiopathic that is, unknown when infectious

causes have been excluded (Feldman et al.,1991; Nelson,2007).

2-2-:Etiology of pneumonia:

Pneumonia can be caused by microorganisms, irritants and unknown

causes, when pneumonias are grouped this way, infectious causes are the most

common type, the symptoms of infectious pneumonia are caused by the

invasion of the lungs by pathogens and by the immune tissue injuries arose

from the infection, although more than one hundred strains of microorganism

can cause pneumonia, only a few are responsible for most cases, the most

common causes of pneumonia are bacteria and viruses, less common causes of

infectious pneumonia are fungi and parasites ( Murray and Bongiorno,2006).

2-2-1:Bacterial pneumonia:

1- Gram positive bacteria:

These bacteria are the most common organisms found in pneumonia,

they include the following:


٥

The most common cause of pneumonia is the gram positive bacterium

S.pneumoniae it accounts for about 20% to 60% of all community acquired

bacterial pneumonias (CAPs) in adults, studies also suggest it causes between

13% to 38% of CAP in children (Smith,2003; Chan et al.,2007).

Staphylococcus aureus, the other major gram positive bacterium

responsible for pneumonia, accounts for about 2% of community acquired

pneumonias and between 10% and 15% of hospital originated pneumonias,it is

the organism most often associated with viral influenza, and can develop about

five days after the onset of flu symptoms, pneumonia from S. aureus most

often occurs in people with weakened immune systems, very young children,

hospitalized patients, and drug abusers who use needles, it is uncommon in

healthy adults(Lutfiyya et al.,2006).

2- Gram negative bacteria:

These bacteria are common infectious agents in hospitalized or nursing

home patients, children with cystic fibrosis, and people with chronic lung

conditions (Mcgehee et al.,2001;Christ-Crain et al.,2006).

Haemophilus influenzae is the second most common organism causing

community acquired pneumonia and accounts for 3% to 10% of all cases

(generally occurring in patients with chronic lung disease, older patients, and

alcoholics)( Gleason and Shaughnessy,2007).

Klebsiella pneumoniae may be responsible for pneumonia in alcoholics

and in other people who are physically debilitated, it is also associated with

recent use of potent antibiotics (Johnstone et al.,2007).

Pseudomonas aeruginosa is a major cause of pneumonia that occurs in

the hospital (nosocomial pneumonia),it is a common pneumonia in patients

with chronic or severe lung disease(Christ-Crain et al.,2006).


٦

Moraxella catarrhalis is found in everyone's nose and mouth, experts

have identified this bacterium as an uncommon cause of certain pneumonias,

particularly in people with lung problems, such as asthma or emphysema

(Murray and Bongiorno,2006).

Neisseria meningitidis is one of the most common causes of meningitis,

but this organism has been reported in pneumonia, particularly in epidemics of

military recruits(Lutfiyya et al.,2006).

Other gram negative bacteria that cause pneumonia include E. coli (a

cause in newborns and also associated with recent antibiotic use), Proteus

(found in damaged lung tissue), and Enterobacter.( Wipf et al.,1999).

2-2-2:Atypical bacterial pneumonia:

Atypical pneumonias are generally caused by tiny bacterial organisms

with special requirements and produce mild symptoms with a dry cough,

hospitalization is uncommon with pneumonia from these organisms, but there

are exceptions(Muller et al.,2007).

They include the following:-

Mycoplasma pneumoniae, cause the un common bacterial pneumonia ,

it is a very small organism that lacks a cell wall, it spreads from prolonged,

close contact and is most often found in school aged children and young

adults, the condition is usually mild and is commonly known as walking

pneumonia estimates of its prevalence in community acquired pneumonias in

adults range from 1.9% to 30%,in one study, it accounted for over a third of

pneumonia cases in children (Murray and Bongiorno,2006; Nisar et al.,2007).

Chlamydia pneumoniae, is now thought to cause 10% of all community

acquired cases of pneumonia ,this atypical pneumonia is most common in

young adults and children, where it is usually mild, in one study, it was the


۷

cause of 14% of cases in a group of children with pneumonia, while less

common in the elderly, it can be very severe in this population (Moine et

al.,1994; Tuazon and Murray,2000).

Legionella pneumophila, is acquired by breathing droplets of

contaminated water, outbreaks have most often been reported in hotels, cruise

ships, and office buildings where people are exposed to contaminated droplets

from cooling towers and evaporative condensers,they have also been reported

after exposure to whirlpools and saunas, legionella is not passed on from

person to person, but it may be much more common than once thought ,some

experts even believe it causes 29% to 47% of all pneumonia cases (Legionella

pneumonia is sometimes categorized as an atypical pneumonia)(

Reingold,1998 ;Tuazon and Murray,2000).

2-3:S.pneumoniae:

2-3-1:History of S.pneumoniae :

S.pneumoniae belongs to the family streptococcaceae, it was however

initially named Diplococcus pneumoniae, and in 1974 that it was renamed

S.pneumoniae according to its growth in chains in liquid media, S.pneumoniae

or Diplococcus pneumoniae, was first isolated and described independently by

Louis Pasteur and George Miller Sternberg in 1880/1881.Sternberg and

Pasteur inoculated rabbits with saliva and isolated the same organism from the

diseased animals, they both grew the bacterial isolate in culture and described

its morphology ,soon after that Albert Fränkel could prove that the

pneumococcus is a common cause of pneumonia (Bogaert et al.,2004).

S.pneumoniae was a major health burden and a mostly deadly disease in


۸

the 19th and the beginning of the 20th century, the discovery of penicillin by

Alexander Fleming and the introduction of antibiotic treatment was a

breakthrough for the cure of pneumococcal pneumonia as well as other

infectious diseases in the early 20th century(Center and Isaacman ,2005).

Unfortunately, due to emergence of antibiotic resistance S. pneumoniae

and other bacterial pathogens continue to be a health problem worldwide

(Tarja,2006).

S.pneumoniae research not only led to a better understanding of

pneumococci and pneumococcal disease but also contributed to major

advances in the field of genetics, in 1928 Frederick Griffith observed that a

non-virulent strain of S. pneumoniae could be transformed into a virulent

strain when incubated together with heat-killed virulent pneumococci using S.

pneumoniae as the model organism, Oswald Avery, Colin MacLeod, and

Maclyn McCarty discovered in 1944 that the transforming factor and thus the

carrier of genetic information is DNA and not proteins, as previously assumed

(Center and Isaacman ,2005).

2-3-2:Biology of S.pneumoniae :

Over 130 years since the first discovery of pneumcoccus, the traditional

phenotypic definition of S.pneumoniae has not changed, in a Gram stain,

pneumococcus appears as an oval shaped, gram positive coccus, 1-2 µm in

diameter, typically in pairs, some times singly or in short chains ,the gram

positive reaction of young cells may be lost when the culture is aged.

Pneumococcus grows as α- hemolytic, centrally depressed colonies on blood

agar, and generally upon primary isolation it is heavily encapsulated, it is

catalase negative and facultatively anaerobic, but can grow aerobically(Wester

et al.,2002).However, 8 % of clinical pneumococcal isolates require an


۹

enriched carbon dioxide (CO2 ) atmosphere if they are cultured on a solid

medium and thus it is recommended that cultures be incubated in a CO2-

enriched atmosphere. The nutritionally fastidious bacteria need blood or serum

to grow, and they are unable to synthesize hemin, by the action of pyruvate

oxidase under aerobic growth conditions (Dominguez and Gali,2001; Hoskins

et al.,2001).Pneumococcus utilizes oxygen to form hydrogen peroxide, which

is toxic to cultured alveolar epithelial cells and other bacterial organisms of the

upper respiratory tract, especially to H. influenzae and peroxide destroys the

labile constituents of the pneumococcal cell and thus, pneumococcus may

destroy itself (Pericone et al., 2000).

Streptococci, and thus also pneumococcus, belong to the heterotrophic

bacterial species that uses organic compounds as a source of carbon, and their

energy yielding metabolism is fermentative, yielding low levels of lactid acid.

Glucose and other carbohydrates are fermented- production of the leucine

amino peptidase (LAP) enzyme is a typical characteristic of all streptococci,

whereas production of pyrrolidonyl arylamidase (PYR) is rare among

streptococci, occurring only in S. pyogenes isolates and in some pneumococcal

isolates (Ruoff et al., 2003).

Published genome analysis suggests that S. pneumoniae has pathways for

catabolism of pentitols(pentose alcoholic sugar) as well as for cellobiose,

fructose, fucose, galactose, galactitol, glucose, glycerol, lactose, mannitol,

mannose, raffinose, sucrose, trehalose, and maltosaccharides, in addition, ten

amino acids and N-acetylglucosamine can be used as nitrogen and carbon

sources (Tettelin et al.,2001).

Colony morphology may vary from small (e.g. unencapsulated) to

clearly depressed and to very large and mucoid colonies, as in the case of

serotypes 1 and 5, and colonies have been described as mucoid, ruffled and

smooth or opaque and transparent (Weiser et al., 1994).Appearance is


۱۰

dependent on surface proteins, capsule and cell wall composition (Watson et

al.,1993).In the phenomenon called phase variation, variants have the same

serotype and are named as transparent or opaque forms of colonies, opaque

variants are larger and whitish, whereas transparent colonies are smaller and

bluish (Weiser et al.,1994).Opaque phenotypes have more capsular

polysaccharide and have been isolated commonly from blood and transparent

colonies have more teichoic acid and they are usually found in the

nasopharynx. Surprisingly, phase variation of the transparent phenotype

increases invasion into human brain vascular micro endothelial cells as much

as six-fold, and this phenomenon might be important in the development of

meningitis.(Tarja,2006).

2-3-3:Pathogenesis of S.pneumoniae:

2-3-3-1:Infection route:

In order to cause disease, the pneumococcus must first adhere and

coloniz in mucosal barrier, then be able to cross that barrier in order to

disseminate within the host, during this whole process, but in particular when

disseminated, the pneumococcus must succeed in circumventing host

immunity(Hanage and Kaijalainen ,2005).

1- Adherence:

The first step the pneumococcus must accomplish is to adhere to the

upper respiratory tract epithelium of its host, the exact procedure how this is

accomplished is still not fully elucidated, adherence can be regarded as a major

element of virulence, in fact, many of the structures shown to be of importance

to adherence also demonstrate properties that are vital for other components of

the pneumococcus ability to be virulent(Martens et al.,2004).The

pneumococcus binds avidly to the surface of respiratory tract cells, especially

up on recognition of salivated sugars (Krivan and Roberts , 1988).It has been

suggested that the enzymes neuraminidases cleaves terminal sialic acids from


۱۱

glycopeptides on respiratory cells, and thus expose receptors of importance to

adherence ,the fact that respiratory viruses like influenza and parainfluenza

demonstrate the same neuraminidase activity further supports the theory that

viral infections and pneumococcal infections can interact synergistically

(Novak and Tuomanen ,1999).

Choline binding protein that has been described to be involved in

adherence is the Choline binding protein A (CbpA), this protein forms a bridge

with glycoproteins on human respiratory cells, this reaction is however

restricted to cytokine activated cells, and has therefore also been suggested to

be important for advancing the pneumococcal disease from colonisation to

invasion CbpA deficient mutants are defective in colonisation of the

nasopharynx and fail to bind to various human cells in vitro, CbpA also has

been reported to bind secretory IgA and complement component C3(McCool

and Weiser,2004).

2-:Colonization:

The first encounter between a pneumococcus and a human usually

occurs in the nasopharyngeal cavity of the human, the most probable outcome

of this exposure is innocent colonization rather than turning ill from it. Human

nasopharyngeal carriage is the principal reservoir of pneumococci and thus

the major source of horizontal spread of this pathogen within the community

(Heffernan and Barrett,2005).Colonization of the nasopharynx by the

pneumococcus occurs frequently, especially among children, it was shown that

60% of preschool children were colonized by one or several serotypes and the

same figure for adults being lower, 2-3.7 % (Henriqus-Normark and

Christensson , 2003;Regev-Yochay and Raz, 2004).The differences in

colonization rates are probably due to the more mature immune system in

adults, there is a clear correlation between rising levels of anti-pneumococcal

surface antibodies and diminished carriage rates (Syrjanen et al., 2001; Simell


۱۲

and Kilpil,2002).Also, vaccination with conjugated vaccine against

pneumococci has resulted in diminished carrier rates of the different serotypes

included in the vaccine. The colonization rates also demonstrate a seasonal

variation, pneumococcal carriage is more frequently seen during the cold

season,the reason for this remains to be explained, although concomitant viral

infections (which are more common during the winter), facilitating

pneumococcal adherence to the respiratory epithelium, are generally believed

to be involved. The pneumococci have also been demonstrated to out-compete

other frequent colonizers in the upper respiratory tract such as Hemophilus

Influenzae and Moraxella catarrhalis, this is accomplished by the production

and release of hydrogen peroxide by the pneumococcus (Regev-Yochay et al.,

2006).

The initial episode pf colonization often occurs at the age of 6 months,

thereafter acquisition of colonization by a new serotype occurs as often as

every four months in infants ,in early childhood, these first colonizing

serotypes can often be detected up to 6 months after acquisition, but the time

can vary both between serotypes and individuals, in adults, the mean carriage

time is shorter, usually 2-4 weeks (Roson and Carratala,2001).

3-:Invasion:

There are two principle procedures that the pneumococcus can use to

continue on the way to cause infection in man, the first way is to use the

normal air filled channels from the nasopharyngeal cavity to other organs such

as the middle ear, the sinuses or the lungs. The most common way for a

pneumococcus to cause pneumonia is by aspiration of the pneumococcus from

the nasopharyngeal cavity down to the lower airways, the other procedure is

direct passage of the pneumococcus through the mucosal barrier in the upper

respiratory airways, either into adjacent tissues or directly as haematogenic

origin (Martens et al.,2004).


۱۳

This latter way is regarded to be uncommon although it has been

suggested to be necessary for penetration of the blood-brain barrier in

meningitis and is also referred to as transmigration. The theory behind

transmigration is that cytokine activated host cells increase the expression of

platelet activating factor (PAF) on the surface. The PAF then works as a

docking station for the phosphoryl choline on the pneumococcus, initiating an

internalisation process of the bacteria, by this process the pneumococcus is

able to penetrate into normally sterile locations, and when the pneumococci

arrive at their final destinations, they will immediately be challenged by the

host immune system, the result of this challenge will either lead to clearance of

the bacteria (which is probably the most common outcome), or to disease, in

the case of the latter outcome, the bacteria might further disseminate to

normally non-infected locations such as the alveoli, the bloodstream or the

middle ear (Ring ,1998; Sandgren and Sjostrom,2004).

4-:Phase variation:

Pneumococci have been shown to have at least two distinctly separated

appearances when grown on a transparent medium, these two appearances are

referred to as either transparent or opaque, how these different morphological

appearances are accomplished remains to be explained, but is generally

considered to depend on protein expression and capsular thickness.

Transparent phenotypes have been demonstrated to express a higher amount of

neuraminidase, a fact that has suggested this as an explanation for the observed

enhanced adhesion of transparent phenotypes in colonization (Melegaro and

Edmunds,2004 ; Millar et al.,2006).Transparent phenotypes also express

higher levels of phosphoryl choline in the cell wall, it has been demonstrated

that the pneumococcus can bind to airway epithelium cells through a platelet

activating factor (PAF) receptor, these receptors are expressed when the cells

are stimulated by cytokines like IL-1, and it has been suggested that the


۱٤

pneumococci attach to the airway epithelium by mimicking PAF, which, like

the pneumococcus contains phosporyl choline. Opaque phenotypes have

generally been- regarded as more virulent, and have been demonstrated to be

more prone to cause invasive disease after intraperitoneal challenge of mice

(Kim ,1998; Lee,2003).

2-3-4: Pathogenicity of S.pneumoniae:

Infection with S.pneumoniae occurs via respiratory droplets from person

to person and in most cases initially leads to asymptomatic carriage of

pneumococci in the upper respiratory tract ,development of disease can occur

by local spread from the nasopharyngeal mucosa leading to sinusitis and otitis

media (Bogaert et al., 2004).Pneumococci can reach the lungs when

aerosolized from the nasopharynx or aspirated directly into the alveoli,

circumventing the ciliated epithelium that is difficult to attach to alternatively,

pre-damage of the respiratory epithelium due to viral infections or chronic

bronchitis favors pneumococcal invasion along the airways (O'Brien et al.,

2000).

Bacteremia can occur as a complication of pneumococcal pneumonia or

without a previous focus of infection, from the bloodstream pneumococci can

invade the meninges and cause meningitis, this is favored by high density

bacteremia but the exact mechanism of invasion is still unclear (Ostergaard et

al., 2006).S.pneumoniae also less frequently causes other diseases such as

endocarditis, pericarditis, osteomyelitis, conjunctivitis, pyogenic arthritis,

necrotizing fascistic and peritonitis (Butler, 2004).

2-3-5:Immunology of S.pneumoniae:

2-3-5-1:Host defense mechanism:

Host defense mechanisms against pneumococcal infection consist of

both non-immunological and immunological mechanisms, the non-specific


۱٥

defense includes normal cough reflex as well as intact mucosal surface and

clearance mechanisms of the upper respiratory tract, the immunological

mechanisms can be further divided into specific and nonspecific defense (Babl

et al.,2001).In the absence of specific opsonizing antibodies, the virulent

organisms can be cleared from the bloodstream during their slow passage

through the sinusoids of spleen (Balachandran et al.,2002).The cytokines

produced by pulmonary macrophages in response to pneumococcal infection

include tumour necrosis factor alpha (TNFα), interleukin-6 and interleukin-1,

stimulate production of acute-phase proteins and attract phagocytic cells such

as Polymorphonuclear leukocytes (PMNLs) and macrophages at the site of

infection (Bergeron et al.,1998).Opsonization, which is followed by

phagocytic uptake and killing of bacteria can be achieved by classical,

alternative and pulmonary surfactant lectin pathways (Virkki et al.,2002).This

does not necessarily require the presence of specific pneumococcal antibodies,

as other factors such as C-reactive protein (CRP), Pneumolysin (Ply), capsular

polysaccharides (CPS) and many other cell-wall components can activate the

complement cascade (Wu et al.,1997).The elimination of pneumococci is,

however, greatly enhanced if the antibodies to the capsular polysaccharide are

present(Vidarsson et al.,1998; Vuori-Holopainen and Peltola,2001).

The specific defence against pneumococcal infection consists mainly of

anti-capsular PS and protein IgG antibodies, binding of antibodies to the PS

capsule of pneumococcus leads to the activation of the complement cascade,

which results in deposition of complement components C3b and iC3b (or CR1

and CR3) to the capsular surface of the bacteria (Esposito et al.,1990; Brown

et al.,2003). The PMNLs and macrophages, which possess receptors for these

complement factors, then uptake and kill the pneumococcus (Gordon et


۱٦

al.,2000).In addition to the amount of antibody present, the

opsonophagocytosis is regulated by the polymorphism of Fc gamma receptors

(FcγRII), the two allotypic forms of FcγRII, H131 and R131, bind IgG1 with

equally high affinity, but only FcγRIIa-H131 can effectively interact with

human IgG2 (Rodriguez et al.,1999).

As the carbohydrates of encapsulated bacteria, such as pneumococcus

mainly elicit IgG2 subclass persons expressing only FcγRIIa-R131 receptors

of low affinity to IgG2 are more vulnerable to pneumococcal infections , the

production of IgG2 subclass begins later in childhood than that of IgG1 and

IgG3, reaching adult levels only by the age of 12 month (Vernacchio et

al.,2000; Yee et al.,2000).Secretory IgA is the main immunoglobulin isotype

at mucosal surfaces and secretions, such as middle ear fluid (MEF), the

occurrence of IgA in nasopharyngeal aspirates and MEF is independent of the

concomitant presence of the antibody in serum (Virolainen et al.,1995).The

IgA may protect against pneumococcal infections by interfering with the

adherence of bacteria to the epithelial cells of the respiratory tract, and by

enhancing surface phagocytosis, in which the leukocytes pin pneumococci

against the alveolar wall in order to engulf them (Novak and Tuomanen,1999).

Pneumococcal antibodies are actively transferred across the placenta,

the transfer rate depends on factors such as the mother’s vaccination status,

antibody isotype and pneumococcal serotype in question, in most studies the

concentration of serotype specific ,these inflammatory mediators

pneumococcal PS antibodies in new-born infants has varied between 30% to

89% of that in the mothers (Munoz et al.,2002).These early antibodies have

been shown to protect against subsequent pneumococcal carriage acquisition

and acute otitis media (AOM)( Salazar et al.,1997).


۱۷

2-3-5-2:Innate immunity:

Innate immunity, including early cytokine release, is initiated upon

recognition of conserved pathogen-associated molecular patterns by various

host cells expressing pattern recognition receptors (Medzhitow and Janeway

,1997).The major pattern recognition receptors in mammalian species are the

Toll-like receptor (TLR) , family of proteins , these proteins share a common

cytoplasmic domain with each other, and with the IL-1 and IL-18 receptors

called the Toll-ILRs, activated Toll-ILRs mediate the eventual translocation of

NF-B and AP-1 into the nucleus via one or a number of distinct adaptor

proteins, most critically MyD88, with resultant transcriptional activation of

numerous proinflammatory cytokine and chemokine-chemokine receptor

genes, at present, 11 different TLRs (TLR1 to -11) have been reported,

showing distinct specificities for microbial and even host ligands and utilizing

different combinations of adaptor proteins (Kawai et al.,1999;Takeuchi et

al.,2000).The requirement for TLR2 for signaling in response to

peptidoglycan, lipoteichoic acid, and lipoproteins has suggested a dominant

role for TLR2 in the induction of innate responses to gram-positive bacteria.

Indeed, a key role for TLR2 in mediating innate immunity, including cytokine

induction in response to a variety of gram-positive bacteria, including S.

pneumoniae, has been demonstrated (Echchannaoui et al.,2002; Gause et

al.,2003).Nevertheless, TLRs other than TLR2 may play a role in responses to

gram-positive bacteria, thus, MyD88-/- mice showed greater lethality and a

more profound defect in macrophage cytokine production in response to

Staphylococcus aureus relative to that in TLR2-/- mice (Takeuchi et al.,2000).

It was recently shown that although TLR2-/- mice are more susceptible to

experimental pneumococcal pneumonia, a substantial part of the inflammatory


۱۸

response was TLR2 independent (Koedel et al.,2003). Additionally,TLR2-/-

mice inoculated intranasally with live S.pneumoniae displayed only a modestly

reduced inflammatory response in the lungs and normal host immunity relative

to that in wild-type mice, despite defective cytokine production from freshly

isolated TLR2-/- alveolar macrophages (Knapp et al.,2004).Less is known

regarding the role of TLRs in shaping the adaptive humoral response to an

intact pathogen, injection of mice with purified antigens in adjuvant

demonstrated a role for MyD88 in mediating an antigen-specific type 1, but

not type 2, in vivo IgG isotype response more recently, a normal pathogen

specific(Schnare et al.,2001).T cells , B7-dependent co stimulation , and

dendritic cells, direct TLR signaling, including the subsequent release of

cytokines, induces dendritic cells to migrate to secondary lymphoid organs,

upregulate T-cell costimulatory molecules, including B7, and release IL-12,

thus leading to the priming of CD4- T cells for type 1 immunity(Abdul khan et

al.,2005).TLRs play an important role in the in vivo anti protein and anti

polysaccharide Ig isotype response to intact S. pneumoniae (Redecke et

al.,2004).

2-3-5-3:Capsule polysacchrides of S.pneumoniae immunogens and

immunity:

Antigens can be assigned to one of two major groups according to their

T-cell activating capacity: T cell -dependent (TD) or T cell -independent (TI)

antigens. Protein antigens, which are classified to the TD group, are normally

immunogenic already in early infancy, the response to TI antigens such as

bacterial polysaccharides develops later in childhood, this may be due to

immaturity of B-cell population in the marginal zone of the spleen, or by the

low expression of CR2 receptors on the marginal zone B cells in young infants


۱۹

(Timens et al.,1998; Toikka et al.,2000).The TI antigens can be further

classified into T cell-independent type 1 (TI-1) and type 2 (TI-2) antigens , the

TI-1 antigens such as lipopolysacharrides (LPS) function as polyclonal B-cell

activators and are totally independent of T-cell help, TI-2 antigens, such as

pneumococcal CPS, can activate antibody producing B cells without antigen-

specific T-cell help (Munoz et al.,2002).The activation is, however, regulated

by CD1 restricted CD8+ helper T cells and natural killer cells (Snapper and

Mond,1996).Unlike the TD antigens, the TI-2 antigens do not induce

immunologic memory, antibody isotype switch or affinity

maturation(Stein,2002).

2-4:Role of specific antibodies:

The concentration of antibodies to CPS increases with age, this

development appears to be serotype specific and coincides with the carriage

acquisition and episodes of pneumococcal infections such as AOM. Several

studies have tried to link the carriage as well as mucosal and invasive

pneumococcal infections to the low pre-existing antibody concentration, or to

the subsequent antibody responses. The nasopharyngeal carriage of

pneumococci induces antibody responses in adults, but the results have been

partly controversial in children under 2 years of age(Musher et

al.,1997;Soininen et al.,2001).The quantity and quality of antibodies required

for protection against pneumococcal infections remains unknown, it may also

be different for each serotype, and vary for mucosal and invasive infections as

well as in different populations and epidemiological settings (Lee et al.,2003).

In addition to the serotype specific IgG antibody concentration, the

distribution of IgG1/IgG2 subclasses, IgG avidity, opsonophagocytic activity

and animal models have been utilized in order to determine the protective


۲۰

immunity. Antibodies to pneumococcal proteins may also have an important-

role in providing protection, and the main proteins and toxins that have been

shown to confer protection in animal models include Pneumococcal surface

protein A (PspA), Pneumococcal surface protein C (PspC), pneumococcal

surface adhesin A (PsaA), Pneumolysin, pneumococcal histidine triad (Pht),

autolysin C, Choline binding protein A (CbpA), 29 kDa C3 protease, and

putative proteinase maturation protein A (Ades et al.,2000; Briles et al.,2000).

2-5:S.pneumoniae immunogens evaluations for vaccination:

Colonization of the upper respiratory tract (URT) is a step prior to

S.pneumoniae infection , most carriage episodes are asymptomatic and last on

the order of weeks to a few months , in principle, colonization may be

prevented or terminated by the innate and/or adaptive immune system or by

competing microbial flora , yet the particular host and pathogen factors

affecting resistance to pneumococcal colonization are still poorly understood

(Krzysztof et al.,2008).Pneumococcal CPS is a major virulence factor of S.

pneumoniae and reduces phagocytosis in the host (Jackson et al.,2003).The

currently licensed pneumococcal vaccine consists of 23 purified CPS

serotypes, CPS vaccine is based on the observation that antibodies against the

capsule protect against disease by enhancing complement dependent

phagocytosis (Wernette et al.,2003).

Pneumococcal vaccination is recommended for all persons 65 years and

older in the United States, however, efficacy studies indicate decreased

protection among this target population despite protective levels of anti-

pneumococcal antibodies .The underlying causes of the reduced vaccine-

efficacy merit investigation. Reports of the diminished vaccine efficacy in- the

elderly may reflect either poor functionality of vaccine induced anti-CPS


۲۱

specific antibodies or inconsistent antibody measurements, the protection is

measured by in vitro analysis of opsonophagocytosis by anti-polysaccharide

antibodies and serves as a surrogate marker of clinical protection (Rubins and

Janoff,2001).

The successes of antipneumococcal therapy using passive transfer of

serotype-specific antibodies and of vaccinations that depend on anticapsular

antibodies showed the importance of humoral immunity as one mechanism of

protection against colonization and disease from S. pneumoniae, for some, but

not all serotypes, such immunity appears to play a role in naturally acquired

protection (Kadioglu et al.,2004; Zhang et al.,2007). However, several lines

of evidence indicate that factors other than acquisition of anticapsular

antibodies play a crucial role in the development of natural protection against

pneumococci (Roche et al.,2007).

The reduction in pneumococcal disease incidence after the first birthday

in the general population occurs simultaneously for many rare and common

serotypes, suggesting the acquisition of one to precede by several years the

age-related rise in anticapsular antibody (Lipsitch et al.,2005). Similar patterns

have been suggested for nasopharyngeal carriage, Experimental and

observational studies in adults have found little or no evidence that higher

anticapsular antibody concentrations are associated with greater protection

from colonization (McCool and Weiser,2004).Mouse studies have similarly

shown that immunity to pneumococcal colonization acquired from prior

exposure to live bacteria or a killed, whole-cell vaccine is antibody

independent, while other studies have shown a similar mechanism for

clearance of longstanding carriage in previously unexposed animals (Van

rossum et al.,2005).


