The Human Microbiota and Chronic Disease. Dysbiosis as a Cause of Human Pathology

Description 

Microbiota-associated pathology can be a direct result of changes in general bacterial composition, such as might be found in periodontitis and bacterial vaginosis, and/or as the result of colonization and/or overgrowth of so called keystone species. The disruption in the composition of the normal human microbiota, or dysbiosis, plays an integral role in human health and human disease.

The Human Microbiota and Human Chronic Disease: Dysbioses as a Cause of Human Pathology discusses the role of the microbiota in maintaining human health. The text introduces the reader to the biology of microbial dysbiosis and its potential role in both bacterial disease and in idiopathic chronic disease states. 
Divided into five sections, the text delineates the concept of the human bacterial microbiota with particular attention being paid to the microbiotae of the gut, oral cavity and skin.  A key methodology for exploring the microbiota, metagenomics, is also described.  The book then shows the reader the cellular, molecular and genetic complexities of the bacterial microbiota, its myriad connections with the host and how these can maintain tissue homeostasis.  Chapters then consider the role of dysbioses in human disease states, dealing with two of the commonest bacterial diseases of humanity – periodontitis and bacterial vaginosis.  The composition of some, if not all microbiotas can be controlled by the diet and this is also dealt with in this section.  The discussion moves on to the major ‘idiopathic’ diseases afflicting humans, and the potential role that dysbiosis could play in their induction and chronicity.  The book then concludes with the therapeutic potential of manipulating the microbiota, introducing the concepts of probiotics, prebiotics and the administration of healthy human faeces (faecal microbiota transplantation), and then hypothesizes as to the future of medical treatment viewed from a microbiota-centric position.

• Provides an introduction to dysbiosis, or a disruption in the composition of the normal human microbiota
  • Explains how microbiota-associated pathology and other chronic diseases can result from changes in general bacterial composition
  • Explores the relationship humans have with their microbiota, and its significance in human health and disease
  • Covers host genetic variants and their role in the composition of human microbial biofilms, integral to the relationship between human health and human disease

Authored and edited by leaders in the field, The Human Microbiota and Human Chronic Disease will be an invaluable resource for clinicians, pathologists, immunologists, cell and molecular biologists, biochemists, and system biologists studying cellular and molecular bases of human diseases.

About the Authors 

Luigi Nibali is a Senior Clinical Lecturer in the Clinical Oral Research Centre, Institute of Dentistry, Queen Mary University London in London, UK.
Brian Henderson is a Professor of Microbial Diseases in the School of Life and Medical Sciences at University College London in London, UK. 

Contents

List of contributors, xvii
Preface, xxi

Section 1 An introduction to the human tissue microbiome, 1
1   The human microbiota: an historical perspective, 3
Mike Wilson
1.1 Introduction: the discovery of the human microbiota: why do we care?, 3
1.2 The importance of the indigenous microbiota in health and disease, 3
1.2.1 The indigenous microbiota and human disease, 4
1.2.2 The indigenous microbiota and human health, 4
1.3 The development of technologies for characterising the indigenous microbiota, 8
1.3.1 Light microscopy, 9
1.3.2 Electron microscopy, 11
1.3.3 Culture ]based approaches to microbial community analysis, 12
