NOVEL FRONTIERS IN THE PRODUCTION OF COMPOUNDS FOR BIOMEDICAL USE
TABLE OFCONTENTS
PART 1 – GENOMICS: THE NEW APPROACH TO THE
Raul Goldschmidt and Karen Bush
The Contribution of Genomics to the Discovery of new Antibiotics..................... 23
David J. Holmes, John P. Throup, Nicola G. Wallis, Martin K. R. Burnham,
Magdalena Zalacain, Sanjoy Biswas, Alison F. Chalker,
Karen A. Ingraham, Andrea Marra, Alex Bryant, Gary Woodnutt, Patrick V.
Warren, James R Brown, Martin Rosenberg
Abstract .................................................................................................................... 23
1. Introduction ........................................................................................................ 24
3.1 Novelty .......................................................................................................... 24
3.5 Amenable to high-throughput screening ....................................................... 26
PART 2 – ANTIBIOTICS....................................................................................33
The antibiotic gallidermin - evolution of a production process .............................. 35
1. The antibiotic gallidermin........................................................................................35
4. Stability test ......................................................................................................... 37
Markus Kempf, Uwe Theobald and Hans-Peter Fiedler
1
EDITORS PREFACE ............................................................................................... v
DISCOVERY OF NEW COMPOUNDS ........................................................ 13
The genomics approach: is it really the solution?......................................................15
Summary................................................................................................................ 15
1.The Traditional Approach..................................................................................15
2. The Biochemical Genetics Approach................................................................ 16
3. The Genomics Approach................................................................................... 17
3.1 Target identification..................................................................................... 17
3.2 Comparative genomics...................................................................................17
4. Perspectives for an Integrated Approach...........................................................20
References............................................................................................................. 21
3. The Properties of an Antibacterial Target........................................................... 24
3.2 Spectrum/Selectivity.................................................................................25
3.3 Expression during infection..................................................................25
3.4 Essential for cell viability.....................................................................26
4. Aminoacyl tRNA synthetases.........................................................................27
5. Two Component Signal Transduction Systems..................................................28
6. Discussion.........................................................................................................29
References..............................................................................................................29
2. The improvement of the production process..................................................... 36
3. Stabilisation of product-formation................................................................... 36
5. Hs-value.............................................................................................................. 37
6. Economic improvement of the production process .......................................... 41
6.2 Development of scale-up procedure ........................................................... 45
6.2.5 Pilot scale fermentations ....................................................................... 51
6.2.6 An optimised scale-up procedure for the production of gallidermin . 52
References .............................................................................................................. 54
1. Resistance to Beta-Lactams............................................................................ 57
Jozsef Aszodi* and Andre Bryskier
4.3. Mosaic Genes ............................................................................................ 65
5. Penetration Barrier ............................................................................................. 66
6. Antibiotic Efflux ............................................................................................... 68
7. The future of β -Lactams.................................................................................... 69
7.1. New Families ............................................................................................. 70
7.2. New Generations...................................................................................... 70
7.3. Potentiators of β-Lactams....................................................................... 72
8. Conclusions....................................................................................................... 74
References ............................................................................................................... 75
Resistance to aminoglycoside antibiotics: Function meets structure................. 85
Gerard D. Wright and Albert M. Berghuis
2. Aminoglycoside modifying enzymes .................................................................. 86
2.1. ANT............................................................................................................. 87
The genetics and biochemistry of resistance to glycopeptide antibiotics ........... 99
P. E. Reynolds
2
6.1 Nutrient sources......................................................................................... 42
6.2.1 Development of a fed-batch process.................................................... 45
6.2.2 Investigation of batch processes.......................................................... 49
6.2.3 Small scale fermentations................................................................... 50
6.2.4 Variation of parameters in shake flask experiments............................ 50
Resistance to β-lactams, a self-regenerating problem................................................ 57
2. Mode of Action of β-Lactams........................................................................... 57
3. Beta Lactamases............................................................................................... 59
3.1. Class Aβ-Lactamases............................................................................... 60
3.2. Class C β-Lactamases............................................................................... 61
3.3. Class D β-Lactamases.............................................................................. 61
3.4. Class B β-Lactamases.............................................................................. 62
4. PBPs................................................................................................................. 62
4.1. Naturally Resistant Pathogens.................................................................... 63
4.2. Intrinsic Resistance Through Acquisition of Resistant PBPs..................... 63
4.4. Point Mutations......................................................................................... 66
Abstract................................................................................................................. 85