۲۲

Acquired immunity was shown to be dependent on the presence of

CD4-T cells at the time of challenge (Trzcinski et al.,2005).Apart from their

role in providing help for the production of antibodies, the role of CD4-T cells

in acquired immunity to extra cellular bacteria remains poorly understood, a

basic question is whether such responses depend on classical antigen

presentation to the T-cell receptor. Two types of observations particularly

raised this concern, first, while wild-type mice inoculated intranasally with a

strain of pneumococcus rapidly cleared bacteria from the lungs and blood

within 2 days, major histocompatibility complex class II-knockout mice in the

same experiment showed persistent infection in both lungs and blood over 3

days, suggesting a nonspecific role for CD4-T cells in early host defense.

Further the pneumococcal toxin pneumolysin caused apparently nonspecific

activation and migration of CD4-T cells in vitro in the absence of antigen

presentation (Zhang et al.,2007;Krzysztof et al.,2008).

Second, immunization of mice with very small quantities of killed

whole pneumococci could protect them against subsequent intranasal

challenge, showing that, in mice, a single exposure to a live attenuated

pneumococcal strain conferred resistance against colonization and invasive

disease and raise concern about the antigenic specificity of the observed

protection (Roche et al.,2007).

2-5-1:Role of adjuvant:

Prophylactic adjuvants must induce a protective immune response

against an infectious agent with minimal short- or long-term side effects.

Protection may require a humoral antibody response, an activated CD4 cell-

mediated response, or a CD8 cytotoxic lymphocyte response, depending on

the agent. Prophylactic adjuvants should direct antigen presentation and


۲۳

modulate the cytokine network to induce the appropriate protective responses.

Adjuvants may produce a number of adverse consequences, both locally at the

injection site and systemically (Edelman 2000).

The mechanisms by which adjuvants promote increased immune

response are slowly becoming more defined as the molecular aspects of

antigen recognition and immune response become more fully understood.

Adjuvants may have up to five of the following mechanisms of action: the

“depot” effect, an antigen presentation effect, an antigen distribution or

targeting effect, an immune activation/ modulation effect, and cytotoxic

lymphocyte induction effect (Cox and Coulter,1997).

One classic mechanism of adjuvant action is the “depot” effect, in which

the adjuvant protects the antigen from both dilution and rapid degradation and

elimination by the host. By localizing and slowly releasing intact antigen, the

adjuvant permits a slow, prolonged exposure of the immune system cells to a

low level of antigen. This prolonged exposure results in continued stimulation

of antibody producing cells, resulting in the production of high levels of

antibody by the host (Harold and Stills,2005).

The significance of the depot effect was demonstrated by Herbert

(1967), who compared antibody production between a single dose of antigen

in a water-in-oil emulsion with daily injections of a small amount of antigen in

saline over 50 days. Although both groups developed high antibody titers, the

antibody levels in the emulsion-injected group remained elevated in contrast to

the saline groups in which the antibody levels declined after cessation of the

daily injections at day 50.

The importance of continued low-level antigen stimulation for the

production of high-affinity antibody is best explained by the antigen selection


۲٤

hypothesis proposed by Siskind and Benacerraf (1969). They proposed that

low-dose antigen exposure resulted in the stimulation of only B cells with

high-affinity receptors, whereas at higher doses, B cells with medium- and

low-affinity receptors would be stimulated (Harold and Stills,2005).

2-6:Role of cytokines:

Cytokines (Greek cyto-, cell; and -kinos, movement) are a category of

signaling molecules that are used extensively in cellular communication, they

are proteins, peptides, or glycoproteins, the term cytokine encompasses a large

and diverse family of polypeptide regulators that are produced widely

throughout the body by cells of diverse embryological origin(Gilman et

al.,2001).The action of cytokines may be autocrine or paracrine, but not

endocrine, the reason for them not being endocrine signals is because the

signal must be released in the general region of the pathogen infected cells, so

other immune molecules which follow the signal will arrive at that site (where

this signal is released)( Tian et al.,2005).Cytokines are critical to the

development and functioning of both the innate and adaptive immune

response, although not limited to just the immune system, they are often

secreted by immune cells that have encountered a pathogen, thereby activating

and recruiting further immune cells to increase the system's response to the

pathogen, cytokines are also involved in several developmental processes

during embryogenesis (David et al.,2007).

2-6-1:Tumor necrosis factor alpha (TNF-α):

Tumor necrosis factor-α is a cytokine involved in systemic

inflammation and is a member of a group of cytokines that stimulate the acute


۲٥

phase reaction, the primary role of TNF-α is in the regulation of immune cells,

is also able to induce apoptotic cell death, to induce inflammation, and to

inhibit tumor genesis and viral replication (Locksley et al.,2001).TNF-α is

produced mainly by macrophages, but they are produced also by a broad

variety of other cell types including lymphoid cells, mast cells, endothelial

cells, cardiac myocytes, adipose tissue, fibroblasts, and neuronal tissue. Large

amounts of TNF-α are released in response to lipopolysaccharide, other

bacterial products, and Interleukin-1 (IL-1).It has a number of actions on

various organ systems, generally together with IL-1 and Interleukin-6 (IL-6)

(Gaur and Aggarwal,2003).

2-6-2:Tumor necrosis factor beta (TNF-β):

This cytokine is produced predominantly by mitogen-stimulated T-

lymphocytes and leukocytes. This factor is secreted also by fibroblasts,

astrocytes, myeloma cells, endothelial cells, epithelial cells and a number of

transformed cell lines. The synthesis of TNF-β is stimulated by interferons-

and IL2, some pre-B-cell lines and Abelson's murine leukemia virus-

transformed pre-B-cell lines constitutively produce TNF-β (Micheau and

Tschopp,2003).TNF-β acts on a plethora of different cells, this activity is not

species-specific, and acts on murine cells but shows a slightly reduced specific

activity, in general, TNF-β binds to the same receptor as TNF-α and TNF-β

and TNF-α display similar spectra of biological activities, although TNF-β is

often less potent or displays apparent partial agonist activity, TNF-β is

cytolytic or cytostatic for many tumor cells. In monocytes TNF-β induces the

terminal differentiation and the synthesis of G-CSF, TNF-β is a mitogen for B-

lymphocytes, in neutrophils TNF-β and TNF-α induces the production of


۲٦

reactive oxygen species, it is also a chemo attractant for these cells, increases

phagocytosis, and also increases adhesion to the endothelium, and promotes

the proliferation of fibroblasts and is involved probably in processes of wound

healing in vivo (Black et al.,1997;Wajant et al.,2003).

2-6-3:Interleukin-6(IL-6):

Interleukin-6 is an interleukin that acts as both a pro-inflammatory and

anti-inflammatory cytokine, it is secreted by T cells and macrophages to

stimulate immune response to trauma, especially burns or other tissue damage

leading to inflammation. In terms of host response to a foreign- pathogen, IL-6

has been shown, in mice, to be required for resistance against the bacterium,

S.pneumoniae (Van der poll et al.,1997). IL-6 is also a "myokine," a cytokine

produced from muscle, and is elevated in response to muscle contraction

(Febbraio and Pedersen ,2005).It is significantly elevated with exercise, and

precedes the appearance of other cytokines in the circulation. During exercise,

it is thought to act in a- hormone-like manner to mobilize extra cellular

substrates and/or augment substrate delivery, additionally, osteoblasts secrete

IL-6 to stimulate osteoclast formation, and smooth muscle cells in the tunica

media of many blood vessels also produce IL-6 as a pro-inflammatory

cytokine, IL-6's role as an anti-inflammatory cytokine is mediated through its

inhibitory effects on TNF-α and IL-1 (Heinrich et al.,2003; Schwantner et

al.,2004).

Interleukin-6 is one of the most important mediators of fever and of the

acute phase response, in the muscle and fatty tissue IL-6 stimulates energy

mobilization which leads to increased body temperature, il-6 can be secreted


۲۷

by macrophages in response to specific microbial molecules, referred to as

pathogen associated molecular patterns (PAMPs), these PAMPs bind to highly

important group of detection molecules of the innate immune system, called

pattern recognition receptors (PRRs), including Toll-like receptors (TLRs),

these are present on the cell surface and intracellular compartments and induce

intracellular signaling cascades that give rise to inflammatory cytokine

production (Culig et al.,2003; Stenvinkel et al.,2005).

2-6-4:Interleukin-10(IL-10):

Interleukin-10, also known as human cytokine synthesis inhibitory

factor (CSIF), is an anti-inflammatory cytokine, this cytokine is produced

primarily by monocytes and to a lesser extent by lymphocytes, this cytokine

has pleiotropic effects in immunoregulation and inflammation (Girndt,2003).It

down-regulates the expression of Th1 cytokines, MHC class II antigens, and

costimulatory molecules on macrophages, it also enhances B cell survival,

proliferation, and antibody production (Tan et al.,1995).

Interleukin-10 is capable of inhibiting synthesis of pro-inflammatory

cytokines like IFN-γ, IL-2, IL-3, TNF-α and GM-CSF made by cells such as

macrophages and the Type 1 T helper cells, it is also displays potent abilities

to suppress the antigen presentation capacity of antigen presenting cells,

however, it is also stimulatory towards certain T cells, mast cells and

stimulates B cell maturation and antibody production

(Girndt,2003;Grimbaldeston,2007).

Cytokines interact in a network that consists of proinflammatory

cytokines (e.g., TNFα, IL-6, IFN-g, and IL-12) and anti-inflammatory


۲۸

cytokines (e.g., IL-10), in patients with pneumonia, cytokines are produced

within the lung at the site of the infection, where they are important for host

defense(Boutten et al.,1996; Dehoux et al.,2004). Indeed, endogenous TNF,

IL-6, and IL-12 are essential for limitation of bacterial growth in lungs ,while

IL-10 hampers antimicrobial defenses (Greenberger et al.,1996).

Effective pulmonary host defense against respiratory pathogenesis

believed to be mainly mediated via phagocytosis by alveolar macrophages and

recruited neutrophils (Gordon et al.,2000).If pneumococci overcome these

defenses and gain entry to the blood stream, systemic protection is afforded by

anti capsular antibodies. Such defenses are orchestrated by a rapid

inflammatory response following infection (Nelson and

Summer,1998).Cytokines have been shown to be important soluble mediators

responsible for coordinating this response , many cytokines are known to be

involved in anti bacterial defenses within the lungs, TNF is capable of

recruiting inflammatory cells to the site of infection both directly and via up

regulation of adhesion molecules (Neumann et al.,1996).TNF is also capable

of stimulating the release of chemokines, cytokines that are directly tactic

chemo for inflammatory cells, macrophage inflammatory protein 1 (MIP-1)

and MIP-2 are two chemokines known to be important in bacterial pneumonia

(Greenberger et al.,1996).Following recruitment of phagocytic cells, TNF can

also promote antimicrobial activity by activating the respiratory burst and by

activating the capacity to deregulate(Klebanoff et al.,2003).These effects of

TNF have been shown to be required for effective in vivo host defense against

a range of microorganisms, including S.pneumoniae (Chen et al.,1992;

Laichalk et al.,1996). IL-1 shares several of TNF’s activities, including the

promotion of cell recruitment and the activation of macrophages at the site of


۲۹

infection (Rogers et al.,2004). In some situations the two cytokines act

synergistically to exert their effects ,IL-6 has been ascribed both pro- and anti-

inflammatory effects,IL-6 can activate monocytes , and it can synergize with

TNF to increase the respiratory burst of neutrophils ,however, regulation of the

inflammatory response by anti-inflammatory cytokines prevents damage to the

host(Taneli,2003).IL-10 contributes by reducing the production of

proinflammatory cytokines and chemokines, and down-regulates the

expression of adhesion molecules (Willems et al.,1994).

These cytokines were chosen for investigation response during

pneumococcal infection, several pneumococcal factors are known to induce

the release of inflammatory mediators, Cell wall , teichoic acids, capsular

polysaccharides, and pneumolysin have been shown to induce cytokine

production from cells in vitro (Katharina,2008).

Chapter three Materials and Methods

۳۰

3- Materials and Methods:

3-1:Materials: 3-1-1: Table 1 kits.

Kit Company Origin

Gram stain AL-Hilal ( K.S.A)

Ziehl-neelsen stain AL-Hilal ( K.S.A)

HISterp (biochemical test) KBOOB, Himedia (India)

Serotype (Antisera) DENKA SEIKEN (JAPAN)

.

McFarland BioMerieux (France)

Total protein concentration Biolab reagent (France)

Interlukin-6 Immunotech (France)

Interlukin-10 BioSource (Belgium

)

TNFα BioSource (Belgium

)

TNFβ R, D. systems ,INC. (U.S.A)

3-1-2: Patients:

A total number of (410) out patients were included in this study, age range (18-

60) years old (males and females) attending the chest unit of AL-sadder Teaching

Hospital in AL-Najaf Governorate who were suspected to have respiratory tract infection

from first February 2008 the end January 2009. Data on the following variables were

recorded: history of cough, color of sputum and fever.

3-1-3:Rabbits:

A total of (85) NewZeland white rabbits were used in the study. Each rabbit

weighed about (1.5-2) kg. and were left for two weeks for adaptation to laboratory

environmental conditions with water and food before experimentation.

3-1-4:Mice:


۳۱

A total of (54) balbC albino mice were used in the study. Each animal weighed

about (30-35)g. Animals were left for two weeks for adaptation to laboratory

environmental conditions with water and food before being used for experimentation.

3-1-5:Solutions:

3-1-5-1:Normal saline:

The solution was prepared by dissolving 0.85 gm sodium chloride (Nacl; BDH

company; U.K; M.W=58.44) in 20 ml D.W; the volume was then completed to 100 ml.

The final concentration was 0.85 %. The solution was then sterilized by autoclaving

(121ºC/ 15 Bar/ 15 minutes). It was used in preparing formalin solution and for titration

purposes.

3-1-5-2:Formal saline (FS):

The solution was prepared by adding 0.5ml of formaldehyde CHO), (BDH

Company), to 99.5ml sterile normal saline, to reach a final concentration of formalin in

this solution to 0.5%. The solution was used as a preservative for immunoglobulin from

trachea and serum immunoglobulin (Pears, 1985) .

3-1-5-3:Tris buffer:

It was prepared by dissolving 12g of tris(NH2C CH2OH)3, BDH Company ,

U.K., M.W=12.4 in a small volume of D.W, the volume was then completed to 1000ml

and the pH was adjusted to 7.0 by Hcl (0.1 N).It was used for the preparation of

polyethylene glycol (PEG) (Jonston and Thorpe,1982).

3-1-5-4:Polyethylenglycol solution:

It was prepared by dissolving 6gm from polyethylenglycol [HO(C2H4O)] BDH

Company , U.K., M.W=6000 in a small volume of tris buffer .and completed to 100ml .

The final concentration was 6%, the pH was adjusted to 7.0. It was used in separation of

immunoglobulin. (Jonston and Thorpe,1982).

3-1-5-5:Sodium deoxycholate solution:

It is prepared by dissolved 2g of sodium deoxycholate powder in 100ml of warm

D.W. and filtered by 2.5mm filter paper.( Macfaddin, 2000).

3-1-5-6:Phenol red inulin broth:


۳۲

It is prepared according to Macfaddin (2000).

Peptone (10g) , Beef extract (1 g) , Sodium chloride (5 g) and Phenol red (0.018 g).

The above reagents were dissolved in one liter of D.W. and the pH was adjusted

to 7.0 and distributed in test tubes (each test tube contain 4.5 ml) and autoclaved at

121Co for 15 min., then 1g of inulin was dissolved in 100ml D.W. and sterilized by

chlorophorm (Smith, 1932). Then 0.5ml from the sterile sugar solution was placed in

each test tube. This broth was used for the identification of S.pneumoniae for its ability

for inulin fermentation.

3-1-5-7:Iodine solution:

It is prepared by dissolved 10g of Iodine and 20g of Potassium iodine in 1liter

of D.W. it is used for capsule detection. (Hall, 1980).

3-1-5-8:α- naphthol reagent:

It was prepared by dissolving 0.5g of α- naphthol powder in 10ml of absolute

ethanol. It was used for capsule detection. (Hall, 1980).

3-1-6: Culture media:

3-1-6-1: General culture media:

Blood agar , Trypticase soy agar and trypticase soy broth , was prepared

according to the manufacturing company recommended method (Hi media , India). Used

for the cultivation of bacteria.

3-1-6-2:- Gentamicin blood agar:

It was prepared by adding 3mg of gentamicin to one litter of sheep blood agar

,the final concentration will be 3mg/l. The medium was used as a selective medium for

S.pneumoniae .(Ruoff and Beighton, 1999).

3-1-6-3:-Skim milk , tryptone, glucose and glycerol medium(STGG):

This medium was used as storage medium. It was prepared as follows:-

Skim milk (3g) , Tryptone (2g) , Glucose (0.5g) and Glycerol (10ml).

The above reagents were dissolved in 100ml of D.W. and each one ml was

distributed in a screw capped tube and autoclaved at 121°C for 20min. S. pneumoniae is

known to survive for 1.5 years at -25°C. in this medium ( Tarja et al., 2004).

3-1-7:Stains:


۳۳

3-1-7-1:Gram stain:

It was prepared according to the manufacturing company instructions(AL-Hilal,

K.S.A).It was used for bacterial staining.

3-1-7-2:Ziehl-Neelsen stain:

It was prepared according to the manufacturing company instructions (AL-

Hilal, K.S.A).Then it was used for acid fast bacilli detection in sputum.

3-1-7-3:Nigrosin stain:

It is prepared by dissolving 0.1g of Nigrosin powder in 10ml of warm D.W. and

filtered by 2.5mm filter paper. It was used for capsule staining. (Cruickshank et al.,

1975).

3-1-8:Lanolin oil:(Hi media , India):

Used as adjuvant. Lanolin is prepared by purifying the wool grease that is a

byproduct from when sheep wool is finished into wool products. Lanolin is a pale yellow

oil whose melting point is around 40Co, and is a oil ester of higher alcohol and higher

fatty acid. In addition to cosmetic and pharmaceutical uses lanolin use as adjuvant

(Thomas, 1968 ; Wittler,1991).

3-2:Methods: 3-2-1:Sputum specimens:

Specimens were obtained before antimicrobial agents were given .(two

replicates in two days for all patients ). Morning sputum was placed in sterile containers

as follows:-

a- The patient was instructed to wash their mouth with normal saline to reduce the

number of bacteria of the oral flora.

b- The patient was directed to breath deeply and cough deeply to bring up sputum

directly into a sterile container.

c- Sputum specimens were transported to the laboratory for examination.(Scheme 1)

(Grange, 1988; Laszalo, 1999).

3-2-2: Sputum Ziehl-Neelsen stain method:


۳٤

This staining method was performed according to Macfaddin(2000).

With a special care, sputum was homogenized for a few minutes

with a clean wooden stick.

a- A loopful from the homogenized sputum was placed onto a clean microscopic

slide and spread on the surface. The smear was dried in air and fixed.

b- The slide was covered with carbol fuchsin for 5 min. with heating without boiling up

to steaming and more stain was added to keep the smear covered. Then the slide was

washed with water.

c- The slide was covered with 20% H2SO4 for 1min., then washed with water.

d- The slide was covered with methylene blue stain for 2 min., then washed with water.

e- Dried and examined under oil lens .

This method is used for Mycobacterium tuberculosis(acid- fast bacilli)

diagnosis.acid- fast bacilli are stained bright red , while other microorganisms are stained

blue.

3-2-3:Sputum gram stain method:

Gram stained sputum preparations were used for polymorpho nuclear

neutrophils (PMNs) and epithelial cells .If the sputum contain too many squamous

epithelial cells (more than 10 cells per lower powered field) (100x) the specimen was

considered not useful since it is overly contaminated with oropharyngeal microorganisms

,sputum samples were evaluated in the microbiological laboratory and Gram stained by

standard technique, sputum samples were considered valuable if no more than 10

squamous epithelial cells and more than 20 neutrophils per low-power field were visible

and were considered positive for pneumonia infections.(Murray,1975; Miriam and

Buenviaje ,1988).

3-2-4:Sputum culture method:

Quantitative sputum cultures were made for each specimen according to sputum

gram stain for pneumonia infections. Sputum specimen were homogenized with an equal

volume of normal saline on a vortex mixer. Blood agar and Gentamicine Blood agar

were inoculated with 0.1 ml of homogenized specimen and spread on the plates with


۳٥

sterile swab. Plates were incubated in (5-10)% CO2 incubator overnight. (Wilson and

Martin , 1972).

3-2-5:S.pneumoniae identification methods:

S.pneumoniae identifications according to morphology staining , culture

characters and biochemical reactions as follows:-

3-2-5-1:Gram stain:

This staining method was performed according to Macfaddin ( 2000).

3-2-5-2: Optochin test:

A size disc of filter paper containing 5µg of optochin (ethylhydrocuprein) was

placed on the surface of a blood agar plate that has been spread confluently with material

from the fresh colony of suspected S.pneumoniae and incubate at 37C0 with (5-10)%

CO2. Growth of S.pneumoniae will be inhibited in a zone extending radialy for at lest

5mm from the margin of the disc.( Macfaddin, 2000).

3-2-5-3: Bile solubility test:

A suspected S.pneumoniae colony was touched with a sterile loop of 2%

sodium deoxycholate solution at pH 7.0 .The plate was incubated for 30min.at 37C0 .,the

colony of S.pneumoniae disappeared, leaving an area of α-haemolysis on the blood agar.(

Macfaddin, 2000).

3-2-5-4: Capsule staining method:

This method was used according to Soensen (1995).

1- A loopful of culture was transferred on a clean and dry slide.

2- Mixed with a loopful of nigrosin stain and allowed to air dry slowly at room

temperature. The slide was gently rinsed with water.

3- The slide was stained with methylen blue stain for 2min and allowed to air dry slowly

at room temperature. The slide was gently washed with water.

4- It was examined under oil immersion objective.

The nigrosin stain provides a dark background to un stained capsule and metylene

blue stain provides blue color to the cells.

3-2-5-5: Inulin fermentation method:


۳٦

This method was carried out according to Macfaddin (2000).

1- Isolates were inoculated into tubes of phenol red inulin broth.

2- The inoculated tubes were incubated at 37C0 for 24hours.

3- Tubes were examined for the presence of a yellow color indicative of acid formation

from the fermentation of inulin and red indicates no inulin fermentation tube .

3-2-5-6: Mice virulence test :

A volume of 0.3 ml of broth culture of S.pneumoniae contain 1x108cfu/ml was

intraperitoneal injected into two mice. Animals were observed for pneumonia onset over

6hours, the liver and lung of dead mice were removed and emulsified with normal saline

and streaked on Gentimicine blood agar plate and incubated in 37C0 at with (5-10)

%CO2 for 24houre. All plates were checked for the presence of S.pneumoniae growth

and the plates with presumptive S.pneumoniae were checked by morphology staining ,

culture characters and biochemical reactions. Two mice were injected with normal saline

intraperitoneally as control. (Sottile and Rytel, 1975).

3-2-5-7: S.pneumoniae biochemical test kit:

KBOOB HISterp Kit(India) for S.pneumoniae identification was used as

described by the manufacturer.

3-2-5-8: Serotype identification test by slide agglutination method:

Serotyping kit (Antisera) for S.pneumoniae serotype identification was used

according to the manufacturing company instructions (DENKA SEIKEN CO.,LTD)

JAPAN.

Principle:

When the reagent mixed with S.pneumoniae cells, which have antigens

corresponding to the reagent, an antigen-specific immunoglobulin reaction occurs to

produse agglutination. The reaction is macroscopically observed to determine each

serotype.

Procedure:

1- A drop of antiserum was mixed by a loop with small amount from fresh colony

2- A small amount from fresh colony was mixed with a drop of normal saline as positive

control.


۳۷

3- The slide was tilted back and forth for 1min. and observed for agglutination. 3-2-5-9: Bacterial storage:

Pure isolates of S.pneumoniae were stored at -25Coin (STGG) medium until used.( Tarja et al., 2004).

(410) Sputum samples

Ziehl-Neelsen stain(negative) Ziehl-Neelsen stain(positive) Gram stain : Neutrophil less than 20 cells Squamous epithelial more than 10 cells Gram stain : Neutrophil more than 20 cells Squamous epithelial less than 10 cells Sputum culture on Gentamicin Blood agar ( no growth) Sputum culture on Gentamicin Blood agar (growth) α- heamolysis on blood agar Gram stain(Gve+) diplococci Optochin test(+) Bile solubility test(+) Capsule stain method(+) Inulin fermentation method(+) Mice virulence(+) S.pneumoniae biochemical test kit(+) Serotype identification test Storage at -25Co


۳۸

Scheme(1):Isolation of S.pneumoniae from sputum of patient infected with respiratory tract

infection.

3-2-6:Pneumonia model in rabbits caused by S.pneumoniae:

Pneumonia model in rabbits caused by S.pneumoniae was used to determine the

most virulent bacterial isolate according to Yershov et al .,(2005).At 24 hours

S.pneumoniae colony from blood agar culture was placed in 5ml tryptone soy broth at

37C0with (5-10)% CO2 for 24 hours, centrifuged at 2500 rpm/3min. Cells were washed

with 1ml normal saline .The number of bacteria in the final suspension was determined

by standard McFarland tube kit (BioMerieux .France) by spectrophotometer at optical

density at 550nm.And confirmed by 10- fold serial tubes dilutions onto blood agar plate

and 0.5ml of freshly prepared S.pneumoniae inoclum containing 1x108 cfu/ml

intranasally layed in 1cm depth on the nasal mucosa. Rabbits were placed in upright

position for 5min. to facilitate the migration of the inoculum to the trachea. Rabbits were

observed for pneumonia onset over 24 hours after infection .The diagnostic criteria for

pneumonia were based on Dennesen et al., (2001).It is:- body temperature , animal

movement , mouth foam , animal breath , bacteremia, and histopathology. Control

rabbits were intranasally administred normal saline.

3-2-7:Antigens preparation :

3-2-7-1:Capsule polysaccharide isolation method:

Capsular polysaccharide antigen was prepared for use in immunization methods , this

method was done according to linker and Russel (1966).

Procedure:-

1- S.pneumoniae was cultured (by swab to obtain heavy growth)on Trypticase Soy Agar

for 24hours at 37C0 with (5-10)% CO2.

2- The growth was harvested by 5ml normal saline and the pH was brought to 8.5 by

10% KOH.

3- The solution was heated at 90C for one hour in a water bath.

4- The solution was cooled and acidified to pH 5 with 5N acetic acid .

5- Centrifuged at 5000 rpm / 10 min. and the pellet was discarded .


۳۹

6- Two volume of(95% ethanol containing 1% sodium acetate) was added to the

supernatant and kept at 40C for 24hours.

7- The formed precipitate was removed by centrifugation at 2500rpm/10min.and the

supernatant was taken.

8- The supernatant was redissolved in (5% sodium acetate with 0.5N acetic acid) for 24

hours at 40C.

9- Two volumes of 95% ethanol was added and kept at refrigerator temperature for 24

hours.

10- It was centrifuged at 5000rpm for 30min.

11- The supernatant was discarded and the pellet was washed with 80%, 95% and

absolute ethanol.

12- Then was dried at 37C0.

Approximately 200mg of the crude capsule polysaccharide was obtained from 20

plates.

3-2-7-1-1:Capsular polysaccharide detection methods:

1- Molisch test:

This method was recommended by Hall (1980).

1- One ml of the respected capsular polysaccharide (carbohydrate) solution was pipetted

into a test tube .

2- Three drops of α-naphthol reagent were added carefully to the solution and mixed

well.

3- One ml of concentrated sulfuric acid (H2SO4) was added carefully to the test tube

down without mixing.

4- A positive result for carbohydrate is indicated by a purple ring forming at the interface

between the denser sulfuric acid and the less dense test solution above.

2- Iodine test:

This method was proposed by Hall (1980).

1- Three ml of the respected capsule polysaccharide(carbohydrate) solution was pipetted

into a test tube .

2- Three drops of iodine solution was added and mixed gently for 1-2 min. .


٤۰

3- A positive result for carbohydrate is indicated by color change the to blue.

3- Total protein concentration calculation:

The biuret method was used to estimate the total protein concentration in the

capsule polysaccharide solution via Biolab reagent(kit) ,France, by spectrophotometric

measurement according to Gornall et al .,(1949).

Reagents:- all are ready to use.

1- R1 NaCl 75 mmol/l.

2- R2 (Biuret reagent) consist of:- NaOH 370 mmol/l , NA-k tartrate 10 mmol/ l , KI

3mmol/ l and Copper II sulfate 3 mmol/ l.