1.4 Culture ]independent approaches to microbial community analysis, 29
1.5 Determination of microbial community functions, 31
1.6 Closing remarks, 32
Take ]home message, 32
References, 33
2  An introduction to microbial dysbiosis, 37
Mike Curtis
2.1 Definition of dysbiosis, 37
2.2 The ‘normal’ microbiota, 38
2.3 Main features of dysbiosis, 45
2.4 Conclusions, 49
Take ]home message, 53
Acknowledgment, 53
References, 53
3  The gut microbiota: an integrated interactive system, 55
Hervé M. Blottière and Joël Doré
3.1 Introduction, 55
3.2 Who is there, how is it composed?, 56
3.3 A system in interaction with food, 58
3.4 A system highly impacted by the host, 61
3.5 A system in interaction with human cells, 62
3.6 Conclusion: an intriguing integrated interactive system deserving further study, 63
Take ]home message, 63
References, 63
4  The oral microbiota, 67
W. G. Wade
4.1 Introduction, 67
4.2 Composition of the oral microbiome, 68
4.2.1 Archaea, 68
4.2.2 Fungi, 68
4.2.3 Protozoa, 68
4.2.4 Viruses, 69
4.2.5 Bacteria, 69
4.3 The oral microbiota in health, 71
4.3.1 Evolution of the oral microbiota, 71
4.3.2 Role of oral bacteria in health, 72
4.4 Role of oral microbiome in disease, 73
4.4.1 Dental caries, 73
4.4.2 Gingivitis, 74
4.4.3 Oral bacteria and non ]oral disease, 74
4.5 Future outlook, 75
Take ]home message, 75
References, 76
5  The skin microbiota, 81
P.L.J.M. Zeeuwen and J. Schalkwijk
5.1 Normal skin, 81
5.2 Skin diseases, 83
5.2.1 Atopic dermatitis, 83
5.2.2 Psoriasis, 84
5.2.3 Acne, 85
5.2.4 Rosacea, 85
5.2.5 Seborrheic dermatitis and dandruff, 86
5.2.6 Primary immunodeficiencies, 86
5.3 Experimental studies, 87
5.4 Dynamics of the skin microbiome, 87
5.5 Axillary skin microbiome transplantation, 89
5.6 Mouse skin microbiome studies, 89
5.7 Concluding remarks, 90
Take ]home message, 90
References, 90
6  Metagenomic analysis of the human microbiome, 95
Luis G. Bermúdez ]Humarán
6.1 Introduction, 95
6.2 The human microbiome, 96
6.3 Changes in microbiota composition during host life cycles, 97
6.4 The human microbiome and the environment, 98
6.5 Disease and health implications of microbiome, 99
6.5.1 The skin microbiota, 99
6.5.2 The airway microbiome, 99
6.5.3 Vaginal microbiome, 100
6.5.4 Gut microbiota and disease, 101
6.5.5 Metabolic disorders (obesity/diabetes), 103
6.6 Conclusions, 105
Take ]home message, 105
References, 106
Section 2 
Microbiota-microbiota and microbiota-host interactions in health and disease, 113
7  Systems biology of bacteria ]host interactions, 115
Almut Heinken, Dmitry A. Ravcheev and Ines Thiele
7.1 Introduction, 115
7.2 Computational analysis of host ]microbe interactions, 118
7.2.1 Analysis of metagenomic data, 118
7.2.2 Metabolic reconstruction through comparative genomics, 119
7.3 Network ]based modeling, 121
7.3.1 Topological network modeling, 121
7.3.2 Constraint ]based modeling, 123
7.3.3 Metabolic reconstructions of human metabolism, 124
7.3.4 Constraint ]based modeling of host ]microbe interactions, 124
7.4 Other computational modeling approaches, 127
7.4.1 Ordinary differential equation (ODE) models, 127
7.4.2 Kinetic modeling, 128
7.5 Conclusion, 129
Take ]home message, 130
Acknowledgments, 130
References, 131
8  Bacterial biofilm formation and immune evasion mechanisms, 139
Jessica Snowden
8.1 Introduction, 139
8.2 Biofilms in human disease, 139
8.3 Biofilm formation, 141
8.4 Immune responses to biofilms, 143
8.4.1 Innate immune responses, 144
8.4.2 Adaptive immune responses, 146
8.4.3 Fibroblasts, epithelial cells and other immune responses, 147
8.5 Biofilm immune evasion strategies, 147
8.6 Vaccines and biofilm therapeutics, 148
8.7 Conclusions, 149
Take ]home message, 149
References, 150
9  Co ]evolution of microbes and immunity and its consequences
for modern ]day life, 155
Markus B. Geuking
9.1 Introduction, 155