1. Introduction....................................................................................................... 85
2.2. AAC.......................................................................................................... 91
2.3. APH........................................................................................................... 93
3. Conclusion........................................................................................................ 96
Acknowledgements............................................................................................... 96
References............................................................................................................. 97
1. Action of Glycopeptides: Vancomycin and Teicoplanin.............................. 99
2. Potential Mechanisms of Glycopeptide Resistance....................................... 100
3. Glycopeptide Resistance in Enterococci ....................................................... 101
3.1. The VanA phenotype.............................................................................. 101
3.2. Similarity and diversity of the resistance operons of VanA-, VanB- and
VanD-type enterococci .................................................................................. 103
3.3. VanC- type resistance ........................................................................... 108
3.3.1.VanE-type Resistance ....................................................................... 109
4. Glycopeptide Resistance in Staphylococci.................................................... 109
5. The Future ................................................................................................... 110
Acknowledgement............................................................................................... 112
References................................................................................................................ 112
3.2.1. VanB- type resistance ..................................................................... 105
3.2.2. VanD - type .................................................................... ..................... 105
β-Lactamases, an old but ever renascent problem ........................................... 117
André Matagne, Moreno Galleni, Nezha Laraki, Gianfrance Amicosante,
Gianmaria Rossolini and Jean-Marie Frère
Abstract............................................................................................................... 117
1 Introduction
2. The target of penicillin and other β-Lactams........................................... 118
3. Resistance mechanisms................................................................................. 119
4. β -Lactamases.................................................................................................. 121
5. Carbapenems and carbapenem-hydrolysing β-lactamases............................ 123
6. Hydrolysis of third-generationcephalosporins : the TEM and SHVvariants124
7. Inhibitor- resistant enzymes............................................................................ 125
8. Overproduction by deregulation of the induction system.............................. 126
Acknowledgements..............................................................................................127
. ....................................................................................................... 118
Fereshteh Naeimpoor and Ferda Mavituna
1.1 Streptomycetes..........................................................................................131
1.2 Streptomyces coelicolor ........................................................................ 133
1.4 Metabolic engineering ............................................................................. 135
1.3 Polyketides, peptide antibiotics and streptomycetes................................ 134
2. Metabolic Flux Analysis in S. coelicolor....................................................... . 135
3. Results and Discussion ............................................................................136
3.2 Effect of DifferentNitrogen Sources on Biomass Yield.........................137
3
Summary............................................................................................................ 99
3.1.1. Peptidoglycan Synthesis: a New Pathway........................................ 102
3.1.2. Peptidoglycan Synthesis: Control of Normal Host Pathway............ 102
9. Conclusion....................................................................................................... 127
References............................................................................................................ 128
Metabolic flux analysis in streptomyces coelicolor: ..............................................131
Abstract.................................................................................................................. 131
1. Introduction..................................................................................................... 131
2.1 Metabolite and Product Excretions........................................................ 136
3.1 Model description................................................................................... 136
3.3 Metabolite excretion with different nitrogen sources ............................... 138
4. Conclusion................................................................................................... 140
Acknowledgements ............................................................................................ 142
Nomenclature ...................................................................................................... 143
References ........................................................................................................... 143
1. Lysine biosynthesis: synthesis ofα -aminoadipic acid a precursor of β-lactam
antibiotics ............................................................................................................... 147
2. Relationships between lysine and penicillin biosynthesis..............................149
3. Metabolic engineering ofthe lysine pathway in P. chrysogenum................... 151
3.2.- Metabolic engineering at the homocitrate synthase level ..................... 152
4.-Futureperspectives......................................................................................... 156
Acknowledgements................................................................................................. 156
References ............................................................................................................ 156
Glycosylation of antibiotics and other agents from Actinomycetes ...................... 161
Summary................................................................................................................ 161
Metabolites: Pathways for Modified Sugars and Cyclitols. ............................... 162
Bioactive Secondary Metabolites.................................................................... 162
2.3. Cyclitols. ..................................................................................................... 164
2.4. Glycosyl transferases ............................................................................... 164
3.Examples......................................................................................................... 165
3.1 The Streptomycin Pathway..................................................................... 165
3.2 Macrolide Sugars.....................................................................................165
3.1 .- Metabolic engineering at the α-aminoadipate reductase level:
Channelling of lysine metabolic flux towards penicillin biosynthesis. ........... 151
Wolfgang Piepersberg
2.2. 6-Deoxyhexoses (6DOH) in Glycosylation of Antibiotics and Other
..