3- R3 Standard 1(Bovine Albumin 6g/dl).

Procedure:-

1- Let stand reagents and specimens at room temperature for 10min.

2- Pipetted into well identified test tube as follow:-

Reagents Blank Standard Assay

R1 1.02ml 1ml 1ml

R2 1ml 1ml 1ml

Standard 20µl

specimens 20µl

3- Mixed well and let stand at room temperature for 10min.

4- Recorded absorbance at 550nm against blank.

Calculation of results:-

Result = absorbance (assay) / absorbance (standard) × Standard concentration.

4-Capsular polysaccharide pathogencity test:

This method was recommended by Tian et al.,(2007).

A normal saline (0.3ml) containing 0.1mg of capsular polysaccharide was

intraperitoneally injected into mice ( two mice for each capsule). Mice were observed for

the over 6 hours after injection.(control mice were normal saline injected).

3-2-7-2: Heat- inactivated S.pneumoniae:


٤۱

The procedure of Benedicte(1999) was followed. The antigen was used in

the immunological method.

Procedure:-

1- S.pneumoniae fresh colony from blood agar was cultured in 5ml trypton soy broth at

37C0 with (5-10)% CO2 for 24 hours.

2- The culture was centrifuged at 3000 rpm/30 min.

3- The supernatant was discarded.

4- The bacterial cells were resuspended in 5ml PBS and recentrifuged at 3000 rpm/30

min .

5- The supernatant was discarded.

6- The bacterial cells were resuspended in 5ml PBS.

7- The bacterial cells were heat inactivated by heating in a water bath at 52C0 for

(5,10,15,20)min. and at 56C0 for (5,10,15,20)min. to chose the best time and temperature

for inactivation.

8- Centrifuged at 3000 rpm/30 min., and the supernatant was discarded.

9- The bacterial pellet was resuspended in 5ml PBS.

10-The inactivated bacteria was verified by subsequent sample plating ,which was

negative for growth.

11- The diluted inactivated bacterial suspension was counted using stander McFarland

tube kit (BioMerieux .France) by spectrophotometer at wave length 550nm.

3-2-8: Immunization protocols:

3-2-8-1: Immune protocol:

Eighteen rabbits were used for the immunization protocols (3 replicates for each

protocol in serotype 1 and 6).(Scheme 2).


٤۲

1-Protocol 1:Intramuscular 1mg capsule for serotype 1 and 6 mix with 1ml lanolin for10 days.

(3Replicates for each serotype) 2-Protocol 2:Intramuscular 1mg capsule for serotype 1 and 6 mix with 1ml lanolin for15 days.

(3Replicates for each serotype) 3-Protocol 3:Intranasal 1×108 cfu/ml of heat killed S.pneumoniae serotype 1 and 6 for 30days.

(3Replicates for each serotype)

Post each protocol

Rabbits

Mucosal (trachea) systemic (serum) Determined specific immunoglobulin titer by

Interfacial capillary tube test Scheme(2):Flow chart for determined of specific immunoglobulin titer in rabbits post three immunization protocols.(3 replicates for each protocol in serotype 1 and 6). 3-2-8-2:Immunization protocol and immune protection:

Thirty rabbits were used in the final immunization protocol (table 2) and

immune protection (table 3). After one week of last immunization dose, rabbits were

challenged with live culture of S.pneumoniae. Rabbits were observed daily for clinical

signs such as:- body temperature , activity ,breathing and bacterial infection. When

rabbits were still alive after 21 days post infection they were considered to have survived

the infection. (Yuanyi.et al.2007).(Scheme 3).

3-2-8-2-1:Table 2 final immunization protocol for 5 replicates:

Rep

licat

es

Immunization dose

sero

type


٤۳

٥ Intramuscular 1mg capsule mix with 1ml lanolin for 15 days. 6

٥ Intramuscular 1mg capsule mix with 1ml lanolin for 15 days. 1

۱۰ Total

3-2-8-2-2:- Table 3 immune protection:

Rep

licat

es

Intr

anas

al h

eter

oolo

gous

Liv

e in

fect

ion

Rep

licat

es

Intr

anas

al h

omol

ogou

s

l

ive

infe

ctio

n

T

otal

rep

licat

es

Immunization dose

Sero

type

5

1×108cfu/ml

5

3×108cfu/ml

10

Intramuscular 1mg capsule mix

with 1ml lanolin for 15 days.

6

5

3×108cfu/ml

5

1×108cfu/ml

10

Intramuscular 1mg capsule mix

with 1ml lanolin for 15 days.

1

1-Protocol 1: Intramuscular 1mg capsule for serotype 1 and 6 mix with 1ml lanolin for10 days. (3Replicates for each serotype)

2-Protocol 2: Intramuscular 1mg capsule for serotype 1 and 6 mix with 1ml lanolin for15 days. (3Replicates for each serotype)

3-Protocol 3: Intranasal 1×108 cfu/ml of heat killed S.pneumoniae serotype 1 and 6 for 30days. (3Replicates for each serotype)

4-Immune protection: 36 days post vaccination and infection.

Post each protocol

Rabbits

Mucosal (trachea) Systemic (serum) IL-6 IL-6 IL-10 IL-10 TNFα TNFα


٤٤

TNFβ TNFβ Scheme(3):Flow chart for determined of cytokines level in rabbits post three immunization protocols and post immune protection.(3 replicates for each protocol in serotype 1 and 6). 3-2-9:Samples:

3-2-9-1: Blood sample:

Blood samples were collected from all immunized and control rabbits. Five ml

blood was drawn aseptically by cardiac puncture in a disposable syringe. It was left at

room temperature till being clotted, and then were centrifuged at 3000 rpm for 5min. The

serum was aspirated from the tube and stored at -25C0 .until used.

3-2-9-2: Mucosal sample:

Mucosal samples were collected from all immunized and control rabbits after

being sacrificed by chloroform.

This method was described by Shnawa and Thwaini(2002) as follows:-

1- Trachea removed and opened using sterile and clean scissor.

2- Opened trachea was washed with normal saline .

3- The tracheal mucosa was scrapped by a sterile surgical scalpel and then it was placed

in another sterile Petri dish containing 5ml of normal saline.

4- By sterile Pasteur pipette the suspension was transferred to sterile plastic test tube.

5- The suspension was centrifuged at 4000rpm for 20min. and the supernatant was

divided in two test tube.

6- The supernatant in test tube one was stored at -25C until used for mucosal

cytokines measurements.

7- While the supernatant in the second tube was used for separation of mucosal

immunoglobulins.

3-2-10:Blood culture:

Blood cultivation was made and the presence or absence of bacteremia in

rabbits treated with live virulent S.pneumoniae and with immune protection method

according to Charles et al .,(2002) was measured as follows:-


٤٥

1- Two ml of rabbit blood was taken by heart puncher and inoculated into 18ml brain

heart infusion broth (in sterile blood culture containers)and incubated at 37C0 for

48hours.

2- Then 0.1ml of the culture was streaked on blood agar plate and incubated at 37C0 for

24hours.

3- The plate were examined for the presence of bacterial growth. The standard

identification methods for the identification of S.pneumoniae were used in case of the

presence of growth.

3-2-11: Histopathological finding:

Liver and lung were removed from treated and control mice and rabbits after

being sacrificed by Chloroform then was fixed in 20 % formalin until stained with

hematoxylin and eosin according to Bancroft and Stevens(1982).

3-2-12: Separation of serum immunoglobulin:

The method used was that of Garvey et al .,(1977):

1- To 1ml of serum, 1ml of PEG 6% was added. The mixture was left in refrigerator for

30min at 4Co .

2- It was centrifuged at 4000 rpm for 20min

3- The supernate was discarded and the precipitate was taken.

4- Five ml of formal saline & 5ml of PEG 6% was added and mixed well and left at

room temperature for 15min.

5- It was centrifuged at 3500rpm for 20min.

6- The supernatant was discarded and the pellet was dissolved in 0.25ml formal saline.

(Scheme 4).

3-2-13: Separation of mucosal immunoglobulin:

This method was performed according to Shnawa and Thwaini(2002):

1-Equal volume of PEG was added to mucosal isolate and was left for 24hours at 4C0.

2-It was centrifuged at 4000 rpm for 20min. and the supernatant was discarded .

3- The pellet was dissolved in 1ml of formal saline and stored at 4C until used.

(Scheme 4).


٤٦

Rabbit serum(A) Rabbit trachea(B)

Equal volume PEG at 4Co for 30min Normal saline

Centrifuged at 4000 rpm for 20min Shake by vortex for 5 min

Pellet Centrifuged at 4000rpm/20 min

Add (5ml formal saline with 5ml PEG) Supernatant

at room temperature for 15min

Centrifuged 3500 rpm for 20min Add equal volume of PEG at 4Co for 24houres

Pellet collection Centrifugation 4000rpm/20min

Dissolved in 0.25ml formal Pellet collection and dissolved in 1ml formal saline

Scheme(4):Flow chart for the separation of: rabbit systemic(A) and mucosal(B)

immunoglobulin.( Shnawa and Thwaini, 2000).

3-2-14:Interfacial capillary tube test for systemic and mucosal for specific

immunoglobulin titer:

The method of Garvey et al .,(1977) was adapted.

3-2-14-1: Interfacial capillary tube precipitation test for systemic specific


1- Eleven sterile test tube were placed in a tube rack.


٤۷

2- Normal saline was placed 0.9ml in a tube 1 and 0.5ml in tube 2 to tube 11.

3- Serum was added 0.1ml to tube 1 mixed and transferred 0.5ml to tube 2, mixed and

transferred 0.5ml to tube 3 ,and so on, until tube 10 and discarding. tube 11 serve as

control. The dilutions will be 1:10 to 1:2560.

4- Capillary tubes from test tube components from tube 1 to tube 11 were half filled.

5- The other half of capillary tubes was filled with prepared antigen (capsule

polysaccharide or heat inactivated bacteria).

6- The capillary tubes were Incubated at 37C0 for 24hours.

7- The results were recorded as the highest dilution that gives positive agglutination

results.

3-2-14-2: Interfacial capillary tube precipitation test for mucosal specific


1- Eleven sterile test tube were set out in the tube rack.

2- Normal saline was placed (0.25ml) in each tubes.

3- Mucosal preparation was added (0.25ml) to tube 1 mixed well and then 0.25ml was

transferred to tube 2, mixed and transferred 0.25ml to tube 3 ,and so on, until tube 10 and

discarding. tube 11 which serve as control.Dilutions will be 1:1 to 1:512.respectivly.

4- Half the capillary tubes from test tube components from tube 1 to tube 11 were filled.

5- The other half of capillary tubes was filled with the prepared antigen (capsule

polysaccharide or heat inactivated bacteria).

6- Capillary tubes were incubated at 37C0 for 24hours.

7- The result was recorded as the highest dilution that gives positive agglutination

reaction.

3-2-15:Cytokines detection:

This tests was intended for quantification of (TNFβ , TNFα , IL-6 and IL-10) in

serum and mucosal secretion of control and immunized rabbits. It is an Enzyme-Linked

Immunosorbant Assay(ELISA) of one immunological step sandwich type assay.

3-2-15-1:Interlukin-6 (IL-6) enzyme immunoassay:

This test was conducted according to the manufacturing company (Immunotech

.A Beckman Coulter Company.France) as follows:-


٤۸

1- The components of the kit were equilibrated at room temperature before use.

2- The components of the kit were prepared as follows:-

Reagents Preparation

Microtiterplates with anti

IL-6: 96 wells

Ready to use

Calibrator (Lyophilized) Adding 2ml of D.W. making final concentration of

10ng/ml.

IL-6 conjugate (Lyophilized) Adding 6ml of D.W .

Diluent's 1 6ml Ready to use (for mucosal IL-6).

Diluent's 2 (Lyophilized) Adding 6ml of D.W. (for serum IL-6).

Washing solution Dilute 50ml in 950ml of D.W.

Substrate (Lyophilized) Adding 2ml of D.W .

Stop solution ( Tracine) 5ml Ready to use

3- The standard solution and the appropriate diluents were prepared as follows:-

Standard con.( Pg/ml) IL-6 Diluents 1 or 2 (µl/ml)

1000 50µl of 10ng/ml calibrator 450

333 150µl of 1000 pg/ml calibrator 300



12.3 150µl of 37pg/ml calibrator 300

0 300

Procedure:-

- 100µl of standards , controls , samples ,was added for wells.

- 100µl of IL-6 conjugate was added per well.

- The wells were incubated for 2hours at room temperature with shaking (350 rpm).

-contents of the wells were aspirated and washed(by aspirating 0.4ml of wash solution)

three cycles by turning the plates upside-down and shaking over the sink ,at the end cycle

the inverted plates firmly tapped on to a clean filter paper .

- 200µl of standard was added to each well.


٤۹

- The plate wells were incubated for 3min. at room temperature in a dark place , with

shaking (350rpm)

- 50µl of stop solution was added to each well.

- The plates were examined at 405nm..

3-2-15-2:Interlukin-10 (IL-10) enzyme immunoassay:

This test was achieved according to the manufacturing company (BioSource

Europe S.A - Belgium) as follows:-




Microtiterplates with anti IL-10: 96 wells ready to use

Conjugate 6ml ready to use

Standards 0 to 5 (Lyophilized) for

systemic and for mucosal.

Adding 1ml of D.W . to each vial.

Solution A (Lyophilized) Adding 10ml of D.W . for mucosal.

Solution B 10ml ready to use .for serum..


Chromogen Dilute 0.2ml in 21ml of substrate buffer

Substrate buffer 21ml ready to use

Stop solution ( H2SO4) 6ml ready to use

Procedure:-

-Pipette 100µl of solution B in to standard wells.

-Pipette 100µl of solution B into serum wells.

-Pipette 100µl of solution A into mucosal wells.

-Pipette 100µl of samples and standards in each wells.

-Incubated at room temperature for 2hours with shaking at 700rpm.

-Aspirate and wash(by Aspirating 0.4ml of wash solution) three cycles by turing the

plates upside-down and shaking over the sink at the end cycle the inverted plates were

firmly tapped on to a clean filter paper .

-Pipette 100µl of solution A into each well.


٥۰

-Pipette 50µl of conjugate solution into each well.

-Incubate at room temperature for 2hours with shaking at 700rpm.

-Aspirate and wash(by 0.4ml of wash solution) three cycles by turing the plates upside-

down and shaking over the sink at the end cycle the inverted plates firmly tapped on to a

clean filter paper .

-Pipette 200µl of chromogeic solution into each wells.

-Incubate at room temperature for 30min. with shaking at 700rpm in dark place.

-50µl of stop solution was added to each wells.

-The plates were examined at 450nm.

3-2-15-3: Tumor Necrosis Factor alpha(TNFα) enzyme immunoassay:

This test was achieved according to the manufacturing company (BioSource

Europe S.A - Belgium) as follows:-




Microtiterplates with anti (TNFα). 96 wells ready to use

Standard 0 (Lyophilized) Adding 2ml of D.W . to vial

Standards 1 to 5 (Lyophilized) for

systemic and for mucosal

Adding 2ml of D.W . to each vial.

Incubation buffer 6ml Ready to use.

Anti -TNFα Dilute with 6ml conjugate buffer.

Conjugate buffer 6ml ready to use.

Chromogen Dilute 0.2ml in 21ml of substrate buffer.

Substrate buffer 21ml ready to use


Stop solution ( H2SO4) 6ml ready to use

Procedure:-

- Pipette 50µl of incubation buffer into each well.

- Pipette 200µl of standard and samples into each well.

- Incubated at room temperature for 2hours with shaking at 700rpm.


٥۱

- Aspirate and wash(by aspirating 0.4ml of wash solution) three cycles by Turing the

plates upside-down and shaking over the sink at the cycle end the inverted plates were

firmly tapped on to a clean filter paper .

- Pipette 100µl of standard 0 into each well.

- Pipette 50µl of Anti -TNFα into each well.

- Incubatedat room temperature for 2hours with shaking at 700rpm.

-Aspiratedand wash(by aspirating 0.4ml of wash solution) three cycles by Turing the

plates upside-down and shaking over the sink at the cycle end the inverted plates firmly

tap on to a clean filter paper .

- Pipette 200µl of chromogen into each well.

- Incubated at room temperature for 30min. with shaking at 700rpm in dark place.

-50µl of stop solution was added to each well.

-The plates were examined at 450nm.

3-2-15-4: Tumor Necrosis Factor beta(TNFβ) enzyme immunoassay:

This test was performed according to the manufacturing company instruction

(R, D systems ,INC. Company . U.S.A) as follows:-




Microtiterplates with antiTNFβ: 96

wells

Ready to use

Calibrator RD5-5. 25ml solution.

Substrate solution A 6ml ready to use.

6ml ready to use .

Mixed together in

equal volume. Substrate solution B

TNFβ Standard 1 (Lyophilized)(for

mucosal)

TNFβ Standard 2 (Lyophilized)(for

serum)

Reconstituted with 5ml of Calibrator RD5-5

produced stock solution 10000pg/ml. (standard) 1

(0pg/ml)


TNFβconjugate (lyophilized) Add 6ml of D.W .


٥۲

Stop solution (H2SO4) 5ml ready to use.

3- The standard solutions were prepared as follow:-

- Set up 7 test tubes.

- Pipette 800µl of calibrator RD5-5 into tube 1 and 500µl of calibrator RD5-5 into

tube 2 to 7.

- Pipette 200µl of stock solution (10000pg/ml) into tube 1.

- Pipette 500µl of tube 1 into tube 2.

- Pipette 500µl of tube 2 into tube 3. so on until tube 7.

- The standard solutions(standard 2 to standard 8) will be :- (2000 , 1000 , 500 , 250 , 125

, 62.5 , 31.2 ) pg/ml. according .

- Calibrator RD5-5 (10000pg/ml) serve as standard 1 (0pg/ml).

Procedure:-

- Add 200µl of standards and samples to each well.

- Incubate at room temperature for 2hous.

- Aspirate and wash(by aspirating 0.4ml of wash solution) three cycles by turing the

plates upside-down and shaking over the sink at the cycle end the inverted plates firmly


- Add 200µl of conjugate solution into each well.

- Incubate at room temperature for 2hous.

- Aspirate and washed(by aspirating 0.4ml of wash solution) three cycles by turing the

plates upside-down and shaking over the sink at the end cycle the inverted plates firmly


- Added 200µl of substrate solution into each well, protect from light.

- Incubate at room temperature for 20min.

- 50µl of stop solution was added to each well.

- The plates were examined at 540nm.

3-2-15-5: Calculation of results :


٥۳

Results were calculated according to equation, the principle of each standard

concentration (pg/ml) and O.D of samples according to standard curve, 4 parameters

logistic curve fitting was used to build up the standard curve. As follows:-

Table(4):TNFβ (systemic).

R = 1130.249 / 1133.536 = 0.9971 B = 1130.249 / 0.7150 = 1580.767 A = 937.5 – 1580.767 × 0.717 = - 195.909 Y* = A with B × Xi*

Table(5):TNFβ (mucosal).

Xi

(O.D

)

Yi (

pg/m

l)

Xi -

Xˉ

Yi -

Yˉ

(Xi -

Xˉ )2

(Yi -

Yˉ )2

(Xi -

Xˉ )(

Yi -

Yˉ )

√∑ (X

i - X

ˉ )2 ∑ (Y

i - Y

ˉ )2

1.381 2000 0.654 1062.5 0.4277 1128906.25 694.875

1138.619

0.819 1000 0.092 62.5 0.0084 3906.25 5.75

0.417 500 - 0.31 - 437.5 0.0961 191406.25 135.625

0.292 250 - 0.435 - 687.5 0.1892 472656.25 299.062

Xˉ=0.727 Yˉ= 937.5 ∑=0.7215 ∑=1796875 ∑=1135.312

Xi

(O.D

)

Yi (

pg/m

l)

Xi -

Xˉ

Yi -

Yˉ

(Xi -

Xˉ )2

(Yi -

Yˉ )2

(Xi -

Xˉ )(

Yi -

Yˉ )

√∑ (X

i - X

ˉ )2 ∑ (Y

i - Y

ˉ )2

1.376 2000 0.6485 1062.5 0.4205 1128906.25 689.0312

1133.536 0.806 1000 0.0885 62.5 0.0078 3906.25 5.53125

0.406 500 - 0.3115 - 437.5 0.0970 191406.25 136.2812

0.282 250 - 0.4355 - 687.5 0.1896 472656.25 299.4062

Xˉ= 0.717 Yˉ= 937.5 ∑=0.7150 ∑=1796875 ∑=1130.249


٥٤

R = 1135.312 / 1138.619 = 0.997 B = 1135.312 / 0.7215 = 1573.544 A = 937.5 – 1573.544 × 0.727 = - 206.466 Y* = A with B × Xi*

Table(6):TNFα (systemic).

Xi

(O.D

)

Yi (

pg/m

l)

Xi -

Xˉ

Yi -

Yˉ

(Xi -

Xˉ )2

(Yi -

Yˉ )2

(Xi -

Xˉ )(

Yi -

Yˉ )

√∑ (X

i - X

ˉ )2 ∑ (Y

i - Y

ˉ )2

3.985 1500 2.182 950 4.761 902500 2072.9

3243.252

2.098 500 0.295 - 50 0.087 2500 - 14.75

0.774 150 - 1.029 - 400 1.058 160000 411.6

0.356 50 - 1.447 - 500 2.093 250000 723.5

Xˉ=1.803 Yˉ= 550 ∑=7.999 ∑=1315000 ∑=3193.25

R= 3193.25 / 3243.252 = 0.984 B= 3193.25 / 7.999 = 399.206 A= 550 – 399.206 × 1.803 = - 169.768 Y* = A with B × Xi*

Table(7):TNFα (mucosal).

Xi

(O.D

)

Yi (

pg/m

l)

Xi -

Xˉ

Yi -

Yˉ

(Xi -

Xˉ )2

(Yi -

Yˉ )2

(Xi -

Xˉ )(

Yi -

Yˉ )

√∑ (X

i - X

ˉ )2 ∑ (Y

i - Y

ˉ )2

3.999 1500 2.045 950 4.182 902500 1942.75

3272.561

2.551 500 0.597 - 50 0.356 2500 - 29.85

0.878 150 - 1.076 - 400 1.157 160000 430.4

0.389 50 - 1.565 - 500 2.449 250000 782.5

Xˉ=1.954 Yˉ= 550 ∑=8.144 ∑=1315000 ∑=3125.8


٥٥

R= 3125.8 / 3272.561 = 0.955 B= 3215.8 / 8.144 = 383.816 A= 550 – 383.816 × 1.954 = - 199.976 Y* = A with B × Xi*

Table(8):IL-6 (systemic).

Xi

(O.D

)

Yi (

pg/m

l)

Xi -

Xˉ

Yi -

Yˉ

(Xi -

Xˉ )2

(Yi -

Yˉ )2

(Xi -

Xˉ )(

Yi -

Yˉ )

√∑ (X

i - X

ˉ )2 ∑ (Y

i - Y

ˉ )2

2.681 1000 1.336 629.75 1.784 396585.062 841.346

1344.940

1.582 333 0.237 - 37.25 0.056 1387.562 - 8.828

0.741 111 - 0.604 - 259.25 0.364 67210.562 156.587

0.378 37 - 0.967 - 333.25 0.935 111055.562 322.252

Xˉ=1.345 Yˉ= 370.25 ∑=3.139 ∑=576238.74 ∑=1311.357

R= 1311.357 / 1344.940 = 0.975 B= 1311.357 / 3.139 = 417.762 A= 370.25 – 417.762 × 1.345 = - 191.639 Y* = A with B × Xi*

Table(9):IL-6 (mucosal).

Xi

(O.D

)

Yi (

pg/m

l)

Xi -

Xˉ

Yi -

Yˉ

(Xi -

Xˉ )2

(Yi -

Yˉ )2

(Xi -

Xˉ )(

Yi -

Yˉ )

√∑ (X

i - X

ˉ )2 ∑ (Y

i - Y

ˉ )2

3.901 1000 2.093 629.75 4.380 396585.062 1318.066

1992.386

1.889 333 0.081 - 37.25 0.006 1387.562 - 3.017

0.990 111 - 0.818 - 259.25 0.669 67210.562 212.066

0.454 37 - 1.354 - 333.25 1.833 111055.562 451.220


٥٦

Xˉ=1.808 Yˉ= 370.25 ∑=6.888 ∑=576238.74 ∑=1978.335

R= 1978.335 / 1992.386 = 0.992 B= 1978.335 / 6.888 = 287.214 A= 370.25 – 287.214 × 1.808 = - 149.032 Y* = A with B × Xi*

Table(10):IL-10 (systemic).

Xi

(O.D

)

Yi (

pg/m

l)

Xi -

Xˉ

Yi -

Yˉ

(Xi -

Xˉ )2

(Yi -

Yˉ )2

(Xi -

Xˉ )(

Yi -

Yˉ )

√∑ (X

i - X

ˉ )2 ∑ (Y

i - Y

ˉ )2

4.598 1976 2.513 1243.25 6.315 1545670.56 3124.28

5068.654

2.588 691 0.503 - 41.75 0.253 1743.062 - 21.000

0.862 204 - 1.223 - 528.78 1.495 279608.28 646.697

0.294 60 - 1.791 - 672.75 3.207 452592.56 1204.895

Xˉ=2.085 Yˉ= 732.75 ∑=11.270 ∑=2279614.46 ∑=4954.872

R= 4954.872 / 5068.654 = 0.977 B= 4954.872 / 11.270 = 439.651 A= 732.75 – 439.651 × 2.085 = - 183.923 Y* = A with B × Xi*

Table(11):IL-10 (mucosal).

Xi

(O.D

)

Yi (

pg/m

l)

Xi -

Xˉ

Yi -

Yˉ

(Xi -

Xˉ )2

(Yi -

Yˉ )2

(Xi -

Xˉ )(

Yi -

Yˉ )

√∑ (X

i - X

ˉ )2 ∑ (Y

i - Y

ˉ )2

4.998 1976 2.591 1243.25 6.713 1545670.56 3221.260

5583.139

3.281 691 0.874 - 41.75 0.763 1743.062 - 36.489

0.992 204 - 1.415 - 528.78 2.002 279608.28 748.223

0.358 60 - 2.049 - 672.75 4.198 452592.56 1378.464

Xˉ=2.407 Yˉ= 732.75 ∑=13.674 ∑=2279614.46 ∑=5311.458

R= 5311.458 / 5583.139 = 0.951


٥۷

B= 5311.458 / 13.674 = 388.434 A= 732.75 – 388.434 × 2.407 = - 202.21 Y* = A with B × Xi*

3-2-16:Statistical analysis: Statistical analysis was made using (graph pad prism version 4) computer

software according to T. test The mean value and standard error (SE) for each value was determined. P. value less than the 0.05 level of significance was considered statistically significant.

Chapter four Results

٥۷

4-Results: 4-1:Sputum gram stain and ziehl-neelsen stain:

The sputum gram stain of the specimens revealed the presence of lymphocytes

190 specimens from a total of 410 specimens (46.34%) were positive, while then

monocyte cells were present in 99 specimens (24.14%),the 3rd group of specimens is that

with dominant neutrophils with 90 positive specimens (21.95%), the number of

specimens positive for acid fast bacilli was 31 specimens (7.56%).(Table 12).

Table(12):Direct gram stain and ziehl-neelsen stain for (410) sputum specimens from patients

infected with respiratory tract infection.

4-2: S.pneumoniae identification : Table(13):Results of identification for S.pneumoniae.

Kit

identification

Mice

virulence

test

Inulin

fermentation

test

Bile

solubility

test

Optochin

test

Capsule

stain

Gram

stain

+ + + + + + G+

diplococci

4-3:Plate culture , microscopic and biochemical characteristics of S.pneumoniae sputum:

From 90 specimens showing neutrophil cells domination ,the positive results for

culture and microscopic and biochemical characteristics for S.pneumoniae was 22

specimens only (24.444%), while 68 specimens (75.555%) were negative results for

colonies by cultivation , microscopic and biochemical characterization for S.pneumoniae

.(Table14).(Figures 1 and 2).

% Numbers of patients Dominant cells types in patients sputum post stained with direct gram stain and ziehl-neelsen stain

46.34 190 Lymphocyte (Direct gram stain)

24.14 99 Monocyte (Direct gram stain)

21.95 90 Neutrophil (Direct gram stain)

7.56 31 Ziehl-neelsen stain positive for acid fast bacilli

100% 410 Total


٥۸

Table(14):Plate culture , microscopic and biochemical characterization of S.pneumoniae sputum isolates:

A(800x) B(800x) Figure(1): Sputum gram stain showing:- A / Neutrophil cells domination without epithelial cells ( pneumonia infection). B / Domination of epithelial cells without neutrophils cells ( Saliva not sputum specimens).