9.2 Symbiosis in eukaryotic evolution, 156
9.3 Evolution of the (innate and adaptive) immune system, 157
9.3.1 Immune proteins, 157
9.3.2 Evolution of adaptive immunity, 158
9.3.3 Two separate adaptive immune systems evolved, 158
9.4 Hygiene hypothesis, 159
9.5 What drives the composition of the microbiota?, 160
9.6 The pace of evolution, 161
Take ]home message, 162
References, 162
10  How viruses and bacteria have shaped the human genome: the implications for disease, 165
Frank Ryan
10.1 Genetic symbiosis, 165
10.2 Mitochondria: symbiogenesis in the human, 167
10.3 Virus symbiogenesis, 169
10.4 HERV proteins, 172
Take ]home message, 174
References, 174
11 The microbiota as an epigenetic control mechanism, 179
Boris A. Shenderov
11.1 Introduction, 179
11.2  Background on epigenetics and epigenomic programming/reprograming, 180
11.3 Epigenomics and link with energy metabolism, 184
11.4 The microbiota as a potential epigenetic modifier, 185
11.5 
Epigenetic control of the host genes by pathogenic and opportunistic microorganisms, 188
11.6  Epigenetic control of the host genes by indigenous (probiotic) microorganisms, 189
11.7 Concluding remarks and future directions, 191
Take home message, 193
References, 193
12 The emerging role of propionibacteria in human health
and disease, 199
Holger Brüggemann
12.1 Introduction, 199
12.2 Microbiological features of propionibacteria, 199
12.3 Population structure of P. acnes, 201
12.4 Propionibacteria as indigenous probiotics of the skin, 202
12.5 Propionibacteria as opportunistic pathogens, 203
12.6 Host interacting traits and factors of propionibacteria, 205
12.7 Host responses to P. acnes, 206
12.7.1 Innate immune responses, 206
12.7.2 Adaptive immune responses, 207
12.7.3 Host cell tropism of P. acnes, 208
12.8 Propionibacterium ]specific bacteriophages, 208
12.9 Concluding remarks, 209
Take home message, 210
References, 210
Section 3 Dysbioses and bacterial diseases: metchnikoff’s legacy, 215
13  The periodontal diseases: microbial diseases or diseases of the host response?, 217
Luigi Nibali
13.1 The tooth: a potential breach in the mucosal barrier, 217
13.2 The periodontium from health to disease, 217
13.3 Periodontitis: one of the most common human diseases, 219
13.4 Periodontal treatment: a non ]specific biofilm disruption, 220
13.5 Microbial etiology, 220
13.6 The host response in periodontitis, 221
13.7 Conclusions, 223
Take home message, 223
References, 223
14 The polymicrobial synergy and dysbiosis model of periodontal disease
pathogenesis, 227
George Hajishengallis and Richard J. Lamont
14.1 Introduction, 227
14.2 A (very) polymicrobial etiology of periodontitis, 229
14.3 Synergism among periodontal bacteria, 230
14.4 Interactions between bacterial communities and epithelial cells, 232
14.5 Manipulation of host immunity, 233
14.6 Conclusions, 237
Take ]home message, 238
References, 239
15 New paradigm in the relationship between periodontal disease and systemic diseases: ef-fects of oral bacteria on the gut microbiota and metabolism, 243
Kazuhisa Yamazaki
15.1 Introduction, 243
15.2 Association between periodontal and systemic diseases, 244
15.2.1 Periodontal disease and diabetes, 244
15.2.2 Periodontal disease and atherosclerotic vascular diseases, 245
15.2.3 Periodontal disease and rheumatoid arthritis, 246
15.2.4 Periodontal disease and non ]alcoholic fatty liver disease, 246
15.2.5 Periodontal disease and pre ]term birth, 247
15.2.6 Periodontal disease and obesity, 248
15.2.7 Periodontal disease and cancer, 248
15.2.8 Periodontal disease and inflammatory bowel disease, 249
15.3 Issues in causal mechanisms of periodontal disease for systemic disease, 249
15.3.1 Endotoxemia (bacteremia), 249
15.3.2 Inflammatory mediators, 251
15.3.3 Autoimmune response from molecular mimicry, 251