4. Conclusion .........................................................................................................166
References .......................................................................................................... 167
1. Introduction......................................................................................................... 169
2. Theory ............................................................................................................. 170
3.3 Lincosamine. ...........................................................................................166
Enzymatic synthesis of amoxicillin....................................................................... 169
A.C. Spiess and V. Kasche
2.1. Solid phase and dissolution..................................................................... 170
2.2. Suspension to suspension conversion...................................................... 172
2.3. Limiting regimes.................................................................................... 173
3.2 Enzyme activity assay............................................................................. 174
3.3 HPLC analysis.......................................................................................... 175
3. Materials and Methods................................................................................... 174
3.1Enzymes and reagent . ............................................................................... 174
4
Metabolic engineering of the lysine pathway for β-lactam overproduction in
Penicillium chrysogenum........................................................................................ 147
Casqueiro, J., Ba%26ntilde;uelos, O., Gutiérrez, S. and Martin, J.F
1. Introduction..................................................................................................... 161
2. Sugars and Cyclitols as Building Blocks in Actinomycete Secondary
3.3 Solubility measurements.......................................................................... 175
3.5 Synthesis of amoxicillin in homogeneous reaction ............................ 175
3.8 Conjugation with fluorescent dyes. ........................................................... 176
3.10 Reactor cycle:....................................................................................... 177
3.12 pH calibration and pH measurement using CLSM............................ 177
3.4 Determination of kcat and Km ............................................................. 175
3.6 Synthesis of amoxicillin in heterogeneous reaction. ................................ 176
3.7 Suspension pictures................................................................................. 176
3.9 CLSM (Confocal laser scanning microscopy) image acquisition. ......... 176
3.1 1 Image processing: ................................................................................... 177
3.13 Bipolar membrane module. Construction and operation.......................177
3.14 Module characterisation........................................................................ 177
4. Results and Discussion .................................................................................. 178
4.1. Solubility and rate of dissolution ............................................................ 178
4.2. Kinetic parameters ofamoxicillin synthesis and hydrolysis .................... 179
4.3. Evaluation of amoxicillin synthesis progress: pH, T, I - dependence ....... 180
4.4. Effect of concentration variation and pure substrate phase ......... 183
4.5. Comparison of soluble and immobilised enzyme ................................... 184
4.8. Mass transfer limit and yield prediction ................................................. 187
4.9. Proposal for integrated reaction separation process............................. 187
5. Conclusion and prospects............................................................................. 189
4.6. Activity and selectivity of immobilised enzymes................................... 185
4.7. pH profiles in immobilised biocatalysts under reaction........................ 186
New Recombinant bi- and trispecific antibody derivatives. .................................... 195
Summary ............................................................................................................ 195
Nico Mertens, Reinilde Schoonjans, An Willems, Steve Schoonooghe,
Jannick Leoen and Johan Grooten
2.1 Cell lines.................................................................................................. 199
2.2 Plasmids and gene assembly.................................................................... 199
2.3 Production and purification of recombinant antibody fragments ............ 200
2.4 T-cell proliferation assay .......................................................................... 200
3. Results and discussion................................................................................... 200
3.1Heterodimerization by CL-CH1 interaction in eukaryotic cells depends on
extension with VI andVH domains............................................................... 200
4.2 Fab-scFv fusion molecule as a model system for intermediate sized BsAb
expression of trispecific molecules.............................................................. 203
heterodimerization......................................................................................... 204
4. Discussion ...................................................................................................... 205
5
References.......................................................................................................... 190
PART 3 - PRODUCTION OF THERAPEUTIC ANTIBODIES...... 193
1.Introduction..................................................................................................... 196
2. Material and Methods...................................................................................... 199
production...................................................................................................... 202
4.3 Fd:L mediated heterodimeration of two scFv molecules leads to efficient
4.4 Influence of linker length and composition on production and
References ......................................................................................................... 206
Advantages of single-domain antigen-binding fragments derived from functional
camel heavy-chain antibodies. ............................................................................... 209
Muyldermans Serge, Conrath Katja, Vu Khoa Bang, Serrao Teresa, Busch