A B Figure(2): Sputum gram stain(2000x) showing :- A /Sputum from patient infected with pneumonia showing:-(1)Neutrophils cells(2)S.pneumoniae ce cells. B/Sputum from patient infected with pneumonia showing neutrophils cells only without S.p bacterial cells.

100% numbers Positive colonies post culture , microscopic and biochemical characteristics.

24.44 22 Positive results for colonies, microscopic and biochemical characteristics for S.pneumoniae.

75.55 68 Negative results for colonies, microscopic and biochemical characteristics for S.pneumoniae.

100% 90 total


٥۹

4-4:Serotype identification for S.pneumoniae isolated from 22 patients infected with pneumonia:

Table(15) indicate that the most dominant serotype is serotype(6) with (8)

isolates (4 males and 4 females) represented by (36.36%) from a total of (22) isolates,

each of the serotypes (3,4,5,7) (8) isolates(5 males and 3 females) (36.36%),the second

group of serotypes were isolated was serotype( 8 , 2 and serotype 1) with (2) isolates

(9.09%) for each of them, (1 male and 1 female for serotype 8 and 2 males only for

serotype 2 and 1).

Table(15):Serotype identification of S.pneumoniae isolated from 22 specimens positive for

S.pneumoniae:

4-5:Mice virulence test:

Mice intraperitoneal (I.P) injection with 0.3ml contained 1×108 cfu/ml live

culture of S.pneumoniae was made to detect the virulence of the isolates.

The results are listed in table (16), the most virulent isolate was serotype 1 which

killed mice at 12 hours post inoculation (used two mice for each isolate), and then

(serotype 2 isolate 1) and (serotype 6 isolate 1) and (serotype 8 isolate 1) each of them

killed mice at 18 hours post inoculation.

Serotype 3,4,5,7 was represented in 8 isolates ,three of them killed mice after 18

hours and one isolate killed mice at 20 hours post inoculation, and four isolates killed

mice after 24 hours. Liver and lung culture for each infected mouse was bacteriologically

positive as well as histopathology investigations.(Figures 3 and 4).

% Female Male No. of isolates Serotype

۳٦.۳٦ 4 4 ۸ ٦

۹.۰۹ 1 1 ۲ ۸

۹.۰۹ 0 2 ۲ ۲

۹.۰۹ 0 2 ۲ ۱

۳٦.۳٦ 3 5 ۸ Other serotype (3,4,5,7)

100% 8 14 ۲۲ Total


٦۰

Table(16):Virulence test for S.pneumoniae serotypes in mice by intraperitoneal route with

1×108 cfu/ml.

++ = Two replicates. Hours= Time for killing.

Serotype1 12hours 18 hours 20 hours 24 hours Liver , Lung culture Isolate 1 ++ ++ Isolate 2 ++ ++

Serotype 2 Isolate 1 ++ ++ Isolate 2 ++ ++

Serotype 6 Isolate 1 ++ ++ Isolate 2 ++ ++ Isolate 3 ++ ++ Isolate 4 ++ ++ Isolate 5 ++ ++ Isolate 6 ++ ++ Isolate 7 ++ ++ Isolate 8 ++ ++

Serotype 8 Isolate 1 ++ ++ Isolate 2 ++ ++

Serotype (3,4,5,7) total of( 8)isolates

++ ++ ++

++

++ ++ ++ ++

All (8) isolates were positive


٦۱

A(800x) B(800x) Figure (3): A / Liver of mouse intraperitoneal injection with 0.3ml of normal saline (control). B/ Liver of mouse intraperitoneal infected with 0.3ml contained 1×108 cfu/ml of live

S.pneumoniae serotype 1 for 12 hours , showing (1) Vascular congestion (2) Cellular swelling and (3) Lysis of cells. (Haematoxylin and Eosin staining).

A(800x) B(800x) Figure (4): A / Lung of mouse intraperitoneal injection with 0.3ml of normal saline (control). B/Lung of mouse intraperitoneal infected with 0.3ml contained 1×108 cfu/ml of live

S.pneumoniae serotype 1 for 12 hours , showing inflammatory cells between alveoli (Haematoxylin and Eosin staining).


٦۲

4-6: Pneumonia model in rabbits caused by S.pneumoniae:

Rabbits were intranasally inoculated with 0.5ml containing 1× 108 cfu/ml live

culture of S.pneumoniae.

The results are listed in table (17) indicating that the most virulent serotype was

serotype 1(isolate 1 and 2) which killed rabbits at day 4 post infection (two rabbits were

used for each isolate).

Then (isolate 1 of serotype 2 , 6 and 8) each of them killed rabbits at day 6 post

infections.

The results indicates that the serotype with low virulence is isolate 8 from serotype

6 which killed rabbits at day 14.

Blood , liver and lung cultures from each of the infected rabbits was positive as well

as histopathology investigations.

As a comparison with serotype 1 rabbits rechalenged intranasal(I.N) with 3×108

cfu/ml live culture of S.pneumoniae serotype 6 to concern the virulence potency, the

results revealed that the bacteria killed the rabbits at day 4.(Figures 5 and 6).

Thus isolate 1 from serotype 1 was chosen in this study because it is the most

virulent one , and isolate 1 from serotype 6 because it is the most dominant serotype.


٦۳

Table(17):Virulence test of S.pneumoniae in rabbits by intranasal route with 1×108 cfu/ml.

++ = Two replicates.

Serotypes

Days causing death

Blood , Liver and lung culture

1 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Isolate 1 ++ ++

Isolate 2 ++ ++

2

Isolate 1 ++ ++

Isolate 2 ++ ++

6

Isolate 1 ++ ++

Isolate 2 ++ ++

Isolate 3 ++ ++

Isolate 4 ++ ++

Isolate 5 ++ ++

Isolate 6 ++ ++

Isolate 7 ++ ++

Isolate 8 ++ ++

8

Isolate 1 ++ ++

Isolate 2 ++ ++

Isolate 1 serotype 6 was rechalenged I.N with 3×108 cfu/ml to concern the virulence potency.

Serotype6

Isolate 1

++ Rabbits I.N with 3×108 cfu/ml ++


٦٤

A(800x) B(800x) Figure (5): A / Liver of a rabbit intranasal injected with 0.5ml of normal saline (control). B /Liver of a rabbit intranasal infected with 0.5ml contained 1×108 cfu/ml of live S.pneumoniae serotype 1. Four days post inoculation ,showing:-(1) Vascular congestion and (2) Cellular swilling and (3) Lysis of cells. (Haematoxylin and Eosin staining).

A(800x) B(800x) Figure (6): A / Lung of a rabbit intranasal injected with 0.5ml of normal saline (control). B /Lung of a rabbit intranasal infected with 0.5ml contained 1×108 cfu/ml of live S.pneumoniae serotype 1.Four days post inoculation showing:- inflammatory cells between and in alveoli.(Haematoxylin and Eosin staining).


٦٥

4-7: Capsule polysaccharide isolation: 4-7-1:Total protein concentration calculation:

The biuret method was used to estimate the total protein concentration in the

capsular polysaccharide solution preparation via Biolab reagent(kit) ,France, by

spectrophotometer according to Gornall et al.,(1949) there are three methods for the

isolation of the capsular polysaccharide, the results indicate that method no.1 was the best

method because it provides the least amount of protein content in the capsular

preparation(Table 18) (figure 7).

Result = absorbent (Assay) / absorbent (standard) × Standard concentration.

Total protein concentration in capsule serotype 1 = 0.011 / 3.5 x 6 = 0.018 g/dl

Total protein concentration in capsule serotype 6 = 0.015 / 3.5 x 6 = 0.025 g/dl

Table(18):Capsule polysaccharide detection parameters.

Capsule

serotype 1

Culture on

blood agar

Molisch test Iodine test Total protein

concentration

pathogenicity

test in mice

parameters _ + + 0.018 g/dl _

Capsule

serotype 6

Culture on

blood agar

Molisch test Iodine test Total protein

concentration

pathogenicity

test in mice

parameters _ + + 0.025 g/dl _

Figure(7):Molisch test (positive). (1) H2SO4 (2) α-naphthol (3) polysaccharides.


٦٦

4-8:Heat - inactivated S.pneumoniae:

Two different temperatures and three degrees for inactivated bacteria (serotype 1

and 6) at 52C0 for (5,10,15,20)min. were used and at 56C0 for (5,10,15,20)min. to chose

the best time and temperature to inactivate bacteria, the results indicate that the best time

and temperature is 52C0 for 20min, therefore it was adapted for further experiments.(Table

19).

Table(19):Heat - inactivated S.pneumoniae parameters. 52C0

Time/ min.

Gram stain

Capsule stain Cultured on blood agar.

Mice virulence .I.P challenge.

5 ++ ++ ++ Not done

10 ++ ++ ++ Not done

15 ++ ++ + Not done

20 ++ ++ _ Save

56C0 Time/ min.

Gram stain

Capsule stain

Cultured on blood agar.

Mice virulence .I.P challenge.

5 ++ ++ _ Save

10 ++ ++ _ Not done

15 + + _ Not done

20 + + _ Not done

4-9:Systemic and mucosal of specific immunoglobulin titer in rabbits post three

different immunization protocols:

The results are shown in table (20) , for protocol 1 specific immunoglobulin titer in

systemic was higher than mucosal washing in both for serotype 6 and 1 was significant at

p value = <0.0001.

Also the results indicate that for protocol 2 specific immunoglobulin titer in

systemic was higher than mucosal washing in both for serotype 6 and 1 and was

significant at p value = 0.0025 and <0.0001 respectively, and serotype 1 systemic was

higher than serotype 6 systemic was significant at p value = 0.0432.

for protocol 3 there was no significant increase in specific immunoglobulin titer

between mucosal washing and systemic response in serotype 1 and serotype 6,while there

was significant increase between specific immunoglobulin titer in serotype 1 mucosal than

serotype 6 mucosal and significant at p value =0.0022.


٦۷

Table(20):Comparison between systemic and mucosal of specific immunoglobulin titer in rabbits post three different immunization protocols for serotype 6 and 1 ( 3 replicates in each serotype and in each protocol).

* = P < (0.05). ** = P < (0.01). *** = P < (0.001). 4-10: Specific immunoglobulin titer and immune protection: 4-10-1:Specific immunoglobulin titer:

Rabbit immunization with intramuscular of capsule 1mg with lanolin 1ml for 15

days in (5 replicates for serotype 6 and 5 replicates for serotype 1).

The results shown in table (21) indicate that serotype 1 gave higher response than

serotype 6, and the statistical analysis proved that specific immunoglobulin titer in

systemic was higher than mucosal washing in both for serotype 6 and 1 with significant p

value = 0.0007.On the other hand there was significant increase between specific

immunoglobulin titer in serotype 1 systemic than serotype 6 systemic with significant p

value =0.0339.

Specific immunoglobulin titer in rabbits post intramuscular capsule 1mg mix with lanolin 1ml for 10 days.

Serotype Mean ± SE Comparison P value 6

systemic 160.00 ± 0.0003356 Serotype 6 (systemic*** / mucosal ) <0.0001 significant*** mucosal 10.667 ± 2.6667 Serotype 1 (systemic**** / mucosal) <0.0001 significant***

1 systemic 213.33 ± 53.333 Serotype 6 systemic / Serotype 1 systemic 0.3739 mucosal 16.000 ± 0.0003331 Serotype 6 mucosal / Serotype 1 mucosal 0.1161

Specific immunoglobulin titer in rabbits post intramuscular capsule 1mg mix with lanolin 1ml for 15 days.


systemic 213.33 ± 53.333 Serotype 6 (systemic** / mucosal ) 0.0025 significant**

mucosal 13.333 ± 2.6667 Serotype 1 (systemic***/ mucosal) <0.0001significant***

1 systemic 320.00 ± 0.0003356 Serotype 6 systemic/ Serotype 1 systemic* 0.0432 significant* mucosal 26.667 ± 5.3333 Serotype 6 mucosal / Serotype 1 mucosal 0.0890

Specific immunoglobulin titer in rabbits post intranasal 1×108 cfu/ml of heat killed S.pneumoniae for 30 days.


systemic 213.33 ± 53.333 Serotype 6 (systemic / mucosal ) 0.5660 mucosal 106.67 ± 21.333 Serotype 1 (systemic/ mucosal) 0.8512

1 systemic 266.67 ± 53.333 Serotype 6 systemic / Serotype 1 systemic 0.5185 mucosal 256.00 ± 0.0003356 Serotype 6 mucosal / Serotype 1 mucosal** 0.0022 significant**


٦۸

Table(21):Comparison between systemic and mucosal of specific immunoglobulin titer in rabbits post intramuscular capsule 1mg mix with lanolin 1ml for 15 days(for serotype 6 and 1)(5 replicates in each serotype).

* = P < (0.05). *** = P < (0.001). 4-10-2:Immune protection:

Rabbits were immunized with intramuscular capsular polysaccharide 1mg mix

with lanolin 1ml for both serotype 6 and 1 , after 15 days post immunization rabbits were

intranasal challenged with homo and heterologouse of live S.pneumoniae.

The results shown in table (22) indicate that immunized rabbits with (capsule 1mg

with lanolin 1ml intramuscularly for 15 days) serotype 1 provided 80% protection and in

same way serotype 6 provided 60% protection.

Also the results shown in table (23) , indicate that serotype 1 provided specific

immunoglobulin titer in systemic higher than serotype 6 systemic p= 0.0425 and specific

immunoglobulin titer in systemic was higher than mucosal washing in both for serotype 6

and 1 with significant p value = 0.0200 and 0.0032 respectively. On the other hand all

survival rabbits post infection with live S.pneumoniae were normal for body temperature ,

movements and breath (Figures 8 and 9).

Serotype Mean ± SE Comparison P value

6

systemic 224.00 ± 39.192 Serotype 6 (systemic*** / mucosal ) 0.0007(significant)***

mucosal 14.400 ± 1.6000 Serotype 1 (systemic*** / mucosal ) 0.0007(significant)***

1 systemic 448.00 ± 78.384 Serotype 6 systemic / Serotype 1 systemic* 0.0339(significant)*

mucosal 25.600 ± 3.9192 Serotype 6 mucosal / Serotype 1 mucosal 0.0694


٦۹

Table(22):Immune protection to intranasal challenge with S. pneumoniae live culture of(A) homologues and (B) heterologues.

S=Systemic M=Mucosal Table (23):Comparison between systemic and mucosal of specific immunoglobulin titer in rabbits post 36 days of immune protection for serotype 6 and 1.(3 replicates for each serotype).

* = P < (0.05). ** = P < (0.01).

Sero

type

Imm

une

dose

Cha

lleng

e do

se

No.

of r

eplic

ates

N

o. o

f pro

tect

ion

Im

mun

e st

ate

prot

ectio

n

Blo

od ,

Liv

er a

nd

Lun

g cu

lture

Liv

er a

nd L

ung

hist

opat

holo

gy

N

o. o

f die

d

Blo

od ,

Liv

er a

nd

Lun

g cu

lture

L

iver

and

Lun

g

hist

opat

holo

gy

Prot

ectio

n

A

6

6

Serotype

6 3×108

5

3

S M 2 60 %

160 16 - - Died at day 7 + + 160 16 - - Died at day 9 + + 320 8 - -

1

1

Serotype

1 1×108

5

4

S M 320 16 - - 1 320 16 - - Died at day 9 + +

80 % 320 16 - - 640 32 - -

B

6

6

Serotype1

1×108

5

0

5 0 % 3 died at day 4 + +

2 died at day 6 + + 1

1

Serotype6

3×108

5

0

5 0 % 3 died at day 4 + +

2 died at day 6 + +

Serotype

Mean ± SE

Comparison

P value

6

systemic 213.33 ± 53.333 Serotype 6 (systemic* / mucosal ) 0.0200 (significant)*

mucosal 13.333 ± 2.6667 Serotype 1 (systemic** / mucosal) 0.0032 (significant)**

1 systemic 400.00 ± 80.000 Serotype 6 systemic / Serotype 1 systemic* 0.0425 (significant)*

mucosal 20.000 ± 4.0000 Serotype 6 mucosal / Serotype 1 mucosal 0.2586


۷۰

A(800x) B(800x) Figure(8): A / Liver of a rabbit intranasal with 0.5ml normal saline (control). B /Liver of a rabbit post 36 day of immune dose and infection(immune protection).No

pathological effects.(Haematoxylin and Eosin staining).

A(800x) B(800x) Figure(9): A / Lung of a rabbit intranasal with 0.5ml normal saline (control). B / Lung of a rabbit post 36 day of immune dose and infection (immune protection). No

pathological effects.(Haematoxylin and Eosin staining).


۷۱

4-11:Cytokines detections: 4-11-1: Systemic and mucosal of cytokines levels in rabbits post three different immunization protocols:

The results show (Tables 24-25-26-27) that protocol 3 gave the highest level in

systemic and mucosal preparation of all cytokine levels , followed by protocol 2 and 1. Table(24): TNFβ levels in rabbits post three different immunization protocols.

Serotype 1 Systemic Mean ± SE Comparison P value Protocol 1 336.42 ± 10.909 Protocol 1 / Protocol 2 0.6371 Protocol 2 280.69 ± 108.80 Protocol 1 / Protocol 3*** 0.0002***

Protocol 3 620.53 ± 18.455 Protocol 2 / Protocol 3* 0.0370*

Serotype 1 Mucosal Mean ± SE Comparison P value Protocol 1 500.25 ± 7.6236 Protocol 1 / Protocol 2*** 0.0003***

Protocol 2 645.10 ± 9.6500 Protocol 1 / Protocol 3*** 0.0001***


Serotype 6 Systemic Mean ± SE Comparison P value Protocol 1 220.24 ± 12.078 Protocol 1 / Protocol 2* 0.0410*

Protocol 2 330.34 ± 35.008 Protocol 1 / Protocol 3** 0.0099**

Protocol 3 498.20 ± 59.042 Protocol 2 / Protocol 3 0.0708 Serotype 6 Mucosal Mean ± SE Comparison P value

Protocol 1 225.50 ± 12.784 Protocol 1 / Protocol 2 0.6494 Protocol 2 238.22 ± 22.560 Protocol 1 / Protocol 3*** <0.0001***


Protocol 1= Intramuscular capsule 1mg mix with lanolin 1ml for10 days. Protocol 2= Intramuscular capsule 1mg mix with lanolin 1ml for 15 days. Protocol 3= Intranasal 1×108 cfu/ml of heat killed S.pneumoniae for 30 days. * = P < (0.05). ** = P < (0.01). *** = P < (0.001). Table(25): TNFα levels in rabbits post three different immunization protocols.

Serotype 1 Systemic Mean ± SE Comparison P value Protocol 1 363.42 ± 7.8726 Protocol 1 / Protocol 2* 0.0229*



Serotype 1 Mucosal Mean ± SE Comparison P value Protocol 1 520.63 ± 12.208 Protocol 1 / Protocol 2* 0.0240*



Serotype 6 Systemic Mean ± SE Comparison P value Protocol 1 230.13 ± 20.818 Protocol 1 / Protocol 2 0.9225 Protocol 2 241.31 ± 106.05 Protocol 1 / Protocol 3*** 0.0003***

Protocol 3 502.23 ± 10.391 Protocol 2 / Protocol 3 0.0705 Serotype 6 Mucosal Mean ± SE Comparison P value

Protocol 1 241.53 ± 1.4499 Protocol 1 / Protocol 2*** <0.0001***



Protocol 1= Intramuscular capsule 1mg mix with lanolin 1ml for10 days. Protocol 2= Intramuscular capsule 1mg mix with lanolin 1ml for 15 days. Protocol 3= Intranasal 1×108 cfu/ml of heat killed S.pneumoniae for 30 days. * = P < (0.05). ** = P < (0.01). *** = P < (0.001).


۷۲

Table(26): IL-6 levels in rabbits post three different immunization protocols.

Serotype 1 Systemic Mean ± SE Comparison P value Protocol 1 316.12 ± 11.917 Protocol 1 / Protocol 2 0.6233 Protocol 2 332.20 ± 27.822 Protocol 1 / Protocol 3* 0.0253*


Serotype 1 Mucosal Mean ± SE Comparison P value Protocol 1 300.22 ± 103.45 Protocol 1 / Protocol 2 0.2230 Protocol 2 450.82 ± 14.848 Protocol 1 / Protocol 3* 0.0232*


Serotype 6 Systemic Mean ± SE Comparison P value Protocol 1 235.51 ± 21.759 Protocol 1 / Protocol 2 0.8471 Protocol 2 240.12 ± 5.3864 Protocol 1 / Protocol 3** 0.0089**


Serotype 6 Mucosal Mean ± SE Comparison P value Protocol 1 249.22 ± 6.2316 Protocol 1 / Protocol 2 0.4688 Protocol 2 260.59 ± 12.788 Protocol 1 / Protocol 3*** <0.0001***


Protocol 1= Intramuscular capsule 1mg mix with lanolin 1ml for 10 days. Protocol 2= Intramuscular capsule 1mg mix with lanolin 1ml for 15 days. Protocol 3= Intranasal 1×108 cfu/ml of heat killed S.pneumoniae for 30 days. * = P < (0.05). ** = P < (0.01). *** = P < (0.001).

Table(27):IL-10 levels in rabbits post three different immunization protocols. Serotype 1 Systemic Mean ± SE Comparison P value




Serotype 1 Mucosal Mean ± SE Comparison P value Protocol 1 430.72 ± 13.149 Protocol 1 / Protocol 2 0.6218 Protocol 2 438.20 ± 4.8705 Protocol 1 / Protocol 3** 0.0085**


Serotype 6 Systemic Mean ± SE Comparison P value Protocol 1 217.82 ± 10.467 Protocol 1 / Protocol 2 0.7575 Protocol 2 228.82 ± 31.556 Protocol 1 / Protocol 3** 0.0085**


Serotype 6 Mucosal Mean ± SE Comparison P value Protocol 1 220.11 ± 14.240 Protocol 1 / Protocol 2 0.1551 Protocol 2 249.12 ± 8.4931 Protocol 1 / Protocol 3* 0.0239*


Protocol 1= Intramuscular capsule 1mg mix with lanolin 1ml for 10 days. Protocol 2= Intramuscular capsule 1mg mix with lanolin 1ml for 15 days. Protocol 3= Intranasal 1×108 cfu/ml of heat killed S.pneumoniae for 30 days. * = P < (0.05). ** = P < (0.01). *** = P < (0.001).


۷۳

4-11-2: Systemic and mucosal of cytokines levels in rabbits post intramuscular

capsule 1mg mix with lanolin 1ml for15 days.

The results indicate (Figure 10) that serotype 1 gave systemic response with no

significant difference between TNFβ / TNFα , TNFβ / IL-6 , TNFα / IL-6 and IL-6 / IL-

10, while with significant difference between TNFβ / IL-10 (p=0.0342) , TNFα / IL-10

(p= 0.0018 ).

The results show that for serotype 1 mucosal preparation there was no significant

difference between TNFβ / TNFα and IL-6/ IL-10.

While there was significant difference between TNFβ / IL-6(p= 0.0004) , TNFβ /

IL-10(p=<0.0001) , TNFα / IL-6(p=0.0060) and TNFα / IL-10(p=0.0037).

On the other hand the results indicate (Figure 11) for serotype 6 systemic response

that there was no significant between TNFβ / TNFα , TNFβ / IL-6 , TNFβ / IL-10 and IL-

6 / IL-10.

While there was significant difference between TNFα/IL-6(p=0.0019) and TNFα

/IL-10(p=0.0305).

Also it shows that for serotype 6 mucosal preparation there was no significant

difference between TNFβ /IL-6 , TNFβ /IL-10 and IL-6/ IL-10.

However there was significant difference between TNFα / TNFβ (p=0.0027 ),

TNFα / IL-6(p=0.0007 ) and TNFα / IL-10(p=0.0001 ).


۷٤

TNFb TNFa IL-6 IL-10 TNFb TNFa IL-6 IL-10 0

100

200

300

400

500

600

700

systemic mucosalFigure(10)Cytokines levels in rabbits post intramuscular serotype 1 capsule 1mg mix with

lanolin 1ml for 15 days.

pg/m

l

TNFb TNFa IL-6 IL-10 TNFb TNFa IL-6 IL-10 0

100

200

300

400

systemic mucosalFigure(11) Cytokines levels in rabbits post intramuscular serotype 6 capsule 1mg mix with

lanolin 1ml for 15 days.

pg/m

l


۷٥

4-11-3: TNFβ levels in rabbits post intramuscular capsule 1mg mix with lanolin 1ml

for15 days.

TNFβ was at it highest level in serotype 1 mucosal than serotype 6 mucosal with

significant increase (p=<0.0001).

In addition, serotype 1 mucosal preparation also was significantly higher with

serotype 1 systemic(p= 0.0004) ,on the other hand there was no significant difference

between serotype 1 systemic preparation and serotype 6 systemic response and between

serotype 6 systemic response and 6 mucosal preparation.

TNFβ (serotype 1 , 6 systemic and serotype 1 , 6 mucosal) increased significantly

than lanolin systemic and lanolin mucosal also with significant increase than control for

both systemic and mucosal preparation.

While there was no significant increase between lanolin systemic and mucosal with

control systemic and mucosal (Figure 12).

0

100

200

300

400

500

600

700

serotype1systemic

serotype6systemicserotype1mucosal

serotype6mucosallanolin systemiclanolin mucosalcontrol systemic

control mucosal

Figure(12)TNFb levels in rabbits post intramuscular capsule 1mg mix with lanolin 1ml for 15 days.

pg/m

l


۷٦

4-11-4: TNFα levels in rabbits post intramuscular capsule 1mg mix with lanolin 1ml

for15 days.

TNFα level for serotype 1 systemically increased significantly than serotype 6

(p=0.0133), and serotype 1 mucosal preparation was increased significantly than serotype

6(p=0.0017).

Also for serotype 1 mucosal preparation increased significantly than serotype 1

(p=0.0018).

On the other hand for serotype 6 mucosal preparation was increased significantly

than serotype 6 (p=0.0192).

Also TNFα (serotype 1 , 6 systemic and serotype 1 , 6 mucosal) increased

significantly than lanolin systemic and lanolin mucosal and there was significant increase

in the treatment group than controls both systemic and mucosal.


control systemic and mucosal (Figure 13).

0

100

200

300

400

500

600

700serotype1systemicserotype6systemicserotype1mucosal

serotype6mucosal

lanolin systemic

lanolin mucosal

control systemic

control mucosal

Figure(13)TNFa levels in rabbits post intramuscular capsule 1mg mix with lanolin 1ml for 15 days.

pg/m

l


۷۷

4-11-5: IL-6 levels in rabbits post intramuscular capsule 1mg mix with lanolin 1ml

for15 days.

Figure (14) show the level of IL-6 post serotype 1 immunization systemically was

increased significantly than for serotype 6 (p=0.0314).

It also shows that serotype 1 mucosal level increased significantly than serotype 6

mucosal(p=0.0006), and serotype 1 mucosal level was increased significantly than for

serotype 1 (p=0.0198).

On the other hand there was no significant difference between serotype 6 systemic

and serotype 6 mucosal.

IL-6(serotype 1 , 6 systemic and serotype 1 , 6 mucosal) increased significantly

than lanolin systemic and lanolin mucosal level.

There was a significant increase in the treatment group than the control for both

systemic and mucosal.


control systemic and mucosal.

0

100

200

300

400

500serotype1systemicserotype6systemicserotype1mucosalserotype6mucosallanolin systemiclanolin mucosal

control systemiccontrol mucosal

Figure(14)IL-6 levels in rabbits post intramuscular capsule 1mg mix with lanolin 1ml for 15 days.

pg/m

l


۷۸

4-11-6: IL-10 levels in rabbits post intramuscular capsule 1mg mix with lanolin 1ml

for15 days.

The results indicate (Figure 15) that IL-10 level in serotype 1 immunized animals

that mucosal was increased significantly than serotype 6 mucosal(p=<0.0001).

Serotype 1 mucosal immunized animals level of increased significantly than

serotype 1 systemic(p=0.0005).

While there was no significant difference between serotype 1 systemic and serotype

6 systemic and between serotype 6 systemic and serotype 6 mucosal.

IL-10(serotype 1 , 6 systemic and serotype 1 , 6 mucosal) increased significantly

than lanolin systemic and lanolin mucosal.

Also there was significant increase in all treatment group than controls for

systemic and mucosal.


control systemic and mucosal.

0

50

100

150

200

250

300

350

400

450serotype1systemicserotype6systemicserotype1mucosalserotype6mucosallanolin systemiclanolin mucosalcontrol systemiccontrol mucosal

Figure(15)IL-10 levels in rabbits post intramuscular capsule 1mg mix with lanolin 1ml for 15 days.

pg/m

l


۷۹

4-11-7:Cytokines levels in rabbits post 36 days of immunization dose and challenge

(immune protection).