15.4 New insights into the mechanisms linking periodontal disease and s­ystemic disease, 252
15.5 Effect of oral administration of P. gingivalis on metabolic change 
and gut microbiota, 252
15.6 Conclusions, 254
Take ]home message, 255
References, 255
16 The vaginal microbiota in health and disease, 263
S. Tariq Sadiq and Phillip Hay
16.1 What makes a healthy microbiota, 263
16.1.1 How does the vaginal microbiota mediate healthiness?, 264
16.1.2 Establishment of the vaginal microbiota, 264
16.1.3 The role of host genetic variation on vaginal health, 264
16.1.4 
Impact of age, menstrual cycle and environmental factors on vaginal health, 265
16.2 The Vaginal Microbiota in Disease, 265
16.2.1 Bacterial vaginosis, 265
16.2.2 
Clinical consequences of altered vaginal microbiota 
(see Figure 1), 268
16.2.3 
Vaginal microbiota and transmission and susceptibility to HIV infection, 269
16.3 Conclusions, 269
Take ]home message, 269
References, 270
Section 4 
Dysbioses and chronic diseases: is there 
a connection?, 273
17 Reactive arthritis: the hidden bacterial connection, 275
John D. Carter
17.1 Introduction, 275
17.2 Reactive arthritis, 276
17.3 Pathophysiology of ReA, 277
17.4 Questions remain, 279
17.5 Conclusion, 280
Take ]home message, 280
References, 280
18 Rheumatoid arthritis: the bacterial connection, 283
Jacqueline Detert
18.1 Preclinical rheumatoid arthritis, 283
18.2 Predisposition to RA, 284
18.3 MCH ]HLA and genetic predisposition to RA, 284
18.4 Molecular mimicry in RA, 285
18.5 Innate immune system and RA, 285
18.6 Bystander activation and pattern recognition receptors, 286
18.7 Antibodies and neoepitopes, 287
18.8 Superantigens, 287
18.9 LPS, 287
18.10 Bacterial DNA and peptidoglycans, 288
18.11 Heat ]shock proteins, 288
18.12 Toll ]like and bacterial infections, 288
18.13 Proteus mirabilis, 288
18.14 Porphyromonas gingivalis and RA, 289
18.15 Gastrointestinal flora and RA, 290
18.16 Smoking, lung infection and RA, 291
18.17 Where to go from here?, 291
Take ]home message, 291
References, 292
19 Inflammatory bowel disease and the gut microbiota, 301
Nik Ding and Ailsa Hart
19.1 The microbiota in inflammatory bowel disease, 301
19.2 Dysbiosis and IBD pathogenesis, 301
19.3 Environmental factors affecting microbiome composition, 302
19.3.1 Diet, 302
19.3.2 Age, 303
19.4 
Genetics and application to the immune system and dysbiosis in IBD, 303
19.5 An overview of gut microbiota studies in IBD, 305
19.6 Specific bacterial changes in IBD, 306
19.6.1 Potentiators, 306
19.6.2 Protectors, 307
19.6.3 
Anti ]inflammatory effects of microbiota (functional dysbiosis), 308
19.7 Functional composition of microbiota in IBD, 308
19.8 Challenges, 310
19.9 Conclusion, 310
Take ]home message, 310
References, 310
20 Ankylosing spondylitis, klebsiella and the low ]starch diet, 317
Alan Ebringer, Taha Rashid and Clyde Wilson
20.1 Introduction, 317
20.2 Clinical features of AS, 317
20.3 Gut bacteria and total serum IgA, 318
20.4 Molecular mimicry in AS, 319
20.5 Pullulanase system and collagens, 320
20.6 Specific antibodies to Klebsiella in AS patients, 321
20.7 The low ]starch diet in AS, 322
20.8 Conclusions, 324
Take ]home message, 325
References, 325
21 Microbiome of chronic plaque psoriasis, 327
Lionel Fry
21.1 Introduction, 327
21.2 Microbiota in psoriasis, 329
21.2.1 Bacteria, 329
21.2.2 Fungi, 330
21.3 Variation of microbiota with site, 331
21.4 Swabs versus biopsies, 331
21.5 Psoriatic arthritis, 331
21.6 Microbiome and immunity, 332
21.7 Evidence that the skin microbiome may be involved in the pathogenesis of psoriasis, 332
21.7.1 Psoriasis and crohn’s disease, 332
21.7.2 Genetic factors, 333
21.7.3 Innate immunity, 333
21.8 New hypothesis on the pathogenesis of psoriasis, 334
Take ]home message, 334
References, 335
22 Liver disease: interactions with the intestinal microbiota, 339