Magnus, Backmann Natasha, Silence Karen, Lauwereys Marc, Desmyter
Aline
1. Introduction .................................................................................................... 209
3. Single antigen-binding domain of camel heavy-chain antibodies................ 210
4. Cloning and selecting the camel variable domains of heavy chain antibodies
............................................................................................................................ 211
2. Functional heavy-chain antibodies in sera of camelids ............................. 210
5.1. Expression yield..................................................................................... 212
5.4. Specificity and affinity ........................................................................... 213
5.5. Enzyme inhibition................................................................................... 213
PART 4 - HETEROLOGOUS PROTEIN PRODUCTION:
NEW PRODUCTION STRATEGIES......................................................... 217
biopharmaceuticals.. ............................................................................................. 219
Acknowledgements............................................................................................ 214
Furin as a tool for the endoproteolytic maturation of susceptible recombinant
M. Himmelspach, B. Plaimauer, F. Dorner and U. Schlokat
1. Introduction................................................................................................... 219
2. Sorting and processing of secretory proteins................................................ 220
2.1. The constitutive and regulated secretory pathways........................... 220
2.2. Endoproteolytic processing of precursor proteins................................. 222
3. The pro-protein convertases......................................................................... 222
3.1. Identification of eukaryotic pro-protein convertases. ............................. 222
3.2. Tissue distribution, sublocalisation and function .................................... 223
4. The endoprotease furin .................................................................................. 224
4.1. Structural organisation .......................................................................... 224
4.3. Substrate specificity................................................................................ 227
5.2. Von Willebrand factor propeptide removal by full length furin ............. 232
4.2. Subcellular localisation and trafficking............................................... 226
5. Improved biotechnological processes by the use of furin...................... 231
5.1. Development of recombinant coagulation factors.............................. 231
5.3. Production of recombinant factor IX using a truncated soluble furin
vitro ............................................................................................................... 236
5.5. Use of furin in transgenic animals....................................................... 238
6
5. Characteristicsm of the single domain antibody fragments....................... 212
5.2. Solubility ................................................................................................. 212
5.3. Stability................................................................................................. 212
5.6. Multivalent constructs............................................................................ 214
5.7. Intrabodies.............................................................................................. 214
References........................................................................................................... 214
derivative........................................................................................................ 236
5.4. Processing of recombinant factor X precursors using furin derivatives in
6. Perspectives .................................................................................................... 239
Acknowledgements............................................................................................ 241
References .............................................................................................................. 241
and anti-leukaemic agents...................................................................................... 249
Mahmoud Mahmoudian
Abstract ..................................................................................................................... 249
1.Introduction...................................................................................................... 250
2. Process development for the generation ofnucleoside 5%26#39;-carboxylic acids ... 250
Acknowledgements ............................................................................................... 264
3. Abacavir (ZiagenTM)........................................................................................ 255
R.P. Singh and M. Al-Rubeai
1. Introduction .......................................................................................................... 267
2. Apoptosis: basic features of cell death.......................................................... 268
Apoptosis and the mitochondria .................................................................... 269
4. Conclusion...................................................................................................... 273
References .................................................................................................................... 273
Gram-positive Bacteria as host cells for Heterologous of production
biopharmaceuticals ................................................................................................. 277
Abstract .............................................................................................................. 277
1. Introduction ......................................................................................................... 278
2.2. Middle stage............................................................................................ 281
2.3. Late stage ................................................................................................... 281
3. Improvement of secretion............................................................................... 284
3.1. early stage secretion improvement.......................................................... 284
3.3. late stage secretion Improvement ......................................................... 285