4-11-7-1:Cytokines levels for control(normal saline) and lanolin. All cytokines levels in post 36 days of immune dose and infection dose (immune

protections) increased significantly than controls (intramuscular of normal saline), and

than intramuscular of lanolin only. The most significant increase was in mucosal than

systemic it was the highest level of all cytokines(Table 28)(Figure 16). Table(28):Cytokines levels in rabbits post 36 days of immune protection compared with control and lanolin.

* = P < (0.05). ** = P < (0.01). *** = P < (0.001).

Serotype Mean ± SE TNFβ Comparison P value 1

Systemic 250.11 ± 7.6346 Serotype 1 systemic*** / control systemic 0.0002***

mucosal 378.21 ± 79.456 Serotype 1 mucosal** / control mucosal 0.0289**

6 Systemic 236.32 ± 21.829 Serotype 6 systemic** / control systemic 0.0046**

Mucosal 225.23 ± 17.293 Serotype 6 mucosal** / control mucosal 0.0052**

Control Systemic 101.15 ± 9.0582 Control Mucosal 110.25 ± 11.503

Serotype Mean ± SE TNFα Comparison P value 1 Systemic 250.42 ± 30.708 Serotype 1 systemic* / control systemic 0.0147*

mucosal 473.11 ± 2.9930 Serotype 1 mucosal*** / control mucosal <0.0001***

6 Systemic 245.21 ± 35.019 Serotype 6 systemic* / control systemic 0.0245*



Serotype Mean ± SE IL-6 Comparison P value 1 Systemic 223.50 ± 20.169 Serotype 1 systemic** / control systemic 0.0037**

mucosal 360.59 ± 4.9512 Serotype 1 mucosal*** / control mucosal <0.0001***


Mucosal 238.80 ± 4.9625 Serotype 6 mucosal*** / control mucosal <0.0001***

Control Systemic 100.62± 0.97140 Control Mucosal 93.518 ± 2.4649

Serotype Mean ± SE IL-10 Comparison P value 1 Systemic 202.60 ± 14.585 Serotype 1 systemic** / control systemic 0.0030**

mucosal 215.31 ± 23.527 Serotype 1 mucosal** / control mucosal 0.0056**





۸۰

TNF b TNF a IL-6 IL-100

100

200

300

400

500 serotype1systemicserotype6systemicserotype1mucosalserotype6mucosallanolin systemiclanolin mucosalcontrol systemiccontrol mucosal

figure(16)Cytokines levels in rabbits post 36 days of immune protection compared withcontrol and lanolin .

pg/m

l

4-11-7-2:Cytokines levels in rabbits post 36 days of immunization and challenge

(immune protection).

Table(29) shows the differences between cytokines levels in systemic and mucosal

preparation of serotype 1 and 6 immunized animals through post 36 days of immune dose

and infection dose(immune protection).

For serotype 1 systemic the results indicate that there was no significant increase

between all cytokines except between TNFβ and IL-10(p=0.0447).

On the other hand for serotype 1 immunized animals mucosal preparation there was

no significant increase between TNFβ and TNFα , IL-6, IL-10.

There was a significant increase between TNFα and IL-6 , IL-10 (p= <0.0001 ,

0.0004) respectively, also between IL-6 and IL-10(p=0.0038).

On the other hand the results shows that there was no significant increase between

all cytokines levels for serotype 6 systemic and was significant between TNFα and IL-

10(p=0.0173), and between IL-6 and IL-10(p=0.016) for serotype 6 mucosal.


۸۱

Table(29):Cytokines levels in rabbits post 36 days of immune protections.

* = P < (0.05). ** = P < (0.01). *** = P < (0.001).

Serotype 1 Systemic Mean ± SE Comparison P value TNFβ 250.11 ± 7.6346 TNFβ / TNFα 0.9927 TNFα 250.42 ± 30.708 TNFβ / IL-6 0.2848 IL-6 223.50 ± 20.169 TNFβ* / IL-10 0.0447* IL-10 202.60 ± 14.585 TNFα / IL-6 0.5044 TNFα / IL-10 0.2323 IL-6 / IL-10 0.4483 Serotype 1 Mucosal Mean ± SE Comparison P value TNFβ 378.21 ± 79.456 TNFβ / TNFα 0.2986 TNFα 473.11 ± 2.9930 TNFβ / IL-6 0.8356 IL-6 360.59 ± 4.9512 TNFβ / IL-10 0.1207 IL-10 215.31 ± 23.527 TNFα*** / IL-6 <0.0001*** TNFα*** / IL-10 0.0004 *** IL-6** / IL-10 0.0038 ** Serotype 6 Systemic Mean ± SE Comparison P value TNFβ 236.32 ± 21.829 TNFβ / TNFα 0.8399 TNFα 245.21 ± 35.019 TNFβ / IL-6 0.5977 IL-6 219.51 ± 19.647 TNFβ / IL-10 0.2269 IL-10 199.68 ± 13.530 TNFα / IL-6 0.5569 TNFα / IL-10 0.2919 IL-6 / IL-10 0.4526 Serotype 6 Mucosal Mean ± SE Comparison P value TNFβ 225.23 ± 17.293 TNFβ / TNFα 0.6576 TNFα 233.55 ± 1.9629 TNFβ / IL-6 0.4927 IL-6 238.80 ± 4.9625 TNFβ / IL-10 0.2696 IL-10 200.76 ± 8.1431 TNFα / IL-6 0.3809 TNFα* / IL-10 0.0173 * IL-6* / IL-10 0.0163 *

Chapter five Discussion

۸۲

5-Discussion: 5-1: Streptococcus pneumoniae isolation and identification:

5-1-1: Sputum gram stain:

The present results indicate (Table 12)(figure 1 and 2) that the sputum

gram stain of the specimens revealed the presence of neutrophils with 90

positive specimens from a total of 410 specimens (21.951%).This means that

the infection is in acute stage (Chandler et al.,2000).

Clinicians are interested in rapid, simple, inexpensive, and readily

available tests that will assist them in prescribing proper medications for life-

endangering infections and, in the present era of prospective payment, will

guide them in the selection of cost-effective treatments (Tarja,2006).

For management of pneumonia, the sputum Gram strain has

traditionally served this function, when comparing it with conventional culture

techniques as the reference standard to determine the cause of pneumonia

(Hahn and Beaty,1970;Ries et al., 1974).

The presence of a large number of neutrophils shown by gram staining and

few of epithelial squamous cells indicates that the sputum specimen was of

good quality for pneumoniae infection (Miriam and Buenviaje ,1988;James et

al.,2001).

Several studies have suggested the greater value and reliability of the

sputum gram's stain in the etiologic diagnosis of pneumonia(Chandler et

al.,2000).These studies generally have been limited to the microbiology

laboratory, where standards such as sputum gram stain ,blood cultures, refined

sputum culture techniques, or serological studies have been used to assign

final diagnosis(Pratter and Irwin.,1995 ).


۸۳

5-1-2: plate culture , microscopic and biochemical characteristics of

S.pneumoniae:

The results show table(13) the identification for S.pneumoniae isolate

from human infected with pneumonia.

To enhance the diagnostic value of a sputum sample and to preclude

assessment of respiratory secretions contaminated by or pharyngeal flora,

microbiologists, infectious disease consultants, and pulmonary disease

specialists recommend that only valid respiratory secretions be processed

(Murray and Washing,1975). Despite the clinical importance and frequent

isolation of S.pneumoniae, there is no one “gold standard” or reference

protocol for its identification, laboratory identification of this pathogen has

been accomplished using one or more assays, including Gram stain

morphology, capsule stain ,colony morphology, hemolysis on sheep blood

agar, optochin susceptibility, solubility in deoxycholate(bile),miniaturized

manual systems such as the API 20 Strep and reaction with specific antisera,

system (Reed et al.,1996; James et al.,2001).Although identification tests

based on phenotypic characteristics of pneumococcus are reliable and

commonly used to identify pneumococcus( Ruoff et al.,2003).Bile solubility

and optochin sensitivity have shown to have almost complete correlation, but

it could not be used as the only protocol to differentiate pneumococcus from

other streptococci (Bayram and Kocoglu,2006).

However, Arbique and coworkers suggest that the optochin, bile

solubility and capsule stain test differentiates S.pneumoniae from other gram

positive bacteria (Arbique et al.,2004).The sputum gram stain and also the

culture were equally helpful in a different sense by indicating which bacteria


۸٤

were the etiological agents of the pneumonia (S.pneumoniae or other bacteria)

(Suzuki et al.,2005).

Therefore it was found in this study that from 90 specimens infected

with bacterial pneumonia only 22 specimens contains S.pneumoniae

(24.444%)(Table 14),that is refer to that other 68 (75.555%)etiological of

pneumoniae was other microorganisms than S.pneumoniae .

5-2: Serotype identification for S.pneumoniae isolated from 22 patients suffering from pneumonia:

Our study showed (table 15) that the most dominant serotype is serotype

6 with 8 isolates(36.36%) then serotype 1,2,8 with 2 isolates to each of

them(9.09%) while other serotypes were 3,4,5,7 with total number of 8

isolates(36.36). These serotypes distribution is similar to the distribution

reported by Twum- Danaso in Riyadh, Saudi Arabia, for the period 2000 and

2001 when he reported that the most dominant serotype distribution was

serotype 6 ,8 ,2 and 3 respectively(Twum-Danso et al.,2003). However,

another study covering three major provinces of Saudi Arabia, including the

central province where Riyadh is located, during 2000 reported a different

distribution of serotypes (Memish et al.,2004).In that study, serotypes 6, 3, 1,

and 2 were the most frequently isolated serotypes.

The present results are in agreement with Eiman et al.,(2008) when she

reported that the most dominant serotype distribution in Kuwait was serotype

6 , 3 ,2 and 8 respectively. Another report by Yenisehirli and Sener (2003) that

is concordant with ours is from Turkey by the fact that the predominant

serotypes were 6,3 and 8 respectively. While Sleeman et al.,(2001)in a study

in England and Wales found a slightly different order of predominance of the


۸٥

serotypes which were 1,2,3,8 and 6 respectively, our results are not in

agreement with these results.

The serotype distribution in many regions depend on narrow

geographical area therefore when people travel frequently between these

countries then facilitates the spread of these serotypes.

5-3: S.pneumoniae virulence:

A comparison was made between all 22 isolates serotypes to found the

most virulent one, our results proved that the most virulent isolate was

serotype 1 which killed mice at 12 hours post intraperitoneal challenge (Table

16)(figures 3 and 4), and killed rabbits at 4 days post intranasal challenge

(Table 17) (figures 5 and 6) with 1×108 cfu/ml live culture of S.pneumoniae.

The present results are in agreement with both Azoulay et al., (2000)

and Abdulrahman et al., (2005), when they reported that serotype 1 was the

most virulent serotype between many serotypes isolated (6,3and8).

The CPS of S.pneumoniae is the single most important virulence factor

of this organism (Gail et al.,2001). The polysaccharide capsule of

S.pneumoniae can be used to define of 8 pneumococcal serotypes, in addition

to acting as the primary virulence determinant for this organism, some

serotypes are mostly associated with nasopharyngeal carriage, whilst others

are more likely to be the causative agents of invasive disease (Hausdorff et al.,

2005; Alannee et al., 2007).Serotype 1 ranks among the most prevalent

invasive serotypes in many countries (Porat et al., 2001 ;Konradsen and

Kaltoft 2002 ; McChlery et al., 2005;Garcia et al., 2006). Serotype 1 has

specific epidemiological features including:- a low colonization frequency,

even in populations in which serotype 1 is a frequent cause of pneumococcal


۸٦

infections (Normark et al., 2001 ;Laval et al.,2006; Nunes et al., 2008) and the

ability to cause outbreaks in communities and in crowded and closed

institutions (Dagan et al., 2000; Leimkugel et al., 2005) then an association

with severe episodes of pneumonia (Byington et al., 2006).

Recently, some authors have speculated that isolates of serotype 1,

which are known to have a highly invasive potential, behave as primary

pathogens, whereas other capsular types demonstrate opportunistic features

(Garau and Calbo, 2007; Hausdorff, 2007).

A common factor in these reports was the low genetic diversity among

the isolates, which has been associated with the short duration of carriage

and/or a low density of this serotype in the nasopharynx, resulting in a reduced

opportunity to exchange genes between strains (Brueggemann and

Spratt,2003; Hausdorff et al., 2005).

It is possible that the association of virulence and time to death with

capsular type occurred because the different CPS endowed pneumococci with

substantially different virulence properties. An alternative explanation was that

there were chance association between different capsular types and genes

responsible for different virulence phenotypes.

The essential role of the capsule in S. pneumoniae virulence was

established through the study of spontaneous mutants, the genetic transfer of

capsular serotypes, and the use of a type 1-specific depolymerase to remove

the capsule prior to infection (Gail et al.,2001).

The chemical structure of the CPS and, to a lesser extent, the thickness

of the capsule determine the differential ability of serotypes to survive in the

blood stream and possibly to cause invasive disease (Alannee et al., 2007).On


۸۷

the other hand pneumococci adhere to nasopharyngeal cells, to lung vascular

endothelial and bronchoepithelial cells, and also to lung resting pneumocytes

via lectin-like interactions or protein-protein interactions, it has been suggested

that pneumococci bind to at least two different cell surface sugar moieties on

noninflamed pulmonary epithelia and vascular endothelia , and the receptors

involved are known to be different for nasopharyngeal cells and for lung cells,

likewise, the platelet-activating factor receptor (PAFr) has been suggested to

serve as a ligand in the lung, whereas the polymeric immunoglobulin receptor

has this role in the nasopharynx, the pneumococcal adherence mechanisms of

resting and activated respiratory cells also differ, however, with the platelet-

activating factor receptor implicated only in activated tissue cells ,it seems,

therefore, that the pneumococcus has a wide range of molecules to utilize for

colonization, but the identity of the host ligand used varies with the

environment (Ogunniyi et al.,2000).

The pneumolysin is essential if the pneumococcus is to successfully

exploit the host tissue cell ligands in the nasopharynx and trachea, as well as

the lung, thus, pneumolysin must be added to the collection of pneumococcal

factors that have been suggested to regulate adherence, including the opacity

locus, permeases, and cell wall structural proteins, although this toxin avidly

binds to eukaryote membranes, it seems unlikely that it acts directly as an

adhesion (Feldman et al.,2002).

The low responses in mice and rabbits to serotype 6 antigens might be

due to the lack of appropriate V genes encoding CPS serotype 6-specific Ab,

since mice and rabbits display strong immune responses to similar antigens of


۸۸

pneumococcal serotypes 1, 2 and 3. Another possible explanation could be that

serotype 6 antigens are rapidly cleared by a mechanism involving recognition

of CPS serotype 6 by lectin-like molecules. This is also suggested by the low

virulence of serotype 6 pneumococci in mice (Alonso et al.,1994).

The conclusion that the overall context of the strain determines the

importance of individual factors is supported by our investigation of the role of

capsule in colonization, in addition to pneumolysin, differences in

pneumococcal serotype also influenced upper and lower respiratory tract

colonization.

5-4: Specific immunoglobulin titer enhancing by three immunization protocols.

In this study we used three different protocols were used to enhance

specific immunoglobulin titer in the rabbits. The results (Table 20) indicate

that protocol 2 was the best protocol for enhancing systemic specific

immunoglobulin and protocol 3 was the best protocol for enhancing mucosal

specific immunoglobulin in the rabbits. Also the results shows that serotype 1

was more enhancing of specific immunoglobulin level from serotype 6 (Table

21).

Carbohydrates are components of several surface molecules including

lipopolysaccharides, teichoic acids and lipoteichoic acids, peptidoglycan and

glycoproteins that are expressed by Gram-negative bacteria and/or Gram-

positive bacteria .CPS consist of several hundred repeating units, and these

repeating units contain one to eight sugars that are usually linked by glycosidic

bonds. Variations in sugar composition, ring forms, linkage positions,


۸۹

anomeric-centre configurations, isomer forms and conformation all contribute

to differences in the immunological epitopes that are present. These variations

result in the generation of the huge diversity of structures that interact

specifically with the immune system(Upreti et al.,2003).

Studies that were carried out several decades ago in mice showed that

carbohydrates are T-cell-independent antigens(Barret,1985).Purified

polysaccharides induce specific IgM responses, without a detectable IgG

response. A failure to induce immunoglobulin class switching from IgM to

most IgG isotypes (excluding IgG3) and a lack of increased antibody

production after rechallenge with antigen are hallmarks of a classic T-cell-

independent immune response. The conjugation of polysaccharides to proteins

seems to allow carbohydrate-specific responses that elicit T-cell help (Lee et

al.,2001).Stimulation of the B cell (with consequent production of

carbohydrate-specific antibody) and activation of the peptide-recognizing

CD4+ T cell result in T-cell help,Th2 helper which promotes immunoglobulin

class switching to IgG and memory responses. Immunoglobulin class

switching and B-cell memory depend on co-stimulation of the B cell through

CD80 and/or CD86 interacting with CD28, through CD40 interacting with

CD40L and perhaps through other interactions between co-stimulatory

molecules. Bacterial polysaccharides are classic antigens for B cells and are

recognized by a B-cell receptor (BCR) that has the correct specificity,

interaction between the polysaccharide and the BCR is sufficient to induce the

signals that are required to stimulate clonal expansion of B cells and antibody

production. However, this pathway by itself does not result in immunological

memory. Polysaccharide – adjuvant interact with a BCR in a similar manner


۹۰

to pure polysaccharides; however, in addition, they elicit T-cell help, through

antigen presentation of the protein component to CD4+ T cells, which provide

the necessary co-stimulation to induce memory B cells and memory T cells.

Therefore, antibody production is achieved, and the consequent

immunological memory results in antigen-specific immunity to the

polysaccharide. This strategy has been exploited to produce pathogen-specific

vaccines that target bacterial polysaccharides.(Sarkis and Dennis,2006).

Host defense mechanisms against S.pneumoniae largely depend on

phagocytosis following opsonization by polysaccharide-specific

immunoglobulin and complement (Balachandran et al.,2002).Since

colonization in the respiratory mucosa is the first step in pneumococcal

pathogenesis, mucosal immune responses may play a significant role. In

addition to inducing systemic immune responses, mucosal vaccination with or

without an effective adjuvant has the advantage of inducing mucosal IgA

antibodies (Kutuna,2005).The important role of type-specific anticapsular

antibodies in host defense against pneumococcal infections is well established,

the early successes of inducing type-specific protective antibodies gave rise to

numerous studies dealing with anticapsular antibodies and the development of

pneumococcal CPS vaccines (Austrian,1979). It has been suggested that

antibodies to the pneumococcal cell wall are not opsonic and fail to protect

mice from infection (Larry et al.,1984).

However, there have been occasional reports of protective antibody

specific for noncapsular pneumococcal antigen , more recently, it has been

demonstrated that monoclonal antibodies and C-reactive protein directed

against phosphocholine ,a cell wall determinant of pneumococcal C-


۹۱

carbohydrate can protect mice from fatal infections with several types of

S.pneumoniae (Yother et al.,1982).

The primary virulence factor of the pneumococcus is the CPS that

surrounds the organism ,the difference in each CPS lies in the composition and

linkage of its sugars, the CPS plays major roles in both colonization and

invasive disease, non-encapsulated strains of S. pneumoniae are unable to

colonize the murine nasopharynx, while stains that produce even 20% of the

normal amount of capsule are still able to colonize as well as the wild type,

therefore, colonization appears to be serotype independent and capsule

dependent (Magee and Yother, 2001). Conversely, the ability to cause invasive

disease is dependent on both the serotype of S. pneumoniae and the presence

of a capsule. Bender and Yother shown that serotype two and three mutants

that have deficiencies in capsule production are a virulent, unlike their highly

virulent parental strains (Bender and Yother, 2001; Hardy et al., 2001).In

addition, serotypes 1 and 2 are both heavily encapsulated strains; therefore,

they are highly invasive (Bruyn et al., 1992).

Further molecular studies revealed that the immune response to CPS is

generally only oligoclonal at the individual level. Lucas et al., (2001)

demonstrated that while 2 volunteers used capsule polysaccharide serotype 3

(CPSS3)-specific variable heavy (VH) and light (VL) segments (VH/VL) pairs

that were identical within each volunteer (VH3-23/Vκ3-15 and VH3-15/Vλ1-

51, respectively), the V genes( found in light chain of antibody) were not

related between the volunteers. Moreover, a larger study performed by Zhou et

al., (2004) using 55 Fab-fragments specific for CPSS1 confirmed that

oligoclonality was found within individuals. Of their six donors, four of them


۹۲

exclusively utilized one specific VH/VL pair, while the other two used 2 pairs

and 5 pairs. Despite this high level of restriction and large sample size, none of

the donors used the same VH/VL pair in response to CPSS2 (Zhou et al.,

2004). In contrast, the VH/VL response to CPSS1 is more conserved at the

population level. Another study by Zhou et al., (2002)showed that two VH/VL

pairs, VH3-23/A23 and VH3-30/L6, were used in three out of seven

volunteers in response to CPSS1 . These results demonstrate the difficulty in

studying the V gene repertoire in response to CPS antigens, as the

characteristics of each repertoire may vary by serotype.

5-5: Immune response to heat killed S.pneumoniae:

The study results indicate that protocol 3 is the best protocol for

enhancing high level of specific immunoglobulin titer in mucosal than other

protocols (Table 20).

Colonization with heat killed pneumococci induced increased levels of

antipneumococcal serum IgG (and mucosal IgA).This serum IgG response

could account for the observed protection from systemic infection and offers

the possibility of long-acting immunity.Trzcinski et al.,2005). To avoid the

risk of live vaccines, the use of killed organisms as vaccine has been

introduced. These vaccines are made from the entire organism, killed by

heating or by adding chemicals such as formaldehyde to make them harmless.

This renders the microbes incapable of causing disease, but preserves some

immunogenic properties of the microorganisms, so that they are still able to

stimulate the immune system. It is a relatively crude approach. The limitations

of these kinds of vaccines are that they are not as potent as live vaccines

(Qazi,2005).


۹۳

Most pathogens enter the host through the mucosal membranes and

seem to induce a local mucosal immune response, mainly represented by

secretory IgA (Brandtzaeg,1995). Studies of carriage of pneumococci in the

upper respiratory tract have shown that such carriage may induce anti-

pneumococcal antibodies (Gwaltney et al.,1975).

In preliminary studies with mice, it was possible to show that a

preparation of whole heat-inactivated pneumococci was immunogenic when

applied to mucosal surfaces and that the nasal mucosa may be the preferred

site for antigen delivery (Aaberge et al.,1995). It has also been shown recently

that nasal immunizations with pneumococcal surface protein A could induce

immunity with the power to protect against challenge with pathogenic

organisms (Wu et al.,1997).Malley et al.,(2001) have previously shown that

intranasal vaccination with killed whole pneumococci given in multiple doses

with or without an adjuvant generates protection from colonization in a

serotype independent manner

In this study, it has been shown that whole heat-inactivated

pneumococci can induce both systemic and mucosal antibodies when applied

by intranasal .

The induction of systemic immunity to pneumococci via the nasal route

suggests that the nasopharyngeal mucosa possesses the necessary structures to

make mucosal immunizations a realistic alternative to the use of potential

vaccines (Brandtzaeg and Haneberg,1997).The superiority of nasal versus

oral, gastric, and rectal routes of antigen presentation was confirmed by the

demonstration of specific IgA antibodies in serum and samples representing

secretion.


۹٤

Furthermore, only intranasal immunizations, in addition to rectal antigen

delivery, induced significant increases in intestinal IgA antibodies, as reflected

in extracts of feces. This was surprising, considering the fact that neither oral

nor gastric immunizations with the same antigen were able to induce

significant increases in such intestinal antibodies. The lack of intestinal

antibodies after oral and gastric immunizations indicates that induction of

intestinal antibodies after intranasal immunization was not due to swallowing

or leakage of antigen from the nose into the intestines. The stimulus for

antibodies to be produced locally in the gut is therefore suggestive of a cellular

link between the nasal induction site and the intestinal effectors site (Haneberg

et al.,1998).

IgG as well as IgA antibodies to pneumococci in lung lavage fluid,

especially after nasal immunization, might indicate that both these antibodies

have a barrier function against invasive pneumococci. pulmonary IgA

antibodies to pneumococci correlated with such antibodies in saliva indicates

that at least some of the IgA is produced locally in the lungs to contribute to

this presumed surface protection. It seems, therefore, that the IgA antibodies in

saliva reflect the IgA antibodies in the lung secretions and that analyses of

salivary IgA would be sufficient for evaluation of mucosal airway antibodies

(Benedicte,1999).

BALB/c mice with CPS, conjugate vaccine, or heat-inactivated

pneumococci also seems to induce serum IgM and no IgG antibodies (Aaberge

et al,.1993; Hvalbye et al.,1995; Aaberge and Løvik,1996).In other strains of

mice, however, IgG antibodies can be induced after parenteral immunization

with a pneumococcal conjugate vaccine, and low levels of IgG antibodies may


۹٥

even be induced in mice after immunization with polysaccharides alone

(Hvalbye et al.,1995).

5-6: Capsule polysaccharide of S.pneumoniae and immune protection:

The present results indicate that when rabbits are immunized

intramuscularly with CPS serotype 1 mix with lanolin (as adjuvant) for 15

days before intranasal infection with live culture of S.pneumoniae provided

80% protection while serotype 6 provided 60% protection (Table 22). And

serotype 1 systemic was significant increase than serotype 6 systemic p=

0.0425 (Table 23).

Colonization of the upper respiratory tract (URT) is a step prior to S.

pneumoniae infection (Bogaert et al.,2004).

Most carriage episodes are asymptomatic and last on the order of weeks

to a few months (Hogberg et al.,2007). In principle, colonization may be

prevented or terminated by the innate and/or adaptive immune systems, or by

competing microbial flora, yet the particular host and pathogen factors

affecting resistance to pneumococcal colonization are still poorly

understood(Van Rossum et al.,2005).

The successes of anti-pneumococcal therapy using passive transfer of

serotype-specific antibodies, and of vaccinations that depend on anticapsular

antibodies showed the importance of humoral immunity as one mechanism of

protection against colonization and disease from S. pneumoniae for some, but

not all serotypes, such immunity appears to play a role in naturally acquired

protection (Weinberger et al.,2005).

Protection against S. pneumoniae is mainly provided by Abs to CPS

(Macleod et al.,1945). Therefore, it has become of extreme importance to


۹٦

understand the mechanism by which protection to CPS is provided by the

immune system.

The Ab response to CPS is considered thymus independent (TI) because

neonatally thymectomized mice and a thymic nude mice are able to mount an

Ab response similar in magnitude to that of conventional thymus-bearing mice

(Humphrey and East,1964;Baker et al,.1973).

The immune response to TD Ags critically depends on the interaction

between CD40L, a molecule which is transiently expressed on the surface of

activated CD4-T lymphocytes, and CD40 expressed on the surface of B

lymphocytes . This interaction activates the resting B lymphocyte to produce

Abs to TD Ags and is important for germinal center creation, memory B

lymphocyte formation, and Ig class switching (Noelle et al.,1991).

The question whether the CD40-CD40L interaction plays a role in the

immune response to TI-2 Ags in general and pneumococcal polysaccharides in

particular remains unsolved. There are contradictory data with regard to the

role of CD40L in the immune response to TI-2 Ags after immunization with

an intact extracellular microorganism or with a polysaccharide-adjuvant. Wu

et al.,(1999) using CD40L knockout mice, reported that the IgG response

against phosphorylcholine (a TI-2 Ag) in mice immunized with a non

encapsulated variant of S. pneumoniae is T lymphocyte and CD40L

dependent, and his conclusions was that neutralization of CD40L reduced the

Ab response to pneumococcal CPS elicited by immunization with whole

bacteria or with a CPS-adjuvant and that the immune response to a CPS-

adjuvant was higher in wild-type mice than in CD40L-deficient.


۹۷

Some pathogenic bacteria form thick capsules that both block immune

responses through inhibition of complement deposition and phagocytosis and

stimulate a weak response resulting from a lack of T-cell involvement.

Contrary to this model, CPS from 23 serotypes of S.pneumoniae have been

successfully used in a multivalent vaccine in the absence of a carrier protein.

Furthermore, serotype 1 pneumococcal polysaccharide (Sp1) has been shown

to activate T cells in vivo and in vitro via an uncharacterized mechanism.