Katharina Brandl and Bernd Schnabl
22.1 Introduction, 339
22.2 Non ]alcoholic fatty liver disease, 339
22.3 Qualitative and quantitative changes in the intestinal microbiota, 340
22.4 Endotoxin, 341
22.5 Ethanol, 342
22.6 Choline, 342
22.7 Alcoholic liver disease, 343
22.7.1 Qualitative and quantitative changes in the intestinal microbiome, 343
22.7.2 Contribution of dysbiosis to alcoholic liver disease, 344
Take ]home message, 346
References, 346
23 The gut microbiota: a predisposing factor in obesity, diabetes
and atherosclerosis, 351
Frida Fåk
23.1 Introduction, 351
23.2 The “obesogenic” microbiota: evidence from animal models, 351
23.3 The “obesogenic” microbiota in humans, 352
23.4 A leaky gut contributing to inflammation and adiposity, 352
23.5 Obesity ]proneness: mediated by the gut microbiota?, 353
23.6 Bacterial metabolites provide a link between bacteria and host metabolism, 353
23.7 Fecal microbiota transplants: can we change our gut bacterial profiles?, 354
23.8 What happens with the gut microbiota during weight loss?, 354
23.9 The “diabetic” microbiota, 355
23.9.1 Type I diabetes and the gut microbiota, 355
23.9.2 Type II diabetes, 355
23.10 The “atherosclerotic” microbiota, 356
23.11 Conclusions, 357
Take ]home message, 357
References, 357
24 The microbiota and susceptibility to asthma, 361
O. Salami and B J. Marsland
24.1 Introduction, 361
24.2 The microenvironment of the lower airways, 361
24.3 Development of the airway microbiota in the neonate, 362
24.3.1 Intrauterine microbial exposure and airway microbiota, 362
24.3.2 Perinatal events and airway microbiota, 363
24.3.3 Breast milk as a source of airway microbiota, 364
24.3.4 Airborne microbiota and airway microbiota, 364
24.4 Upper airway microbiota, 364
24.5 What constitutes a healthy airway microbiota, 365
24.6 Microbiota and asthma, 365
24.7 Dietary metabolites and asthma, 366
24.8 Conclusion, future perspectives and clinical implications, 367
Take ]home message, 367
References, 367
25 Microbiome and cancer, 371
Ralph Francescone and Débora B. Vendramini ]Costa
25.1 Introduction, 371
25.2 Microbiome and cancer: where is the link?, 374
25.3 Microbiome and barrier disruption, 376
25.4 Microbiome and different types of cancer, 377
25.4.1 Colon cancer, 377
25.4.2 Skin cancer, 378
25.4.3 Breast cancer, 379
25.4.4 Liver cancer, 379
25.4.5 Local microbes affecting distant cancers, 381
25.5 Microbiota and metabolism: the good and the bad sides, 382
25.6 Chemotherapy, the microbiome and the immune system, 384
25.7 Therapeutic avenues, 385
25.7.1 Modulation of bacterial enzyme activity, 385
25.7.2 Antibiotics, 386
25.7.3 Pre ] and probiotics, 386
25.7.4 Fecal transplantation, 386
25.8 Unresolved questions and future work, 387
Take ]home message, 387
References, 387
26 Colorectal cancer and the microbiota, 391
Iradj Sobhani and Séverine Couffin
26.1 Introduction, 391
26.2 Colon carcinogenesis and epidemiological data, 392
26.2.1 Human carcinogenesis model, 392
26.2.2 Age ]related risk in the general population, 393
26.2.3 Gene ] and familial ]related risks, 393
26.2.4 Environment ]related risk, 394
26.3 The microbiota, 394
26.4 Bacteria and CRCs links, 395
26.4.1 Historical data, 395
26.4.2 Clinical data, 396
26.4.3 Experimental data and mechanisms involved, 397
26.5 Hypotheses and perspectives, 402
Take ]home message, 405
References, 405
27 The gut microbiota and the CNS: an old story witha new beginning, 409
Aadil Bharwani and Paul Forsythe
27.1 Introduction, 409
27.2 The microbiota ]gut ]brain axis: a historical framework, 410
27.3 The microbiota ]gut ]brain axis: an evolutionary perspective, 411
27.4 The gut microbiota influence on brain and behavior, 413
27.5 Microbes and the hardwired gut brain axis, 415
27.5.1 The vagus, 416
27.5.2 The enteric nervous system, 417