3. Apoptosis and its control during industrial scale cell culture processes ........ 271
Lieve Van Mellaert and Jozef Anné
2. The general secretion pathway.................................................................... 279
2.1. Early stage ................................................................................................... 279
2.4. Intrinsic features of secretory proteins.................................................. 283
3.2. middle stage secretion Improvement .................................................... 285
3.3.1. SPase activity.................................................................................... 286
3.3.3. Protein folding.................................................................................. 287
4. Examples of Gram-positive bacteria as host cells for the production of
heterologous proteins......................................................................................... 287
4.1. High-level production of biopharmaceutical compounds ...................... 288
B.brevis.......................................................................................................... 289
4.2. Gram-positive bacteria as live vaccine delivery systems................. 291
4.2.1. Gram-positive bacteria used as antigen delivery systems ............... 291
7
Development of bioprocesses for the generation of anti-inflammatory, anti-viral
4. Production of the anti-leukaemic agent 506U78............................................ 258
References............................................................................................................ 264
Apoptosis and bioprocess technology..................................................................... 267
3.3.2. Proteolytic breakdown...................................................................... 286
4.2.2.Approaches for antigen presenatation............................................... 293
Clostridium tetani........................................................................................... 295
4.3. Other application areas.............................................................................. 296
Multiple Pathways of Exoprotein Secretion in Gram-negative bacteria ................. 301
1. Introduction.............................................................................................................. 301
2. The General Secretory Pathway...................................................................... 303
3. The Type I or ABC secretion pathway................................................................ 306
4. The type III or Contact Secretion Pathway...................................................... 306
5. Progress and technical problems ......................................................................... 308
6. A specific example: the Klebsiella oxytoca pullulanase secreton...................308
References ................................................................................................................. 309
5. Conclusions and perspectives........................................................................ 296
References ................................................................................................................ 297
Anthony P Pugsley
Alterations of Metabolic Flux Distributions in Recombinant Escherichia coli in
Response to Heterologous Protein Production ........................................................ 313
Jan Weber and Ursula Rinas
Summary............................................................................................................. 313
1. Introduction..................... .. .. . . . . . . . . .. . . . . . .. .. . . . . ... . . . .... .. .. .. ........ .. . . ............... ......... 313
2.Methodology.................................................................................................... 315
2.1 Metabolic Flux Analysis......................................................................... 315
2.2 Underdetermined Networks ............................................................................... 316
2.3 Why Linear Programming?........................................................................ 317
2.4 Properties of the Metabolic Network ....................................................... 318
2.5 Optimal amino acid drain for protein production..................................... 319
3.1 Production of hfgf-2 by temperature shift..................................................... 320
3.2 Temperature-induced production of human insulin ...................................326
3.3 Effect of a stable and unstable recombinant protein on the host metabolism
......................................................................................................................... 328
Embden-Meyerhof-Parnas Pathway............................................................ 332
PEP Carboxykinase and PEP Carboxlyase ............................................... 332
By-products................................................................................................. 332
TCA Cycle ............................................................................................... 332
Glyoxylate shunt ........................................................................................ 332
Transhydrogenase....................................................................................... 332
Oxidative Phosphorylation......................................................................... 333
Methylglyoxal Pathway .............................................................................. 333
Ammonium, Glutamate and Glutamine ...................................................... 333
3.Applications...................................................................................................... 320
Pentose Phosphate Pathway........................................................................ 333
8
4. Summary and Concluding Remarks............................................................ 329
Appendix............................................................................................................... 332
Bioreaction Network of E. coli....................................................................... 332
Phosphotransferase System........................................................................ 332
4.2.3. Heterologous antigens presented by Gram-positive bacteria .......... 294
RNA .............................................................................................................. 334