Velez et al., (2009) in his study report that Sp1 utilizes the major

histocompatibility complex (MHC) class II pathway in antigen-presenting

cells (APCs) for processing and presentation. APCs internalize and process

Sp1 through a nitric oxide-dependent mechanism and, once inside the cell, it

associates with MHC class II proteins in an H-2M-dependent manner that

leads to in vivo T-cell activation. These results establish that Sp1 activates T

cells which can lead to abscess formation in mice through an H-2M-dependent

polysaccharide antigen presentation pathway in APCs, potentially contributing

to pneumococcal polysaccharide vaccine efficacy through the recruitment of

T-cell help.

5-7:Capsule polysaccharide and heat killed S.pneumoniae activated

cytokines production:

The results show (Tables 24-25-26-27) that protocols 2 and 3 gave the

highest level in systemic and mucosal preparation of all cytokines levels ,then

protocol 1.And the results show that protocol 2 enhancing high levels of all

cytokines in mucosal and systemic but the cytokines levels in mucosal site

were significant increase than in systemic for serotype 1 and 6 (Figures 10 to

15).


۹۸

Tumor necrosis factor-alpha, synthesized by activated

monocyte/macrophage Cells (Pennica et al.,1984) and TNF-beta originating

from stimulated T cells (Gray et al.,1984) reveal a 36% identity and 51%

homology in the overall amino acid sequence share common receptor binding

domains and exert similar cytolytic and cytostatic effects on various tumor

targets in vitro and in vivo. The roles of both TNF species have been described

to extend to modulation of hematopoiesis, by sharing similar inhibitory effects

on colony growth of granulocyte-macrophage, erythrocyte, and granulocyte-

erythrocyte-megacaryocyte- macrophage precursors (Pennica et al.,1984).

In patients with unilateral pneumonia, much higher cytokine

concentrations have been found in bronchoalveolar lavage fluid obtained from

the infected lung than in lavage fluid from the uninvolved lung or in plasma

(Boutten et al.,1996). This suggests that during clinical pneumonia cytokines

are produced at the site of the infection. Mouse studies have indicated that

locally produced cytokines are required for an effective host defense against

bacterial pneumonia (Greenberger et al.,1995).

Capsular polysaccharides of Gram-positive organisms are known to

trigger inflammatory cytokine release (Soell et al.,1995) and with S.

pneumoniae the more virulent the serotype the higher the level of secreted

cytokine (Arva and Andersson,1999).Tumour necrosis factor (TNF) levels as

high as those induced by lipopolysaccharide (LPS) in Gram-negative bacteria

have been reported (Simpson et al.,1994). In one such study, it was found that

100–1000 fold more Gram-positive organisms were needed than Gram-

negative to induce the same concentration of cytokine IL-1β and IL-6 release

(Chow et al.,1999). However, this did not correlate with the severity of


۹۹

disease, indicating that different mechanisms are responsible for Gram-

positive and Gram-negative sepsis. β2 integrins and TLR2 have been

implicated in cytokine responses in Gram-positive infections, whereas CD14

and TLR4 are instrumental in Gram-negative (Matsuguchi et al.,2000).

Systemic responses to bacterial antigens results from interaction of multiple

plasma factors (immunoglobulins, complement, binding proteins) and multiple

cell-surface receptors (Fc receptors, scavenger receptors, TLR and CD14. In

this study we have used the measurement of the cytokines IL-6 and TNF as an

indicator of the overall pattern of proinflammatory cytokines release and IL-10

as an indicator of the overall pattern of antiinflammatory cytokine after

stimulation with an heat killed bacteria and CPS.

Tumor necrosis factor is a central mediator of the inflammatory

response and yields prognostic value in septic patients, whereas the

contribution of anti-inflammatory cytokines, such as IL-10, to an adverse

outcome is controversial (Latifi et al.,2002).

Interleukin-10 able to counterbalance the potentially harmful

inflammatory effects of TNF-α and other proinflammatory molecules.

However, it has recently been proposed that IL-10 excess is able to induce

immunosuppression in bacterial sepsis and increases mortality by impairing

bacterial clearance in pneumococcal pneumonia (Van der Poll et al.,1996).

Large interindividual differences in the degree of TNF-α and IL-10

inducibility have been observed. Single nucleotide polymorphisms, especially

the biallelic TNF-α-308 gene promoter, the lymphotoxin-α , and the IL-10–

1082 gene promoter polymorphism, have been associated with different

cytokine production (Louis et al.,1998).


۱۰۰

Epidemiologic family studies have shown a genetic predisposition for

infection-related mortality. Susceptibility for invasive pneumococcal disease

has been associated with the mannose binding lectin gene, but no genetic

linkage has been found for sepsis severity (Roy et al.,2002).

Several authors showed increased IL-10 blood levels in patients with

severe sepsis or septic shock (Rodriguez-Gaspar et al.,2001), but an influence

of IL-10 polymorphisms on the severity of sepsis has not been evaluated. In

community-acquired pneumonia, the risk for septic shock has been associated

with the TNF-α polymorphism (Waterer et al.,2001).

The innate immune response to severe bacterial infections is

orchestrated by the proinflammatory cytokines TNF, IL-1, IL-6, and IL-8

(Bone et al.,1997). An anti-inflammatory reaction involving IL-10 parallels

the excessive production and may induce a state of immunosuppression in

patients with sepsis. There is considerable evidence in the literature that IL-10

can either improve or worsen health status depending on the model evaluated.

Mice lacking any IL-10 response due to an inherited deficiency have higher

lethality due to septic shock, possibly because of uninhibited proinflammatory

reactions (Latifi et al.,2002). In contrast, treatment with exogenous IL-10 in a

model of pneumococcal pneumonia led to an impaired host defense (Van der

Poll et al.,1996). IL-10 influences the immune response via downregulation of

proinflammatory cytokine release and inhibition of class II major

histocompatibility complex expression, resulting in impaired bacterial

clearance (Zanotti and Kumar,2002 ).

In addition, IL-10 influences adaptive immunity in sepsis via

downregulation of monocyte expression due to intracellular sequestration


۱۰۱

(Fumeaux and Pugin,2002). these data indicate that IL-10 is necessary to

counterbalance proinflammatory reactions.

The immune response is not only dependent on the structure of the

antigen but on its size too. Thus, it is possible that CPS could behave

differently from the particulate form found in the bacteria. To test this we

bound CPS onto lanolin and investigated the cytokine release from blood and

mucosal stimulated with these.

5-8:Cytokines levels post 36 days of immunization dose and challenge

(immune protection):

The present results indicate that all cytokines levels after 36 days of

immunizing dose and infection dose (immune protections) increased

significantly than controls (intramuscular of normal saline), and than

intramuscular of lanolin only. The most significant increase was in mucosal

than systemic it was the highest level of all cytokines(Table 28)(Figure

16).Also these results shows that the differences between cytokines levels in

systemic and mucosal preparation of serotype 1 and 6 immunized animals

through post 36 days of immune dose and infection dose(immune protection)

table(29).

Different patterns of cytokine release are associated with different

receptor recruitment. CPS produced approximately similar concentrations of

IL-6 and TNF, and produced similar concentrations of IL-6 and IL-10

whereas LPS and whole bacteria elicited large quantities of TNF compared to

IL-6 and IL-10. This predominant IL-6 response, in the case of LPS

particularly, is in agreement with recent studies which indicate that TNF and


۱۰۲

IL-6 may play a significant role in host defence against bacterial infection

(Jagger et al.,2002) whereas previously it was considered merely a marker for

the severity of the bacterial challenge. The larger IL-6/TNFa ratio observed

with LPS (i.e. Gram negative bacteria) compared with that from Gram-positive

bacteria can probably be attributed to the activation of different primary TLR

initially. TLR4 has been identified as the primary receptor for enteric LPS

whereas TLR2 is implicated as the receptor for Gram-positive cell wall

components (Dekkers et al.,2000).

Many models are being suggested currently as to how different TLR

proteins, in conjunction with additional receptors, stimulate macrophages in

defense against specific organisms (Matsuguchi et al.,2000).Cauwells et

al.,(1997), working with a whole blood system and comparing LPS, whole,

encapsulated R6x S. pneumoniae and purified pneumococcal capsule

concluded that as many as two receptors, in addition to CD14, appear to lead

to cytokine production by Gram positive bacteria, but not by Gram-negative

organisms. However these different signalling pathways might converge

intracellularly. Specific antibodies to the CPS play an important role in

fighting infection and enhancing phagocytosis at a local level. Once infection

becomes systemic the inflammatory response leading to sepsis and death is a

major problem and antibodies to CPS may have an insignificant role in

influencing this.

Conclusion and Recommendations

۱۰۳

Conclusions:

1- Streptococcus pneumoniae serotype 6 was the most dominant serotype

and serotype 1 was the most virulent serotype in AL-Najaf Governorate.

2- A single dose of 1mg of Streptococcus pneumoniae capsular

polysaccharide serotype 1 with 1ml lanolin combination demonstrated by

intramuscular route seem to enhance immunogenicity when provided 80%

of immune protection in rabbits while serotype 6 provided 60% of immune

protection.

3- A single dose of 1mg of Streptococcus pneumoniae capsular

polysaccharide serotype 1 with 1ml lanolin combination demonstrated by

intramuscular route enhance systemic immunoglobulin higher than mucosal

and enhances TNFα , TNFβ , IL-6 and IL-10 in mucosal higher than in

systemic site.

Conclusion and Recommendations

۱۰٤

Recommendations:

1- Prepare a safe combined polysaccharide vaccine containing the local

serotypes for the favor of the Streptococcus pneumoniae pneumonia in

patients.

2- Elucidate the possible T-depended epitope in Streptococcus pneumoniae

serotype that activate T- cell.

3- Interpret the high cytokines release by mucosal T- cell and lower

antibody release by murine B cell system in the rabbits immunized with

Streptococcus pneumoniae serotype 1 and 6 polysaccharides whatever the

nature of the immunization protocol.

۱۰٥

References: - Aaberge, I. S.; North, R. J.; Groeng, M. and Lovik. M. (1993). Antibody

response to pneumococcal polysaccharide vaccine in young, adult and

oldmice. Scand. J. Immunol. 38:17-30.

- Aaberge, I. S.; Groeng, E.C. I. L.; Haugen, R.; Dalseg, M.; Lovik, B. and

Haneberg, A. (1995).Induction of systemic and mucosal antibodies to

pneumococcal antigens by mucosal immunization. J. Cell. Biochem.

Suppl.19A:242.

- Aaberge, I. S. and Lovik, M. (1996).The antibody response after immunization

with pneumococcal polysaccharide vaccine in splenectomized mice: the

effect of re-immunization with pneumococcal antigens. AP.MI.S. 104:307-

317.

- Abdul khan; Quanyi Chen; Zheng-Qi Wu James ,C. and Clifford ,M.

Snapper1.(2005). Both innate immunity and type 1 humoral immunity to

Streptococcus pneumoniae Are mediated by MyD88 but differ in their

relative levels of dependence on Toll-Like receptor. Inf. and Imm. Vol.

73. No.1 p: 298-307.

- Abdulrahman, M.D.; Kingsley, T.; Fahad Al Zamil, F. and Abdelmageed

Kambal, A.B. (2005). Streptococcus pneumoniae serotypes / serogroups

causing invasive disease in Riyadh, Ann. Saudi. Med.25(2):94-99.

- Ades, E.; Paton, J.C.; Briles, D.E. (2000). Intranasal immunization of mice with

a mixture of the pneumococcal proteins PsaA and PspA is highly protective

against nasopharyngeal carriage of Streptococcus pneumoniae. Infect.

Immun. 68: 796-800.

- Alannee, S. R. J.; McGee, L.; Jackson, D.; Chiou, C. C.; Feldman, C.; Morris, A.

J.; Ortqvist, A.; Rello, J. and Luna, C. M. (2007) .Association of serotypes

of Streptococcus pneumoniae with disease severity and outcome in adults:

an international study. Clin. Infect. Dis. 45, 46-51

۱۰٦

- Alonso, D.E.; Velasco, A. F. M.; Verheul, A. M. P. and Vanstejn, STEIJN, H.

A. T. (1994). Epitope specificity of rabbit immunoglobulin G (IgG) elicited

by pneumococcal type 23F synthetic oligosaccharide - and native

polysaccharide-protein conjugate vaccines: Comparison with human anti-

polysaccharide 23F Ig Infect. Immun. Vol. 62, No.3. p:799-808. - Anderton, J. M., Rajam, G., Romero Steiner, S., Summer, S., Kowalczyk, A. P.,

Carlone, G. M., Sampson, J. S. and Ades, E. W. (2007). E-cadherin is a

pneumococcal surface adhesin A(PsaA) of Streptococcus pneumoniae.

Microb Pathog 42, 225-236.

- Arbique, J.C.; Poyart, C.; Trieu-Cuot, P.; Quesne, G,; Carvalho, G.; Steigerwalt,

A.G. and Morey, R.E. (2004). Accuracy of phenotypic and genotypic

testing for identification of Streptococcus pneumoniae and description of

Streptococcus pseudopneumoniae sp. nov. J. Clin. Microbiol. 42: 4686-96.

- Arva, E. and Andersson, B. (1999). Induction of phagocyte-stimulating and Th1-

promoting cytokines by in vitro stimulation of human peripheral blood

mononuclear cells with Streptococcus pneumoniae. Scand. J. Immunol.

49:417-23.

- Austrian, R. (1979).Pneumococcal vaccine: development and prospects. Am. J.

Med. 67:547.

- Azoulay, V.; Rieux, M.; Muffat, P. and BEDOS, E. (2000) Relationship

between capsular type, Penicillin susceptibility and virulence of human

Streptococcus pneumoniae isolates in mice. Anti. Micr. And Chemo. Vol.

44, No. 6. p: 1575-1577.

- Babl, F.E.; Polton, S.I. and Klein, J.O.(2001)Constancy of distribution of

serogroups of invasive pneumococcal isolates among children: experience

during 4 decades. J.Clin. Infect. Dis. 32: 1156-1161.

۱۰۷

- Baker, P. J.; Reed, N. D.; Stashak, P. W.; Amsbaugh, D. F. and Prescott, B.

(1973).Regulation of the antibody response to type 3 pneumococcal

polysaccharide. I. Nature of regulatory cells. J. Exp. Med. 137:1431.

- Balachandran, P.; Brooks-Walter, A.; Virolainen-Julkunen, A.; Hollingshead, SK.

And Briles, D.E. (2002) Role of pneumococcal surface protein C in

nasopharyngeal carriage and pneumonia and its ability to elicit protection

against carriage of Streptococcus pneumoniae. Infect. Immun. 70(5): 2526-

34.

- Bancroft, J. D. and Stevens, A. (1982). Theory and practice of histological

techniques. Churchill living stone, New York. 117. - Barocchi, M. A., Censini, S. and Rappuoli, R. (2007). Vaccines in the era of

genomics: the pneumococcal challenge. Vaccine 25, 2963-2973.

- Barrett, D. J. (1985).Human immune responses to polysaccharide antigens: an

analysis of bacterial polysaccharide vaccines in infants. Adv. Pediatr. 32,

139-158.

- Bayram, A. and Kocoglu, E. (2006). Real-time polymerase chain reaction assay

for detection of Streptococcus pneumoniae in sputum samples from patients

with community-acquired pneumonia. J. Microbiol. Immunol.Infec. 39(6):

452-7.

- Bender, M. H. and Yother, J. (2001). Cps B is a modulator of capsule-associated

tyrosine kinase activity in Streptococcus pneumoniae. J. Biol. Chem. 276,

47966-47974.

- Benedicte , K. R. (1999).Intranasal immunization with heat-inactivated

Streptococcus pneumoniae protect mice against systemic pneumococcal

infection. Infect. Immun. vol:67 no.9 p:4320-4325.

-Bentley, S. D.; Aanensen, D. M; Mavroidi, A; aunders,D; abbinowitsch, E.;

ollins,M .Donohoe,K. and Murphy,L.(2006). Genetic Analysis of the

۱۰۸

Capsular Biosyn- thetic lcus from all 90 pneumococcal serotypes. Pl. Genet.J.

325: 891-44.

- Bergeron, Y.; Oullet, N.; Deslauriers, A-M.; Simard, M.; Olivier, M. and

Bergeron, M.G. (1998) Cytokine kinetics and other host factors in response

to pneumococcal pulmonary infection in mice. Infect. Immun. 66: 912-922.

- Black, R.A.; Rauch, C.T.; Kozlosky, C.J.; Peschon, J.J.; Slack, J.L.; Wolfson,

M.F.; Castner, B.J.; Stocking, K.L.; Reddy, P.; Srinivasan ,S.; Nelson, N.;

Boiani, N.; Schooley, K.A. and Gerhart, M. (1997). A metalloproteinase

disintegrin that releases tumour-necrosis factor-alpha from cells. Nature. 385

(6618): 729-33. - Black, S.; Shineeld, B.; and Fireman,H. (2000). Efcacy, safety and immu-

nogenicity of heptavalent pneumococcal conjugate vaccine in children.

Pediatr. J.Infect. Dis. 19:187-195.

- Bogaert, D.; De Groot, R. and Hermans, P. W. (2004). Streptococcus pneumoniae

colonization: the key to pneumococcal disease. J.Infect. Dis. 4, 144-154.

- Bone, R.C.; Grodzin, C.J. and Balk, R.A. (1997). Sepsis: a new hypothesis for

pathogenesis of the disease process. Chest. Infec .J. 112:235-243.

- Boutten, A. M. S.; Dehoux, N.; Seta, J.; Ostinelli, P.; Venembre, B.; Crestani,M.

C.; Dombret, G.; Durand. and Aubier.G. (1996). Compartmentalized IL-8

and elastase release within the human lung in unilateral pneumonia. Am.

J.Respir. Crit. Care Med. 153:336-342.

- Brandtzaeg, P. (1995). Molecular and cellular aspects of the secretary

immunoglobulin system. AP.MI.S 103:1-19.

- Brandtzaeg, P. and Haneberg, B. (1997). Role of nasal-associated lymphoid tissue

in the human mucosal immune system. Mucosal. Immunol. 5:4-08. - Briles, D.; Hollingshead, J. and King, A. (2000). Immunization of humans with

recombinant pneumococcal surface protein A elicits antibodies that passively

protect mice from fatal infection with Streptococcus pneumoniae bearing

۱۰۹

heterologous. J.Infect. Dis. 182:1694-1701.

- Brown, E.J.; Hosea, S.W. and Frank, M.M. (2003) The role of antibody and

complement in the reticule endothelial clearance of pneumococci from the

bloodstream. J. Infect. Dis. 4: S797-805. - Brueggemann, A. B.; Griffiths, D. T.; Meats, E.; Peto, T.; Crook, D. W. and

Spratt, B. G. (2003). Clonal relationships between invasive and carriage

Streptococcus pneumoniae and serotype and clone-specific differences in

invasive disease potential. J. Infect. Dis. 187, 1424-1432.

- Brueggemann, A. B. and Spratt, B. G. (2003). Geographic distribution and clonal

diversity of Streptococcus pneumoniae serotype 1 isolates. J. Clin. Microbiol.

41, 4966-4970. - Bruyn, G. A.; Zegers, B. J. and Van Furth, R. (1992) Mechanisms of host defense

against infection with Streptococcus pneumoniae. Clin. Infect. Dis. 14, 251-

262.

- Butler, J. C. (2004). Epidemiology of Pneumococcal Disease. In The

Pneumococcus. Washington D.C.: ASM Press. P: 148-168.

- Byington, C. L.; Korgenski, K.; Daly, J.; Ampofo, K.; Pavia, A. and Mason, E. O.

(2006). Impact of the pneumococcal conjugate vaccineon pneumococcal

parapneumonic empyema. Pediatr. J.Infect. Dis. 25,250-254. - Carvalho, M.G.; Steigerwalt, A.G.;Thompson, T.; Jackson, D. and Facklam,

R.R.(2003).Confirmationof nontypeable Streptococcus pneumoniae-like

organisms isolated from outbreaks of epidemic conjunctivitis as

Streptococcus pneumoniae. J. Clin. Microbiol. 41: 4415-7.

- Cauwels, A.; Wan, E.; Leisman, M. and Tuomanen, E. (1997). Co-existence of

CD14 dependent and independent pathways for stimulation of human

monocytes by gram-positive bacteria. Infect. Immun. 65:3255-60. - Center, K. J. and Isaacman, D. J. (2005). Streptococcus pneumoniae description of

the pathogen, disease epidemiology, treatment, and prevention.

Pharmacotherapy 25, 1193-1212.

۱۱۰

- Chan, E.Y.; Ruest, A.; Meade, M.O. and Cook, D.J. (2007).Oral decontamination

for prevention of pneumonia in mechanically ventilated adults: systematic

review and meta-analysis. BM.J. 26, 18-25. - Chandler, L.J.; Reisner, B.S.; Woods, G.L. and Jafri, A.K. (2000). Comparison of

four methods for identifying Streptococcus pneumoniae. Diagn. Microbiol.

Infect. Dis. 37: 285-287

- Charalambous, B.M.; Batt, S.L.; Peek, A.C.; Mwerinde, H.; Sam, N. and

Gillespie, S.H. (2003). Quantitative validation of media for transportation

and storage of Streptococcus pneumoniae.J. Clin. Microbiol.41: 5551-6.

- Charles, P.E.; Piroth, L. and Desbiolles,N.(2002).New model of pneumoniae in

immunocompetent rabbits. Crit. Care. Med.J. 30:2278-2283.

- Chen, W.; Havell, E. A. and Harmsen, A. G. (1992). Importance of endogenous

tumor necrosis factor alpha and gamma interferon in host resistance against

Pneumocystis carinii infection. Infect. Immun. 60:1279-1284.

- Chow, J.C.; Young, D.W.; Golenbock, D.T.; Christ, W.J. and Gusovsky, F.

(1999).Toll-like receptor 4 mediates LPS-induced signal transduction. J. Biol.

Chem.274:10689-92.

- Christ-Crain, M.; Soltz, D. and Bingisser, R. (2006). Procalcitonin guidance of

antibiotic therapy in community-acquired pneumonia. Am. J. Respir. Crit.

Care Med.;174:84-93.

- Cox, J.C. and Coulter, A.R. (1997). Adjuvants-a classification and review of the

modes of action. Vaccine. 15:248-256.

- Cruckshank, R.; Daguid, J.P.; Marmion, B.P. and Swain, R.H.A. (1975). Medical

microbiology vol. 2( 12 th ed) P:459. The English language bock society

London.

- Cryz, J.R.; Furer, E. and Germenar, R. (1985). Purification and Vaccine Potential

of Klebsiella Capsular Polysaccharides Infect. Immun. Vol. 50, No. 1. p:

225-230.

۱۱۱

- Culig, Z.; Bartsch, G. and Hobisch, A. (2003). Interleukin-6 regulates androgen

receptor activity and prostate cancer cell growth. Mol. Cell. Endocrinol.J.

197 (1-2): 231-8. - Dagan, R.; Englehard, D. and Piccard, E. (1992). Epidemiology of invasive

pneumococcal infections in Israel. JAMA. 268:3328-3332.

- Dagan, R.; Gradstein, S.; Belmaker, I.; Porat, N.; Siton, Y.; Weber, G.; Janco, J.

and Yagupsky, P. (2000). An outbreak of Streptococcus pneumoniae

serotype 1 in a closed community in southern Israel. Clin. Infect. Dis. 30,

319-321.

- David, F.; Farley, J.; Huang, H.; Lavoie, J.P. and Laverty, S. (2007)Cytokine and

chemokine gene expression of IL-1beta stimulated equine articular

chondrocytes. Vet. Surg. 36(3):221-7.

- Dehoux, M. S.; Boutten, A.; Ostinelli,J.; Seta, N. and (2004). Compartmentalized

cytokine production within the human lung in unilateral pneumonia. Am.

J.Respir. Crit. Care Med. 150:710-716. - Doit, C.; Loukil, C.; Geslin, P. and Bingen, E. (2002) Phenotypic and genetic

diversity of invasive pneumococcal isolates recovered from French children.

J. Clin. Microbiol. 40: 2994-8.

- Dekkers, P.E.P.; Juffermans, N.P.; Ten Hove, T.; De Jonge, E.; Van Deventer,

S.J.H. and Van der Poll, T. (2000). Endotoxin down-regulates monocyte and

granulocyte IL-6 receptors without influencing gp 130 expression in humans.

J. Infect. Dis. 181:1055-61.

- Dennesen, P.J; Vanderv, N. and kessels, A.G.(2001). Resolution of infectious

parameters after antimicrobial therapy in patients with ventilator-associated

pneumonia. AM. J. respi. 163:1371-1375.

- Dominguez, J. and Gali,N. (2001). Detection of Streptococcus pneumoniae

antigen by a rapid immuno chromatographic assay in urine samples. Chest

.Infect.J.119(1): 243 -9.

۱۱۲

- Echchannaoui, H.; Frei, K. C.; Schnell, S. I.; Leib, W. and Zimmerli, R. (2002).

Toll-like receptor 2-deficient mice are highly suceptible to Streptococcus

pneumoniae meningitis because of reduced bacterial clearing and enhanced

inflammation. J. Infect. Dis. 186:798-806.

- Edelman, R. (2000). An overview of adjuvant use. In: O’Hagan DT, ed. methods

in molecular medicine: Vaccine adjuvants - preparation methods and research

protocols. Totawa. N.J. Humana Press. P: 127.

- Eiman, M.; Mokaddas, O.; Rotimi, O. and John Albert, M. (2008).

Implications of Streptococcus pneumoniae Penicillin Resistance and

Serotype Distribution in Kuwait for disease treatment and prevention Clin.

Vacc. Imm.J. p:203-207. - Eskola, J.; Kilpi, R. and Palmu, A. (2001). Efficacy of a pneumococcal conjugate

vaccine against acute otitis media. N. Engl. J. Med. 344:403-409.

- Esposito, A.L.; Clark, C.A. and Poirier, W.J. (1990)An assessment of the factors

contributing to the killing of type 3 Streptococcus pneumoniae by human

polymorph nuclear leukocytes in vitro. APMIS.J. 98: 111-121.

- Febbraio, M.A. and Pedersen, B.K. (2005). Contraction-induced myokine

production and release: is skeletal muscle an endocrine organ. Exerc. Sport.

Sci. Rev.j. 33 (3): 114-9.

- Feldman, C.; Kallenbach, J.M.; Levy, H.; Thorburn, J.R.; Hurwitz, M.D. and

Koornhof, H.J. (1991). Comparison of bacteraemic community-acquired lobar

pneumonia due to Streptococcus pneumoniae and Klebsiella pneumoniae in an

intensive care unit. Respiration.J. 58 (5-6): 265-70.

- Feldman, C. R.; Anderson, R.; Cockeran, T.; Mitchell, P.; Cole,A. and Wilson, R.

(2002). The effects of pneumolysin and hydrogen peroxide, alone and in

combination, on human ciliated epithelium in vitro. Respir. Med. 98:580-585.

۱۱۳

- Fumeaux, T. and Pugin, J. (2002). Role of interleukin-10 in the intracellular

sequestration of human leukocyte antigen-DR inmonocytes during septic

shock. Am. J. Respir. Crit. Care. Med.166:1475-1482.

- Gail, G.; Hardy, M.; Ashallad, D.; Magee, E.; Christyl, L. and Janet, Y. (2001)

Essential role for cellular phosphoglucomutase in virulence of type 3

Streptococcus pneumoniae Infect. Immun. Vol. 69, No. 4. p:2309-2317.

- Garau, J. and Calbo, E. (2007). Capsular types and predicting patient outcomes in

pneumococcal bacteremia. Clin. Infect. Dis. 45, 52-54.

- Garcia, S.; Levine, O. S.; Cherian, T.; Gabastou, J. M. and Andrus, J. (2006).

Pneumococcal disease and vaccination in the Americas: an agenda for

accelerated vaccine introduction. Panam. J. Public. Health. 19, 340-348.

- Garvey, J.S.; Gemer, N.E. and Sussdrof, D.H.(1977). Method in immunology. (3rd

ed.)P 517-534.Addison –Wosley publishing company Inc. Masachusetts.

- Gause, W. C.; Urban, J. F.; and Stadecker, M. J. (2003).The immune response to

parasitic helminthes: insights from murine models. Trends. Immunol.24:269-

277.

- Gaur, U. and Aggarwal, B.B. (2003). Regulation of proliferation survival and

apoptosis by members of the TNF superfamily. Biochem. Pharmacol. 66 (8):

1403-8.

- Gilman, A.; Goodman,L.S.; Hardman, J.G. and Limbird, L.E. (2001). Goodman &

Gilman's the pharmacological basis of the rapeutics. New York: McGraw-Hill.

- Girndt, M. (2003). Humoral immune responses in uremia and the role of IL-10.