27.6 Hormonal pathways to the brain, 418
27.7 Microbes and immune pathways to the brain, 420
27.8 Metabolites of the microbiota: short ]chain fatty acids, 421
27.9 Clinical implications of the microbiota ]gut ]brain axis, 422
27.10 Conclusion, 422
Take ]home message, 423
References, 423
28 Genetic dysbiosis: how host genetic variants may affect microbial b­iofilms, 431
Luigi Nibali
28.1 The holobiont: humans as supra ]organisms, 431
28.2 Genetic variants in the host response to microbes, 432
28.2.1 Bacterial recognition pathway, 432
28.2.2 Bacterial proliferation, 433
28.3 Genetic dysbiosis, 434
28.3.1 Genetic dysbiosis of oral biofilm, 435
28.3.2 Genetic dysbiosis of gut biofilm, 435
28.3.3 Genetic dysbiosis of skin biofilm, 436
28.3.4 Genetic dysbiosis of vaginal biofilm, 437
28.4 Summary and conclusions, 438
Take ]home message, 438
References, 438
Section 5 Mirroring the future: dysbiosis therapy, 443
29 Diet and dysbiosis, 445
M. Estaki, C. Quin and D.L. Gibson
29.1 Introduction, 445
29.2 Coevolution of the host ]microbiota super ]organism, 445
29.3 Gut microbiota in personalized diets, 446
29.4 The evolution of diet, 447
29.5 Plasticity of the microbiota and diet, 447
29.6 Interaction among gut microbiota, host and food, 448
29.7 Consequences of diet ]induced dysbiosis for host health, 450
29.8 The role of gut microbes on the digestion of macronutrients, 451
29.8.1 Carbohydrates, 451
29.8.2 Proteins, 451
29.8.3 Lipids, 452
29.9 Diet induces dysbiosis in the host, 452
29.9.1 Protein, 453
29.9.2 Carbohydrates, 453
29.9.3 Lipids, 454
29.10 The effect of maternal diet on offspring microbiota, 456
29.11 The effects of post ]natal diet on the developing microbiota of neonates, 457
29.11.1 Breast milk, 457
29.11.2 Formula, 458
29.12 Conclusion, 459
Take ]home message, 459
Host-food, 460
References, 460
30 Probiotics and prebiotics: what are they and what can they do for us?, 467
Marie José Butel, Anne ]Judith and Waligora ]Dupriet
30.1 The gut microbiota, a partnership with the host, 467
30.2 Probiotics, 467
30.2.1 Probiotics, a story that began a long time ago, 467
30.2.2 What are probiotics?, 468
30.2.3 How do probiotics work?, 468
30.2.4 Safety of probiotics, 469
30.3 Prebiotics, 470
30.3.1 What are prebiotics?, 470
30.3.2 How do prebiotics work?, 471
30.4 Synbiotics, 471
30.5 Pro ], pre ], and synbiotics in human medicine today, 471
30.5.1 Pro ] and prebiotics and infectious diarrhea, 471
30.5.2 Pro ] and prebiotics and inflammatory bowel diseases, 472
30.5.3 Pro ] and prebiotics and irritable bowel syndrome, 473
30.5.4 Pro ] and prebiotics and allergy, 474
30.5.5 Pro ] and prebiotics and obesity and diabetes, 475
30.5.6 Other indications, 475
30.5.7 Pre ] and probiotics in pediatrics, 476
30.6 Concluding remarks, 477
Take-home message, 478
References, 47831 The microbiota as target for therapeutic intervention in pediatric intestinal diseases, 483
Andrea Lo Vecchio and Alfredo Guarino
31.1 Introduction, 483
31.2 Use of probiotics in pediatric intestinal diseases, 484
31.2.1 Acute diarrhea, 484
31.2.2 Inflammatory bowel diseases, 486
31.2.3 Irritable bowel syndrome, 487
31.2.4 Infant colic, 487
31.2.5 Necrotizing enterocolitis, 488
31.3 Fecal microbiota transplantation for treatment of intestinal diseases, 488
31.3.1 Preparation and administration, 488
31.3.2 Advantages and barriers, 490
31.3.3 The use of FMT in specific intestinal diseases, 490
31.4 Conclusion, 492
Take ]home message, 493
References, 493
32 Microbial therapy for cystic fibrosis, 497
Eugenia Bruzzese, Vittoria Buccigrossi, Giusy Ranucci and Alfredo Guarino
32.1 Introduction: pathophysiology of cystic fibrosis, 497
32.2 Intestinal inflammation in CF, 498
32.3 Dysbiosis in CF, 499
32.4 Microbial therapy in CF, 502
32.5 Conclusion, 504
Take home message, 504
References, 504
Index, 000