Blood Purif. 20 (5): 485-8.

- Gleason, P.P. and Shaughnessy, A.F. (2007)Stepes new drug reviews telithromycin

(Ketek) for treatment of community-acquired pneumonia. Am. Fam.

Physician.;76.p:55-78.

۱۱٤

- Gordon, S.; Irving, G.; Lawson, R.; Lee, M. and Read, R.C. (2000) Intracellular

trafficking and killing of Streptococcus pneumoniae by human alveolar

macrophagesare influenced by opsonins. Infect. Immun. 68: 2286-2293.

- Gornall, A.C.; Bardawill, C.J. and David, M.M. (1949) test book of clinical

chemistry 3rd ED. J.BIO. chem.177-751.

- Grange, J.M. (1988). Mycobacteria and human disease. Edward Arnold, London.

- Gray, P.W,.; Aggarwal, B.B.; Benton, C.V.; Bringman, T.S.; Henzel, W. Jarrett,

J.A.; Leung, D.W.; Moffat, B. and Svedersky, L.P. (1984). Cloning and

expression of cDNA for human lymphotoxin, a lymphokine with tumour

necrosis activity. Nature.3. 12:721-730.

- Greenberger, M. J.; Strieter, R. M.; Kunkel, S. L.; Danforth, J. M. and Standiford,

T. (1995). Neutralization of IL-10 increases survival in a murine model of

Klebsiella pneumonia. J. Immunol. 155:722-729.

- Greenberger, M. J.; Kunkel, S. L. R. M.; Strieter, N. W.; Lukacs, J.; Bramson,J.;

Gauldie, F. L.; Graham, M.; Hitt, J. M.; Danforth, A. and Standiford, T.

J.(1996). IL-12 gene therapy protects mice in lethal Klebsiella pneumonia.J.

Immunol. 157:3006-3012.

- Grimbaldeston, M. (2007). Mast cell–derived interleukin 10 limits skin pathology

in contact dermatitis and chronic irradiation with ultraviolet B. Natur. Imm. 8:

1095-1104.

- Gwaltney, J. M.; Sande, J. M. A. R. and Hendley, J. O. (1975). Spread of

Streptococcus pneumoniae in families. II. Relation of transfer of S.pneumoniae

to incidence of colds and serum antibody. J. Infect. Dis. 132: 62-68.

- Hahn, H. H. and Beaty, H. N. (1970). Trantracheal aspiration in the evaluation of

patients with pneumonia. Ann. Intern. Med.72:183-187.

- Hall, P.(1980).Laboratory manual (chemistry for the allied health science).p.136

Baur.

۱۱٥

- Hanage, W. P.; Kaijalainen ,T. H. and Jon ,M.K. (2005). Invasiveness of

Serotypes and Clones of Streptococcus pneumoniae among Children in

Finland. J. Infect. Dis. 73: 431-435.

- Haneberg, B. R.; Dalseg, E.; Wedege, E. A.; Hoiby, I. L.; Haugen, F.; Oftung, S.

R.; Andersen, L. M.; Naess, A.; Aase, T. E.; Michaelsen, L. and Holst, J.

(1998) .Intranasal administration of a meningococcal outer membrane vesicle

vaccine induces persistent local mucosal antibodies and serum antibodies wit

hstrong bactericidal activity in humans. Infect. Immun. 66:1334-1341.

- Hardy,G. G.; Magee, A. D.; Ventura, C. L.; Caimano, M. J. and Yother, J. (2001).

Essential role for cellular phosphoglucomutase in virulence of type 3

Streptococcus pneumoniae. Infect. Immun. 69, 2309-2317.

- Harold, F. and Stills, J.(2005). Adjuvants and antibody p roduction: dispelling the

myths associated with Freund’s complete and other adjuvants, ILAR. J.

Volume 46, Number 3.

- Hausdorff, W. P.; Feikin, D. R. and Klugman, K. (2005). Epidemiological

differences among pneumococcal serotypes. Lancet. Infect. Dis. 5,83-93.

- Hardy, G. G.; Caimano, M. J. and Yother, J. (2000).Capsule biosynthesis and

basic metabolism in Streptococcus pneumoniae are linked through the

cellular phosphoglucomutase. J. Bacteriol. 182:1854-1863.

- Hausdorff, W. P. (2007). The roles of pneumococcal serotypes 1 and 5 in

paediatric invasive diseases. Vaccine. J. 25, 2406-2412.

- Heffernan, R. T. and Barrett, N. L. (2005). Declining incidence of invasive

Streptococcus pneumoniae infections among persons with AIDS in an era of

highly active antiretroviral therapy, 1995-2000. J. Infect. Dis. 191(12): 2038-

45.

- Heinrich, P.C.; Behrmann, I.; Haan, S.; Hermanns, H.M.; Müller-Newen, G. and

Schaper, F. (2003). Principles of interleukin-6-type cytokine signalling and

its regulation. Biochem. J. 374: 1-19.

۱۱٦

- Henrichsen, J. (1995). Six newly recognized types of Streptococcus pneumoniae. J

Clin Micro-biol 33: 2759-62.

- Henriques Normark, B. and Christensson, B. (2003). Clonal Analysis of

Streptococcus pneumoniae nonsusceptible to Penicillin at Day-Care Centers

with Index Cases, in a Region with low incidence of resistance emergence of

an invasive type 35B Clone among Carriers. J. Infect. Dis 9: 337-344. - Henriques, B.; Kalin, M. and Ortqvist, A. (2000). Molecular epidemiology of

Streptococcus pneumoniae causing invasive disease in 5 countries. J. Infect.

Dis. 182, 833-839.

- Herbert, W.J. (1967). Some investigations into the mode of action of the water-in-

mineral-oil emulsion antigen adjuvants. In: Symposium series of

Immunobiology standardization. Vol. 6. Basel NY: Karger. P: 213-220.

- Hogberg, L. P.; Geli, H.; Ringberg, E.; Melander, M.; Lipsitch, W. and Ekdahl,

K.(2007). Age and serogroup-related differences in observed durations of

nasopharyngeal carriage of penicillin-resistant pneumococci. J. Clin.

Microbiol.45:948-952. - Hoskins, J.; Alborn, W. E. and Arnold, J. (2001). Genome of the bacterium

Streptococcus pneumoniae strain R6. J. Bacteriol 183, 5709-5717.

- Humphrey, J. H. D. M. and East, J. (1964). Studies on globulin and antibody

production in mice at birth. J.Immunol. 7:419.

- Hvalbye, B. I. S.; Aaberge, C.; Peeters, S. and Lovik, M. (1995). Pneumococcal

polysaccharide protein-conjugate vaccine in a mouse model: immune

response and protection. Scand. J. Immunol. 41:622.

- Jackson, L.A.; Neuzil, K.M.; Yu, O.; Benson, P.; Barlow, W.E.; Adams, A.L.;

Hanson, C.A.; Mahoney, L.D.; Shay, D.K. and Thompson, W.W. (2003).

Effectiveness of pneumococcal polysaccharide vaccine in older adults. N.

Engl. J. Med. 348:1747-1755.

۱۱۷

- Jagger, M. P.; Huo, Z. and Riches, P. G. (2002) Inflammatory cytokine

(interleukin 6 and tumour necrosis factor alpha) release in a human whole

blood system in response to Streptococcus pneumoniae serotype 14 and its

capsular polysaccharide. Clin. Exp. Immunol. 2. 130:467-474.

- James, A.; Kellogg, D.; David, A. and Bankert, W. (2001).Identification of

Streptococcus pneumoniae Revi. J. Vol. 39, No. 9. p: 3373-3375.

- Jonhnston, A. and Thorpe, R.(1982). Immunochemistry in practice black well

scientific puplication. p:150.

- Johnson, S.; Opstad, N.L.; Douglas, J.M. and Janoff, E.N. (1996). Prolongedand

preferential production of polymeric immunoglobulin A in response to

Streptococcus pneumoniae capsular polysaccharides. Infect. Immun.

64:4339-4344.

- Johnstone, J.; Marrie, T.J.; Eurich, D.T. and Majumdar, S.R. (2007) Effect of

pneumococcal vaccination in hospitalized adults with community-acquired

pneumonia. Arch. Intern. Med.167:1938-1943.

- Kadioglu, A.; Coward, W. M.; Colston, J.; Hewitt,C. R. and Andrew, P. W.

(2004).CD4-T-lymphocyte interactions with pneumolysin and pneumococci

suggest a crucial protective role in the host response to pneumococcal

infection. Infect. Immun. 72:2689-2697.

- Kaetzel, C.S.; Robinson, J.K.; Chintalacharuvu, K.R.; Vaerman, J. and Lamm,

M.E. (1991). The polymeric immunoglobulin receptor (secretory

component)mediates transport of immune complexes across epithelial cells:

Alocal defense function for IgA. Proc. Natl. Acad. Sci. USA. 88:8796-8800. - Katharina, B.(2008). Recognition and clearance of Streptococcus pneumoniae by

the innate immune system. Department of microbiology, tumor and cell

biology Karolinska Institutet, Stockholm, Sweden .Ph.D. thesis.145p.

۱۱۸

- Kawai, T.; O. Adachi, T.; Ogawa, K.; Takeda,M. and Akira, S. (1999).

Unresponsiveness of MyD88-deficient mice to endotoxin. J. Immunol.

11:115-122.

- Kim, J. W. (1998). Association of intrastrain phase variation in quantity of

capsular polysaccharide and teichoic acid with the virulence of Streptococcus

pneumoniae. J. Infect. Dis. 177(2): 368-77.

- Klebanoff, S. J.; Vadas, M.; . Harlan, A. J. and Sparks, M.L.(2003). Stimulation

of neutrophils by tumournecrosis factor. J. Immunol. 136:4220-4225.

- Klugman, K. P. (2002). Bacteriological evidence of antibiotic failure in

pneumococcal lower respiratory tract infections. Eur. Respir. J. Suppl .36,

3s-8s. 85(16): 6157-61.

- Knapp, S.; Wieland, C. W.; van’t Veer, O.; Takeuchi, S.; Akira, S.; Florquin, R.

and van der Poll, T. (2004). Toll-like receptor 2 plays a role in the early

inflammatory response to murine pneumococcal pneumonia but does not

contribute to anti-bacterial defense. J. Immunol. 172:3132-3138.

- Koedel, U.; Angele, B. T.; Rupprecht, H.; Wagner, A.; Roggenkamp, H.W.;

Pfister,C. and Kirschning, C. J. (2003).Toll-like receptor 2 participates in

mediation of immune response to experimental pneumococcal meningitis. J.

Immunol. 170:438-444.

- Konradsen, H. B. and Kaltoft, M. S. (2002). Invasive pneumococcal infections in

Denmark from 1995 to 1999: epidemiology, serotypes, and resistance. Clin.

Diagn. Lab. Immunol. 9, 358-365.

- Krivan, H. C. and Roberts, D. D. (1988). Many pulmonary pathogenic bacteria

bind specifically to the carbohydrate sequence GalNAc beta 1-4Gal found in

some glycolipids. Proc Natl Acad Sci U S A.

- Krzysztof, T.; Claudette, M.; Thompson, A.; Amit, S. and Alan, B.(2008).

Protection against nasopharyngeal colonization by Streptococcus pneumoniae

۱۱۹

is mediated by antigen-specific CD4-T Cells. Inf. and Imm. Vol. 76, No. 6, p:

2678-2684.

- Kutuna, T. (2005). Systemic and mucosal immune response in mice after

intranasal immunization with pneumococcal polysaccharides conjugate

vaccine with recombinant cholera toxin B subunit as an ajuvant. Nagoya.

Medical. J.49(2) p:135-148.

- Kwapinski, J. B. G. (1972). Methodology of immunochemical and immunological

research PP.300. Wiley-Interscience .New York.

- Laichalk, L. L.; Kunkel, S. L.; Strieter, R. M.; Danforth, J. M. Bailie, M. B. and

Standiford, T. J. (1996). Tumor necrosis factor mediates lung antibacterial

host defense in murine Klebsiella pneumonia. Infect. Immun. 64:5211–

5218.

- Larry, S.; Mcdaniel ,G.; Geraldine, S.; Kearney, E. and David, E. (1984).

Monoclonal antibodies against protective Pneumococcal antigens can

protect from fatal infection with S.pneumoniae. Cellular immunobiology

unit of the tumor institute, department of microbiology, and the

comprehensive cancer center, University of Alabama in Birmingham,

Birmingham, Alabama 35294.

- Laszalo, A. (1999). Tuberculosis: Laboratory aspects of diagnosis Canadian.

medical association Journal. 160 (12): 1725- 1729

- Latifi, S.Q.; O’Riordan, M.A. and Levine, A.D. (2002).Interleukin-10 controls

the onset of irreversible septic shock. Infect. Immun. 70:4441-4446.

- Laval, C. B.; Andrade, A. L. S. S.; Pimenta, F. C.; Andrade, J. G.; Oliveira, R.

M.; Silva, S. A.; Lima, E. C.,; Fabio, J. L.; Casagrande, S. T. and

Brandileone, M. C. (2006). Serotypes of carriage and invasive isolates of

Streptococcus pneumoniae in Brazilian children in the era of pneumococcal

vaccines. Clin. Microbiol. Infect. 12, 50-55.

۱۲۰

- Lee, C. J.; Lee, L. H.; Lu, C. S. and Wu, A. (2001). Bacterial polysaccharides as

vaccines - immunity and chemical characterization. Adv. Exp. Med. Biol.

491, 453-471.

- Lee, C.J. (2003). Protective immunity of pneumococcal glycoconjugates, Infor.

Healthcare. 29: 333 - 349.

- Lee, L.H.; Frasch, C.; Falk, L.A.; Klein, D.L. and Deal, C.D. (2003). Correlates

of immunity for pneumococcal conjugate vaccines. Vaccine, In press.

Infect. Immun. 68:126-129.

- Leimkugel, J.; Forgor, A. A.; Gagneux, S.; Pfluger, V.; Flierl, C.; Awine, E.;

Naegeli, M.; Dangy, J. P. and Smith, T. (2005). An outbreak of serotype 1

Streptococcus pneumoniae meningitis in northern Ghana with features that

are characteristic of Neisseria meningitidis epidemics. J. Infect. Dis. 192,

192-199.

- Linker,A. and Russel, S. (1966) A new polysaccharide resembling alginic acid

isolating. J. Biol. Chime. Vol. 241, No. 16, Issue of August 25, pp. 3845-

3851, Printed in U.S.A. - Lipsitch, M. (1999). Bacterial vaccines and serotype replacement: lessons from

Haemophilus inuenzae and prospects for Streptococcus pneumoniae.

Emerg. J. Infect. Dis. 5:336-345.

- Lipsitch, M..; Whitney ,C. G..; Zell, E. T.; Kaijalainen, R.; Dagan,M. and

Malley, R.(2005). Are anticapsular antibodies the primary mechanism of

protection against invasive pneumococcal disease. Emerg. Infect. Dis 2:15-

22.

- Locksley, R.M.; Killeen, N. and Lenardo, M.J. (2001). The TNF and TNF

receptor superfamilies: integrating mammalian biology. Cell. 104 (4): 487-

501.

- Louis, E.; Franchimont, D,.; Piron, A.; Gevaert, Y.; Schaaf-Lafontaine, N.;

Roland, S.; Mahieu, P.; Malaise, M.; De Groote, D. and Louis, R.

۱۲۱

(1998).Tumour necrosis factor (TNF) gene polymorphism influences TNF-

alpha productionin lipopolysaccharide (LPS)-stimulated whole blood cell

culturein healthy humans. Clin. Exp. Immunol.113:401-406.

- Lucas, A.H., Moulton, K.D., Tang, V.R. and Reason, D.C. (2001). Increased

immunogenicity and induction of class switching by conjugation of

complement C3d to pneumococcal serotype 14 capsular polysaccharide.

Infect. Immun. 69, 853-64.

- Lue, C.; Tarkowski, A. and Mestecky, J. (1988). Systemic immunization with

pneumococcal polysaccharide vaccine induces a predominant IgA2

response of peripheral blood lymphocytes and increases of both serum and

secretory anti-pneumococcal antibodies. J. Immunol. 140:3793-3800. - Lutfiyya, M.N.; Henley, E. and Chang, L.F. (2006) Diagnosis and treatment of

community-acquired pneumonia. Am. Fam. Physician.73:442-450.

- Macfaddin, J. F.(2000). Biochemical tests for identification of medical Bacteria.

3rd-ed, williium and Wilkins, U. S. A.

- Macleod, C. M.; Hodges, R.; Heidelberger,G. M. and Bernhard, W. G.

(1945).Prevention of pneumococcal pneumonia by immunization with

specific capsular polysaccharide. J. Exp. Med. 82:445.

- Magee, A. D. and Yother, J. (2001). Requirement for capsule in colonization by

Streptococcus pneumoniae. Infect. Immun. 69, 3755-3761.

- Malley, R.; Lipsitch, M.; Stack, A.; Saladino, R.; Fleisher, G. and Pelton, S.

(2001). Intranasal immunization with killed unencapsulated whole cells

prevents colonization and invasive disease by capsulated pneumococci.

Infect. Immun. 69:4870-4873.

- Martens, P. W.; Lundgren, S.W.; Konradsen, B. and Benfield, H.B. (2004).

Serotype- specific mortality from invasive Streptococcus pneumoniae disease

revisited. BMC Infect. Dis.4(21).

۱۲۲

- Matsuguchi, T.; Musikacharoen, T.; Ogawa, T. and Yoshikai, Y. (2000).Gene

expression of toll-like receptor 2, but not toll-like receptor 4, is induced by

LPS and inflammatory cytokines in mouse macrophages. J. Immunol.

165:5767-72.

- McChlery, S. M.; Scott, K. J. and Clarke, S. C. (2005). Clonal analysis of invasive

pneumococcal isolates in Scotland and coverage of serotypes by the licensed

conjugate polysaccharide pneumococcal vaccine :possible implications for

UK vaccine policy. Eur. J. Clin. Microbiol. Infect.Dis. 24, 262-267.

- McCool, T. L. and Weiser, J. N. (2004). Limited role of antibody in clearance of

Streptococcus pneumoniae in a murine model of colonization. Infect. Immun.

72(10): 5807-13.

- McGehee, J.L.; Podnos, S.D.; Pierce, A.K. and Weissler, J.C. (2001) Treatment of

pneumonia in patients at risk of infection with Gram-negative bacilli. Am. J.

Med. 84:597-602.

- Medzhitow, R. and Janeway,C. A. (1997). Innate immunity: the virtues of a

nonclonal system of recognition. Cell 91:295-298.

- Melegaro, A. and Edmunds, W. J. (2004). The 23-Valent pneumococcal

polysaccharide vaccine. Part II. A cost-effectiveness analysis for invasive

disease in the elderly in England and Wales. European Journal of

Epidemiology. 19(4): 365-375.

- Memish, Z. A.; Bulky, H. H.; Shibl, A. M.; Barrozo,C. P. and Gary, G. C. (2004).

Streptococcus pneumoniae in Saudi Arabia: antibiotic resistance and

serotypes of recent clinical isolates. Int. J. Antimicrob. Agents. 23:32-38.

- Mestecky, J. and McGhee, J.R. (1987). Immunoglobulin A (IgA): molecular and

cellular interactions involved in IgA biosynthesis and immune response. In

Advances in immunology. Volume. 40. F.J. Dixon, editor. Academic Press.

San.Diego, CA. 153-245.

۱۲۳

- Micheau, O. and Tschopp, J. (2003). Induction of TNF receptor I-mediated

apoptosis via two sequential signaling complexes. Cell. 114 (2): 90-181.

- Millar, E. V.; O'Brien, K. L. and Greet, L.Y. (2006). Effect of community-wide

conjugate pneumococcal vaccine use in infancy on nasopharyngeal carriage

through 3 Years of Age: A cross-sectional study in a high-risk population.

43: 8-15.

- Miriam, B. and Buenviaje, M.D. (1988). Quantitative sputum culture and gram

stain: pulmonary for infection vs. colonization. Phil. J. Microbiol. Infect. Dis.

1989; 18(1):28-35. - Moine, P.; Vercken, J. S.; Chevret, C.; Chastang, B. and Gajdos, P. (1994). Severe

community-acquired pneumonia. Etiology epidemiology, and prognosis

factors. French study group for community-acquired pneumoniain the

intensive care unit. Infect. Dis. 14:112-117.

- Muller, B. Harbath, S. and Stolz, D.(2007) Diagnostic and prognostic accuracy of

clinical and laboratory parameters in community-acquired pneumonia. BMC

Infect. Dis.7:10.

- Munoz, F.M.; Englund, J.A. and Cheesman, C.C. (2002)Maternal immunization

with pneumococcal polysaccharide vaccine in the third trimester of gestation.

Vaccine. 20: 826-837.

- Murray, M.T. and Bongiorno, P.B. (2006).Pneumonia: Bacterial, mycoplasmal,

and viral. In JE Pizzorno Jr, MT Murray, eds., Textbook of Natural Medicine,

3rd ed., vol. 2, pp. 2039–2044. Edinburgh: Churchill Livingstone.

- Murray, P. R.(1975).Microscopic and bacteriologic analysis of expectorated

sputum. Mayo. Clin. Proc. 50:339-344.

- Murray, P.R. and Washing, J.A. (1975).Microscopic and bacteriologic analysis of

expectorated sputum .Mayo. Clin. Proc .50:399-442.

- Musher, D.M.; Groover, J.E. and Reichler, M.R. (1997) Emergence of antibody to

capsular polysaccharides of Streptococcus pneumoniae during outbreaks of

۱۲٤

pneumonia: association with nasopharyngeal colonization. Clin Infect. Dis.

24: 441-446.

- Nelson, F.R. (2007). Basic Pathology (8th ed.). Philadelphia: Saunders. ISBN 1-

4160-2973-7.

- Nelson, S. and Summer, W. R. (1998).Innate immunity, cytokines and pulmonary

host defense. Infect. Dis. Clin. N. Am. 12:555-567.

- Neumann, B. T.; Machleidt, A.; Lifka, K.; Pfeffer, D.; Vestweber, T. W.; Mak, B.;

Holzmann, F. and Krönke, M. (1996). Crucial role of 55-kilodalton TNF

receptorin TNF-induced adhesion molecule expression and leukocyte organ

infiltration. J. Immunol. 156:1587–1593.

- Nisar, N.; Guleria, R.; Kuman, S.; Chand, C. T. and Ranjan, B. N. (2007)

Mycoplasma pneumoniae and its role in asthma. Postgrad. Med. J.83:100-

104.

- Noelle, R. J. J.; Daum, W. C.; Bartlett, J.; McCann, M. and Shepherd, D. M.

(1991).Cognate interactions between helper T cells and B cells. V.

Reconstruction of T helper cell function using purified plasma membranes

from activated Th1 and Th2 T-helper cells and lymphokines. J. Immunol.

146:1118.

- Normark, B. M. H.; Kalin, M.; Ortqvist, A.; Akerlund, T.; Liljequist, B. O.;

Hedlund, J.; Svenson, S. B.; Zhou, J. and Spratt, B. G. (2001). Dynamics of

penicillin-susceptible clones in invasive pneumococcal disease. J. Infect. Dis.

184, 861-869.

- Novak, R. and Tuomanen, E. (1999) Pathogenesis of pneumococcal pneumonia.

Semin. Respir. Infect. 14: 209-217.

- Nunes, S.; Sa –Lea, R.; Pereira, L. C. and Lencastre, H. (2008).Emergence of a

serotype 1 Streptococcus pneumoniae lineagecolonising healthy children in

Portugal in the seven-valent conjugate vaccination era. Clin. Microbiol.

Infect. 14, 82-84.

۱۲٥

- O'Brien, K. L.; Walters, M. I.; Sellman, J.; Quinlisk, P.; Regnery, H.; Schwartz,

B. and Dowell, S. F. (2000). Severe pneumococcal pneumonia in previously

healthy children: the role of preceding influenza infection. Clin. Infect. Dis.

30,784-789.

- Ogunniyi, A. R.; Folland, D.; Briles, S.; Hollingshead, S. and Paton, J.

(2000).Immunization of mice with combinations of pneumococcal virulence

pro-teins elicits enhanced protection against challenge with Streptococcus

pneumoniae. Infect. Immun. 68:3028-3033.

- Ostergaard, C.; O'Reilly, T.; Brandt, C.; Frimodt-Moller, N. and Lundgren, J. D.

(2006). Influence of the blood bacterial load on the meningeal

inflammatory response in Streptococcus pneumoniae meningitis. BMC

Infect. Dis. 6, 78.

- Pears, A. (1985). Histochemistry (^ed) pp: 450 Churchill-Livingston, New York.

- Pennica, D.; Nedwin, G.E.; Hayflick, S.; Seeburg, P.H. and Derynck, R.

(1984).Human tumor necrosis factor: Precursor structure, expression and

homology to lymphotoxin. Nature. 312:724.

- Pericone, C. D.; Overweg, K.; Hermans, p. and Weiser, N.(2000).Inhibitory and

bactericidal effects of hydrogen peroxide production by Streptococcus

pneumoniae on other inhabitants of the upper respiratory tract. Infect.

Immun. 68 (7): 3990-3997.

- Porat, N.; Trefler, R. and Dagan, R. (2001). Persistence of two invasive

Streptococcus pneumoniae clones of serotypes 1 and 5 in comparison to that

of multiple clones of serotypes 6B and 23F among children insouthern Israel.

J. Clin. Microbiol. 39, 1827-1832.

- Pratter, M. R. and Irwin, R. S. (1995). Clinical value of the Gram-stain smears of

respiratory secretions. Chest.J. 88:163-164.

۱۲٦

- Qazi, K. R.(2005).Heat shock proteins as vaccine adjuvants. Department of

Immunology, The Wenner-Gren Institute, Stockholm University, Stockholm,

Sweden. Ph.D. thesis. 71 p.

- Redecke, V.; Hacker, H.; Datta, S. K.; Fermin, A. P.; Pitha, M. D. H. and Raz, E.

(2004). Cutting edge: activation of Toll-like receptor 2 induces a Th2

immune response and promotes experimental asthma. J. Immunol.

172:2739-2743.

- Reed, W.V.V.; Byrd, G.S.; Gates, R.H.; Howard, R.S. and Weaver, M.J.

(1996).Sputum Gram's stain in community-acquired pneumococcal

pneumonia-A meta-analysis. West. J. Med. 165:197-204.

- Regev-Yochay, G. and Raz, M.(2004). Nasopharyngeal carriage of

Streptococcus pneumoniae by adults and children in community and family

settings. Clin. Infect. Dis. 38(5): 632-9.

- Regev-Yochay, G. T.; Thompson, K. C.; Malley, R. and Lipsitch, M. (2006).

Interference between Streptococcus pneumoniae and Staphylococcus

aureus: in vitro hydrogen peroxide-mediated killing by Streptococcus

pneumoniae. J. Bacterol. 188(13): 4996-5001.

- Reingold, A.L. (1998). Role of Legionellae in acute infections of the lower

respiratory tract. Rev. Infect. Dis. 10:1018-28.

- Ries, K.; Levison, M. E. and Kaye, D. (1974). Tran tracheal aspiration in

pulmonary infection. Arch. Intern. Med. 133:453-458.

- Ring, A. W. (1998). Pneumococcal trafficking across the blood- brain barrier.

Molecular analysis of a novel bidirectional pathway. J. Clin. 102(2): 347-

360. - Robbins, J. B.; Austrian, R. C.; Rastogi, J.; Lee, S. C.; Schiffman,G. J. and Hen-

richsen. R. (1983). Considerations for formulating the second generation

pneumococcal capsular polysaccharide vaccine with emphasis on the cross-

reactive types within groups. J. Infect. Dis. 148:1136-1159.

۱۲۷

- Roche, A. M.; King, S. J. and Weiser, J. N. (2007).Live attenuated Streptococcus

pneumoniae strains induce serotype-independent mucosal and systemic

protection in mice. Infect. Immun. 75:2469-2475.

- Rodriguez, M.E.; van der Pol, W.L.; Sanders, L. and van der Winkel, J.

(1999).Crucial role of Fc gammaRIIa (CD32) in assessment of functional

anti-Streptococcus pneumoniae antibody activity in human sera. J. Infect.

Dis. 179: 423-433.

- Rodriguez-Gaspar, M.; Santolaria, F,; Jarque-Lopez, A.; Gonzalez-Reimers, E.;

Milena, A. and De la Vega, M.J. (2001).Prognostic value of cytokines in

SIRS general medical patients.Cytokine.J.15:232-236.

- Rogers, H. W.; Tripp,C. S.; Schreiber, R. D. and Unanue, E. R. (2004).

Endogenous IL-1 is required for neutrophil recruitment and macrophage

activation during murine listeriosis. J. Immunol. 153:2093-2101.

- Roson, B. and Carratala, J. (2001). Etiology reasons for hospitalization, risk

classes, and outcomes of community acquired pneumonia in patients

hospitalized on the basis of conventional admission criteria. J. Bacterol. 33:

158-165.

- Roy, S.; Knox, K.; Segal, S.; Griffiths, D.; Moore, C.E.; Welsh, K.I.; Smarason,

A.; Day, N.P.; McPheat, W.L. and Crook, D.W. (2002).MBL genotype and

risk of invasive pneumococcal disease: a case-control study. Lancet.

359:1569-1573.

- Rubins, J.B. and Janoff, E.N. (2001).Pneumococcal disease in the elderly: what's

preventing vaccine efficacy. Drugs. Aging. 18:305-311. - Ruoff, K.L.; Whiley, R.A.; Beighton, D.; Murray, P.R.; Baron, Ellen, J.;

Jorgensen, J. H. and Pfaller, A. (2003) Streptococcus. In: (ed) Manual of

clinical microbiology, 8th edition. ASM Press, Washington D.C., 405-421.

۱۲۸

- Ruoff, W. and Beighton. (1999). In Murray, Baron, Pfaller, Tenover and Yolken

(ed.), Manual of clinical microbiology, 7th ed. American Society for

Microbiology, Washington, D.C.

- Russell, M.W.; Kilian, M. and Lamm, M.E. (1999). Biological activities of IgA

in mucosal immunology. 2nd edition. P.L. Ogra .editors. Academic Press.

San Diego. CA. 225-240.

- Russell, M.W.; Reinholdt, J. and Kilian, M. (1989). Anti-inflammatory activity

of human IgA antibodies and their Fab fragments: inhibition of IgG

mediated complement activation. Eur. J. Immunol. 19:2243-2249.

- Salazar, J.C.; Daly, K.A. and Giebink, G.S. (1997). Low cord blood

pneumococcal immunoglobulin G (IgG) antibodies predict early onset acute

otitis media in infancy. Am. J. Epidemiol. 145: 1048-1056. - Sandgren, A.; Sjostrom, K.; Olsson-Liljequist, B.; Christensson, B.;

Samuelsson, A.; Kronvall, G. and Henriques Normark, B. (2004). Effect

of clonal and serotype-specific properties on Streptococcus pneumoniae on

human respiratory epithelium in vitro. Infect. Immun. 57, 2006-2013.

- Sandgren, A. and Sjostrom, K. (2004). Effect of clonal and serotype-specific

properties on the invasive capacity of Streptococcus pneumoniae. J. Infect.

Dis. 189(5): 785-96.

- Sarkis, K. M. and Dennis, L. (2006). The love–hate relationship between bacterial

polysaccharides and the host immune system. Nature. Reviews. Immunology.

6, 849-858.

- Schnare, M.; Barton, G. M.; Holt, A. C.; Takeda, K. S. and Medzhitov, R.

(2001).Toll-like receptors control activation of adaptive immune responses.

Nat. Immunol. 2:947-950.

- Schwantner, A.; Dingley, A.; Ozbek Suat, J.; Rose-John Stefan, G. and Joachim,

L. (2004). Direct determination of the interleukin-6 binding epitope of the

interleukin-6 receptor by NMR spectroscopy. J. Biol. Chem.279 (1): 571-6.

۱۲۹

- Shnawa, I.M.S. and Thwaini ,Q.N.A.(2002). Lapin mucosal humoral versus

systemic humoral and cellular immune response post to intratesticular

administration of heat killed campylobacter fetus bacteria .J. Babylon. Sci.

Appl.3:58-542.

- Simell, B.; Kilpi,T. M.(2002). Pneumococcal carriage and otitis media induce

salivary antibodies to pneumococcal capsular polysaccharides in children. J.

Infect. Dis. 186(8): 1106-14.

- Simpson, S.Q.; Singh, R. and Bice, D.E. (1994).Heat-killed pneumococci and

pneumococcal capsular polysaccharides stimulate tumour necrosis factor α

production by murine macrophages. Am. J. Respir. Cell Mol Biol.;10:284-9.

- Siskind, G.W. and Benacerraf, B. (1969). Cell selection by antigen in the immune

response. Adv. Immunol. 10:1-50. - Sjostrom, K.; Spindler, C.,; Ortqvist, A.; Kalin, M.; Sandgren, A.; Kuhlmann-

Berenzon, S. and Henriques-Normark, B. (2006). Clonal and capsular types

decide whether pneumococci will act as a primary or opportunistic

pathogen. Clin. Infect. Dis. 42, 451-459.

- Sleeman, K.; Knox, K.; . George, R.; Miller, E. and Waight, P. (2001). Invasive

pneumococcal disease in England and Wales: vaccination implication. J.

Infect. Dis. 183:239-246.

- Smith, M. L. (1932). The effect of heat on sugar solution used for culture media.

Biochemical J., 26 : 1467-1468.

- Smith, M.D. (2003). Rapid diagnosis of bacteremic pneumococcal infections in

adults by using the binax now Streptococcus pneumoniae urinary antigen

test: A prospective, controlled clinical evaluation. J. Clini. Microbiol. 41(7):

2810-2813.

- Snapper, C.M. and Mond, J.J. (1996) A model for induction of T cell-independent

humoral immunity in response to polysaccharide antigens. J. Immunol. 157:

2229-2233.

۱۳۰

- Soell, M.; Diab, M.; Haan-Archipoff, G.; Beretz, A.; Herbelin, C.; Poutrel, B. and

Klein, J.P. (1995).Capsular polysaccharide types 5 and 8 of Staphylococcus

aureus bind specifically to human epithelial (KB) cells, endothelial cells and

monocytes and induce release of cytokines. Infect. Immun.63:1380-6.

- Soensen, U.B.S. (1995). Pneumococcal polysaccaride antigens: capsules and C-

polysaccaride. Danish. Med. Bull. 42: 47-53.

- Soininen, A.; Pursiainen, H.; Kilpi, T.M. and Käyhty, H. (2001). Natural

development of antibodies to pneumococcal capsular polysaccharides

depends on the serotype: association with pneumococcal carriage and acute

otitis media in children. J. Infect. Dis. 184: 569-576

-Sottile, M. I. and Rytel, W. M. (1975).Application of counter

immunoelectrophoresis in the identification of S.pneumoniae in clinical

isolates .J. Clinical. Microbiology.vol.2.no.3.p:173-177.

- Stein, K.E. (2002). Thymus-independent and thymus-dependent responses to

polysaccharide antigens. J. Infect. Dis. 165: S49-52.

- Stenvinkel, P.; Ketteler, M. and Johnson, R.J. (2005). IL-10, IL-6, and TNF-alpha:

central factors in the altered cytokine network of uremia--the good, the bad,

and the ugly. Kidney. Int. J. 67 (4): 1216-33

- Suzuki. N.; Seki, M.; Nakano, Y.; Kiyoura, Y.; Maeno, M. and Yamashita, Y.

(2005). Discrimination of Streptococcus pneumoniae from viridians group

streptococci by genomic subtractive hybridization. J. Clin. Microbiol. 43:

4528-34.

- Syrjanen, R. K.; Kilpi, T. M. and bowefg, K.R. (2001). Nasopharyngeal carriage

of Streptococcus pneumoniae in finnish children younger than 2 yearsold. J.

Infect. Dis.184(4)451-9.

- Takeuchi, O.; Takeda, K.; Hoshino, K.; Adachi, O.; Ogawa, T. and Akira,

S.(2000).Cellular responses to bacterial cell wall components are mediated

through MyD88-dependent signaling cascades. Int. Immunol. 12:113-117.

۱۳۱

- Tan, J. C.; Braun, S.; Rong, H.; DiGiacomo, R.; Dolphin, E.; Baldwin, S.;

Narula, S. K.; Zavodny, P. J. and Chou, C. C. (1995). Characterization of

recombinant extracellular domain of human interleukin-10 receptor. J. Biol.

Chem. 270 (21): 12906-11.

- Taneli, Puumalainen. (2003). An eleven Valent diphtheria and tetanus

conjucation pneumococcal vaccine immunogenicity and safety in Filipino

infants .University of Helsinki faculty of medicine department of paediatrics

Helsinki, Finland Ph.D. thesis. 124 p.

- Tarja, K.; Esa, R.; Pentti U. and Elja, H. (2004). Survival of Streptococcus

pneumoniae, Haemophilus influenzae and Moraxella catarrhalis frozen in

Skim Milk-Tryptone-Glucose-Glycerol Medium. J. Clin. Micr. p.412-

414.Vol. 42, No. 1. - Tarja, K. (2006).The identification of Streptococcus pneumoniae . University of

Oulu, Faculty of Medicine, Department of Medical Microbiology

Department of Nursing Science and Health Administration Oulu, Finland.

Ph.D. thesis. 75p.

- Tettelin, H.; Nelson, K.E.; Paulsen, I.T.; Eisen, J.A.; Read, T.D.; Peterson, S.;

Heidelberg, J.; DeBoy, R.T.; Haft, D.H.; Dodson, R.J. and Durkin, A.S.

(2001). Complete genome sequence of a virulent isolate of Streptococcus

pneumoniae. Science. 293: 498-506.

- Thomas, J. (1968). Studies on haemorrhagic septicaemia oil adjuvant vaccine. I.

Methods of production. Kajian. Vet. Malaysia–Singapore. 1, 152-158.

- Tian, B.; Nowak, D.E. and Brasier, A.R. (2005) A TNF-induced gene

expression program under oscillatory NF-kappaB control. BMC. Genomics.

Sep. 28;6:137.

- Tian, H.; Avi, G. and Boes, M.(2007). Pneumococcal capsular polysaccharide

vaccine protection agenst serotype 1 Streptococcus pneumoniae in mice. J.

infec. immun. vol.75 no.4 p:164-1650.

۱۳۲

- Timens, W.; Boes, A.; Rozeboom-Uiterwijk, T. and Poppema, S. (1998).

Immaturity of the human splenic marginal zone in infancy. Possible

contribution to the deficient infant immune response. J. Immunol. 143:

3200-3206.

- Toikka, P.; Irjala, K. and Juven, T. (2000). Serum procalcitonin, C-reactive

protein and interleukin-6 for distinguishing bacterial and viral pneumonia in

children. Pediatr.J.Infect. Dis.19: 598-602.

- Trzcinski, K.; Thompson, C.; Malley, R. and Lipsitch, M. (2005). Antibodies to

conserved pneumococcal antigens correlate with, but are not required for

protection against pneumococcal colonization induced by prior exposure in

a mouse model. Infect. Immun. 73:7043-7046.

- Tuazon, C.U. and Murray, W. (2000). Atypical pneumonias. In Pennington JE,

ed. Respiratory infections: diagnosis and management. NewYork: Raven

Press. 341-63.

- Tuomanen, E.I.; Austrian, R. and Masure, H.R. (1995). Pathogenesis of

pneumococcal infection. N. Engl. J. Med. 332:1280-1284.

- Twum-Danso, K.; Al-Mazrou, A.; Kambal, M. A. M. and Al-Zamil. F. A.

(2003).Penicillin resistance in serogroups /serotypes of Streptococcus

pneumoniae causing invasive infections in central Saudi Arabia. Saudi.

Med. J. 24:1210-1213.

-Upreti, R. K.; Kumar, M. and Shankar, V. (2003).Bacterial glycoproteins:

functions, biosynthesis and applications. Proteomics.J. 3, 363-379.

- Van der Poll, T.; Marchant, A.; Keogh, C.V.; Goldman, M. and Lowry, S.F.

(1996).Interleukin- 10 impairs host defense in murine pneumococcal

pneumonia. J. Infect. Dis. 174:994-1000.

- Van der Poll, T.; Keogh, C.V.; Guirao, X.; Buurman, W.A.; Kopf, M. and

Lowry, S.F. (1997). Interleukin-6 gene-deficient mice show impaired

defense against pneumococcal pneumonia. J. Infect. Dis. 176 (2): 439-44.

۱۳۳

- Van Rossum, A. M.; Lysenko, E. S. and Weiser, J. N. (2005). Host and bacterial

factors contributing to the clearance of colonization by Streptococcus

pneumoniae in a murine model. Infect. Immun. 73:7718-7726.

- Velez, D.; Lewis, J.; Kasper, L. and Cobb, A. (2009). Type 1 Streptococcus

pneumoniae carbohydrate utilizes a nitric oxide and MH II-dependent

pathway for antigen presentation. Immunology. Volume 127, Number 1, p:

73-82(10).

- Vernacchio, L.; Romero-Steiner, S. and Martinez, J.E. (2000). Comparison of an

opsonophagocytic assay and IgG ELISA to assess responses to

pneumococcal polysaccharide and pneumococcal conjugate vaccines in

children and young adults with sickle cell disease. J. Infect. Dis. 181: 1162-

1166.

- Vidarsson, G.; Sigurdardottir, S.T. and Gudnason, T. (1998). Isotypes and

opsonophagocytosis of pneumococcus type 6B antibodies elicited in infants

and adults by an experimental pneumococcus type 6B- tetanus toxoid

vaccine. Infect. Immun. 66: 2866-2870.

- Virkki, R.; Juven, T.; Rikalainen, H.; Svedstrom, E.; Mertsola, J. and Ruuskanen,

O. (2002). Differentation of bacterial and viral pneumonia in children.

Thorax. 57: 438-441.

- Virolainen, A.; Jero, J.; Käyhty, H.; Karma, P.; Leinonen, M. and Eskola, J.

(1995).Nasopharyngeal antibodies to pneumococcal capsular

polysaccharides in children with acute otitis media. J. Infect. Dis. 172:

1115-1118.

- Vuori-Holopainen, E. and Peltola, H. (2001). Reappraisal of lung tap: review of

an old method for better etiologic diagnosis of childhood pneumonia. Clin.

Infect. Dis. 32: 715-726.

- Wajant, H.; Pfizenmaier, K. and Scheurich, P. (2003). Tumor necrosis factor

signaling. Cell Death. Differ. 10 (1): 45-65.

۱۳٤

- Waterer, G.W.; Quasney, M.W.; Cantor, R.M. and Wunderink, R.G. (2001)Septic

shockand respiratory failure in community-acquired pneumonia have

different TNF polymorphism associations. Am. J. Respir. Crit.Care.

Med.163:1599-1604. - Watson, D.A.; Musher, D.M.; Jacobson, J.W. and Verhoef, J. (1993) A brief

history of the pneumococcus in biomedical research: a panoply of scientific

discovery. Clin. Infect. Dis. 17: 913-24.

- Weinberger, D. M.; Dagan, R. N.; Givon-Lavi, G.; Regev-Yochay, R.; Malley, M.

and Lipsitch, M. (2005).Epidemiologic evidence for serotype-specific

acquired immunity to pneumococcal carriage. J. Infect. Dis.55:582-612. - Weiser, J.N.; Austrian, R.; Sreenivasan, P.K. and Masure, H.R. (1994) Phase

variation in pneumococ-cal opacity: relationship between colonial

morphology and nasopharyngeal colonization. Infect. Immun. 62: 2582-9.

- Wernette, C.M.; Frasch, C.E.; Madore, D.; Carlone, G.; Goldblatt, D.; Plikaytis,

B.; Benjamin, W.; Quataert, S.A. and Hildreth, S. (2003). Enzyme-linked

immunosorbentassay for quantitation of human antibodies to pneumococcal

polysaccharides. Clin. Diagn. Lab. Immunol. 10:514-519. - Wester, C. W.; Ariga, D.; Nathan, C.;Rice, T.W.; Pulvirenti, J.; Patel, R.; Kocka,

F.; Ortiz, J. and Weinstein, R.A. (2002) Possible overestimation of penicillin

resistant Streptococcus pneumoniae colonization rates due to

misidentification of oropharyngeal strepto-cocci. Diagn. Microbiol. Infect.

Dis. 42: 263-8.

- WHO Position Paper. (2007).Weekly Epidemiological Record, No. 12,; 82:93-

104.

- Willems, F.; Marchant, A.; Delville, J. and Gérard, P. C. (1994). Interleukin-10

inhibits B7 and intercellular adhesion molecule-1 expression on human

monocytes. Eur. J. Immunol.24:1007-1009.

۱۳٥

- Wilson, M.J.B. and Martin, D.E. (1972).Quantitative sputum culture as a means of

excluding false positive reports in the routine micrpbiology laboratory. J.

Clin. Path. 25:697-700.

- Wipf, J.E.; Lipsky, B.A. and Hirschmann, J.V. (1999).Diagnosing pneumonia by

physical examination: relevant or relic. Arch. Intern. Med. 159 (10): 1082-7.

- Wittler, R.(1991)To Heinrich Frings GmbH & Co. KG, Bonn, Fed. Rep. of

Germany). - Wizemann, T. M.; Heinrichs, J. H. and Adamou, J. E. (2001).Use of a whole

genome approach to identify vaccine molecules affording protection against

Streptococcus pneumoniae infection. Infect. Immun. 69:1593-1598.

- World Health Organization. (1998). WHO meeting on maternal and neonatal

pneumococcal immunization. Wkly. Epidemiology. Rec. 73:187-188.

- Wu, H.Y.; Nahm, M.H.; Guo, Y.; Russell, M.W. and Briles, D.E. (1997).

Intranasal immunization of mice with PspA (pneumococcal surface protein

A) can prevent intranasal carriage, pulmonary infection, and sepsis with

Streptococcus pneumoniae. J. Infect. Dis. 175: 839-846.

- Wu, Z. Q.; Vos, Q.; Shen, Y.; Lees, A.; Wilson,S. and Briles, W. (1999). In vivo

polysaccharide-specific IgG Isotyperesponses to intact Streptococcus

pneumoniae are T-cell dependent and require CD40- and B7-ligand

interactions. J. Immunol. 163:659.

- Yee, A.; Phan, H.M.; Zuniga, R.; Salmon, J. and Musher, D.M. (2000).

Association between FcgammaRIIa-R131 allotype and bacteremic

pneumococcal pneumonia. Clin. Infect. Dis. 30: 25-28.

- Yenisehirli, G. and Sener, B. (2003). Antibiotic resistance and serotype

distribution of Streptococcus pneumoniae strains isolated from patients at

Hacettepe University Medical Faculty. Mikrobiyol. Bul. 37:1-11.

۱۳٦

- Yershov, A.L.; Jordan, B.S. and Gujmon, B. (2005).Relationship between the

inoculums dose of Streptococcus pneumoniae and pneumonia onset in rabbit

model. J. European respiratory 25:693-700.

- Yother, J.; Volanakis, J. E. and . Briles, D. E. (1982). Human C-reactive protein is

protective against fatal Streptococcus pneumoniae infection in mice. J.

lmmunol.128:2374.

- Yuanyi, L.; Marcelo, G. and Miriam, E. (2007).Immunization with recombinant

sao protein protection agenst streptococcus suis infection.J.Clin. and

Vacc.immun. vol.14 no.8 p:937-943.

- Zanotti, S.and Kumar, A. (2002).Cytokine modulation in sepsis and septic shock.

Expert. Opin. Investig. Drugs. 11:1061-1075.

- Zhang, Q.; Bagrade, L.; Bernatoniene, J.; Clarke, E. and Paton, J. C. (2007). Low

CD4 T cell immunity to pneumolysinis associated with nasopharyngeal

carriage of pneumococci in children. J. Infect. Dis. 195:1194-1202.

- Zhou, J.; Lottenbach, K. R.; Barenkamp, S. J.; Lucas, A. H. and Reason, D. C.

(2002). Recurrent variable region gene usage and somatic mutation in the

human antibody response to the capsular polysaccharide of Streptococcus

pneumoniae serotype1 . Infect. Immun. 70, 4083-4091.171.

- Zhou, J.; Lottenbach, K.R.; Barenkamp, S.J. and Reason, D.C. (2004). VH and

VL fragments response to capsular polysaccharide of Streptococcus

pneumoniae . Infect. Immun. 72. 3505-14.

:ألخالصهارتادوا وحده األمراض ألص�دريه ف�ي مستش�فى الص�در مريضا ٤۱۰ تضمنت هذه الدراسة

تراوح�ت ۲۰۰۹إلى نهاي�ة ك�انون الث�اني ۲۰۰۸النجف للمدة بين بداية شباط محافظةالتعليمي في

.من الذكور و اإلناث سنه ٦۰-۱۸أعمارهم بين

۱۹۰عين�ه كان�ت هن�اك ٤۱۰م�ن ب�ين إنرام بينت النتائج األولية للقشع المصبوغ بصبغه ك

(عين��ه ۹۹وحي��ده الن��واة بينم��ا كان��ت خالي��ا %46.341)(اللمفي��ه للخالي��ا عين��ه فق��ط موجب��ه

%21.951) ( عينه فقط ۹۰إذ بلغت العدالتوكانت ألمجموعه ألثالثه من الخاليا هي 24.146%)

) (Acid fast bacilliالحامض��يه للعص��يات الموجب��ه للص��بغه الموجب��ه ، كم��ا بلغ��ت ع��دد العين��ات

عين��ه فق��ط ۲۲كان��ت ألعدل��ه عين��ه الت��ي كان��ت موجب��ه لخالي��ا ۹۰. وم��ن (%7.560)عين��ه ۳۱

لبكتري��ا والكيموحيوي��ه و ألمجهري��ه هالم��ز رعي�� مطابق��ة للمواص��فات (24.444%)

Streptococcus pneumoniae مواص�فات غي�ر مطابق�ة لل %75.555)(عين�ه 68بينم�ا كان�ت

. Streptococcus pneumoniaeلبكتريا والكيموحيويهو ألمجهريه هالمز رعي

1 ال�نمط ح�ين ك�ان النج�ف ف�ي مدين�ة في انتشارا األكثركان ٦النمط نتائج الدراسة أنبينت

ض�راوة األكث�ر ألن�ه ه ل�ذلك ت�م اختي�ار األخرى األنماطمن بين في الفئران واألرانب ضراوة األكثر

م�ن ب�ين كاف�ه س�يادة األكث�ر 6 ال�نمط م�ن ۱ ألعزل�هك�ذلك ت�م اختي�ار األخ�رى األنم�اطن كافه من بي

.األخرى طاألنما

ن أ، بين�ت النت�ائج األران�بف�ي متخصصةالستثاره استجابة مناعية طرق ثالث استعمالتم

من��اعي م��ن المحف��ز ال 1mlم��ن المحفظ��ة م��ع 1mg( الحق��ن ف��ي ألعض��له بم��زج الثاني��ة ألطريق��ه

lanolin (هي األفضل في تحفي�ز الكلوبي�ولين المتخص�ص ف�ي لمده خمسه عشر يوم لتمنيع األرانب

بكتري�ا مقتول�ة حراري�ا لم�ده cfu/ml 108×1( الحق�ن ف�ي األن�ف الثالث�ة ألطريق�ه تبينما كان�الدم .

تائج بان النمط بينت الن كما .ألهوائيه القصبةكلوبيولين المتخصص في في تحفيز ال األفضلهي شهر)

في المناعية االستجابةبأن مستوى اإلحصائيوبين التحليل ٦من النمط أعلى مناعية استجابةوفر ۱

لك��ال =p ۰.۰۰۰۷وبف��رق معن��وي ٦و ۱ولك��ال النمط��ين ألهوائي��ه ألقص��بهمنه��ا ف��ي أعل��ىال��دم

وبف��رق معن��وي ف��ي ال��دم ٦م��ن ال��نمط أعل��ى مناعي��ة اس��تجابةمس��توى ۱وف��ر ال��نمط كم��االنمط��ين،

p=0.0339 .

م��ن 1mgتمني��ع األران��ب بطريق��ة( الحق��ن ف��ي ألعض��له بم��زج إن نت��ائج الدراس��ة أش��ارت

لم�ده خمس�ه عش�ر ي�وم لتمني�ع األران�ب) lanolinم�ن المحف�ز المن�اعي 1mlم�ع ۱المحفظة للنمط

۳٦بع�د ري�ة ألران�ب المختبف�ي ا الحمايةمن %60وفر ٦في حين النمط %80 حمايةوفر مستوى

ووف�ر إذ ( ال�دفاع المن�اعي) Streptococcus pneumoniaeببكتري�ا األص�ابه يوم من التمني�ع و

، وكان�ت مس�توى p=0.0425ف�ي ال�دم وبف�رق معن�وي ٦من ال�نمط أعلى مناعية استجابة ۱النمط

p=0.0032ولكال النمطين وبفرق معن�وي ألهوائيه ألقصبهمنه في أعلىفي الدم المناعية االستجابة

،p=0.0200 .على التوالي

الثالث�ة ألطريق�هبينت النت�ائج أن و تمنيع كذلك تم قياس مستوى السايتوكينات في ثالث طرق

على واألولى الثانية ألطريقهثم ولكافه السايتوكينات ألهوائيه والقصبةمستوى في الدم أعلى أعطت

ع�دم وج�ود ف�رق معن�وي ب�ين ۱ف�ي ال�دم ولل�نمط الثاني�ة ألطريق�هلنت�ائج م�ن خ�الل ، وبينت ا التوالي

وب��ين IL-6و TNFα مس��توى وب��ين IL-6و TNFβ مس��توى وب��ين TNFαو TNFβ مس��توى

بف��رق معن��وي IL-10و TNFβ مس��توى ووج��ود ف��رق معن��وي ب��ين IL-10و IL-6 مس��توى

p=0.0342 مس��توى ، وب��ين TNFα وIL-10 بف��رق معن��ويp=0.0018 وبين��ت النت��ائج ف��ي ،

-IL مس�توى وب�ين TNFβو TNFα مستوى عدم وجود فرق معنوي بين ۱للنمط ألهوائيه ألقصبه

وب��ين p=0.0004بف��رق معن��وي IL-6و TNFβ مس��توى ووج��ود ف��رق معن��وي ب��ين IL-10و 6

بفرق معنوي IL-6 و TNFα مستوى وبين p=<0.0001بفرق معنوي IL-10و TNFβ مستوى

p=0.0060 مس�توى وب�ين TNFα وIL-10 بف�رق معن�ويp=0.0037 بين��ت أخ�رى، م��ن جه�ة

مس��توى وب��ين TNFαو TNFβ مس��توى ف��ي ال��دم ع��دم وج��ود ف��رق معن��وي ب��ين ٦النت��ائج لل��نمط

TNFβ وIL-6 مس��توى وب��ين TNFβ و IL-10 مس��توى وب��ين IL-6و IL-10 ووج��ود ف��رق

IL-10و TNFα مس�توى وب�ين p=0.0019بف�رق معن�وي IL-6 و TNFα مس�توى معنوي بين

عدم وجود فرق معن�وي ألهوائيه ألقصبهفي ٦للنمط ، كذلك بينت النتائج p=0.0305بفرق معنوي

IL-10 و IL-6 مس��توى وب��ين IL-10و TNFβ مس��توى وب��ين IL-6و TNFβ مس��توى ب��ين

مس��توى وب��ين p=0.0027بف��رق معن��وي TNFβ و TNFα مس��توى ووج��ود ف��رق معن��وي ب��ين

TNFα و IL-6 بف��رق معن��ويp=0.0007 مس��توى وب��ين TNFα وIL-10 بف��رق معن��وي

p=0.0001 .

معنوي�ة أكث�رك�ان يوم م�ن (ال�دفاع المن�اعي) ۳٦بينت النتائج أن مستوى السايتوكاينات بعد

ألقص�بهفي معنوية األكثرفقط ، وكانت ) lanolin( المحفز المناعي مع المعاملةومن السيطرةمن

بينت النتائج عدم وجود فرق معنوي بين آخر، ومن جانب وكيناتتمنه في الدم ولكافه الساي ائيهألهو

۱لل�نمط p=0.0447بف�رق معن�وي IL-10 و TNFβ مس�توى كل الس�يتوكينات ع�دى ب�ين مستوى

p=<0.0001بف�رق معن�وي IL-10 و IL-6و TNFα مس�توى في الدم ووجود فرق معنوي ب�ين

ألقص�بهف�ي ۱لل�نمط p=0.0038بف�رق معن�وي IL-10و IL-6التوالي وبين على p=0.0004و

كل السايتوكاينات لل�نمط مستوى بينت النتائج عدم وجود فرق معنوي بين أخرىومن جهة ، ألهوائيه

وب�ين p= 0.0173بف�رق معن�وي IL-10و TNFα مس�توى في الدم ووج�ود ف�رق معن�وي ب�ين ٦

.ألهوائيه ألقصبهفي ٦للنمط p=0.016ي بفرق معنوIL-10 و IL-6 مستوى

streptococcus pneumoniae isolated from patients infected ... and... · prof. dr. jaafar k.n....

Documents