Lipids ............................................................................................................. 334
Lipopolysaccharide.................................................................................... 334
Peptidoglucane ......................................................................................... 334
Glycogen ............................................................................................................ 334
One-carbon unit and Polyamine .............................................................................. 334
Biomass...................................................................................................... 335
Miscellaneous .................................................................................................... 335
Fibroblast Growth Factor hFGF-2............................................................. 335
Objective .................................................................................................................. 335
recombinant protein .............................................................................................. 339
References.......................................................................................................... 335
Dynamics of proteolysis and its influence on the accumulation of intracellular
Rozkov A., Yang S. and Enfors S.-O
Summary .................................................................................................................... 339
1.Introduction..................................................................................................... 339
2.Materials and Methods..................................................................................... 341
2.1 Microorganism ....................................................................................... 341
2 .2 Media and Cultivation: ........................................................................... 341
2.3 Product concentrations ........................................................................... 342
2.4 Determination of the proteolysis rate constant .................................. 342
3. Results and Discussion.................................................................................. 342
4. Conclusions.................................................................................................... 346
PART 5 – ARTIFICIAL ORGANS AND XENOGRAFTING........... 349
The impact of transgenesis and cloning on cell and organ xenotransplantation to
humans ................................................................................................................... 351
Summary ............................................................................................................ 351
1. Why xenografting ? ...................................................................................... 352
3. The mechanisms ofxenograft rejection........................................................... 354
4. The preventive treatments of xenograft rejection........................................... 355
5. The biosafety of xenografting......................................................................... 358
6. The acceptability of xenografting ....................................................................... 359
7.Conclusion and perspectives........................................................................ 361
References.............................................................................................................. 361
Abstract ...................................................................................................................... 365
1. Introduction ................................................................................................... 365
2. Materials and Methods.................................................................................. 367
Louis-Marie Houdebine , Bernard Weill
Reinforced Bioartificial Skin In The Form Of Collagen Sponge And Threads....... 365
Eun Kyung Yang, Young Kwon Seo and Jung Keug Park
9
Amino Acids............................................................................................. 333
Pretein........................................................................................................ 334
Nucleotides................................................................................................. 334
DNA........................................................................................................... 334
References........................................................................................................... 346
2.1 Collagen scaffolds ...................................................................................... 367
2.1.1 Extraction of Type I Collagen Solution ............................................... 367
2.1.2 Fabrication of Macroporous Collagen Sponge..................................... 367
2.1.3 Fabrication of Collagen Threads and Mesh ........................................ 368
2.1.5 Reinforcement by Incorporating with Collagen Mesh........................ 370
2.1.6 Measurement of Mechanical Strength............................................... 370
2.1.7 Morphological Analysis...................................................................... 370
2.2.1 Primary Cell Culture ............................................................................... 371
2.2.2 Culture of Bioartificial Skin............................................................... 371
2.2.3 Morphology Examination .................................................................. 372
2.1.4 Reinforcement by Cross-linking Treatments ......................................... 369
2.2 Bioartificial skin.............................................................................................. 371
3. Results and Discussion.......................................................................................... 372
3.1 Collagen scaffolds...................................................................................... 372
3.2 Bioartificial skin ........................................................................................... 377
Acknowledgement................................................................................................. 379
PART 6 - ANTITUMOUR COMPOUNDS ................................................381
combinatorial biosynthesis........................................................................................... 383
Towards the generation of novel antitumour agents from actinomycetes by
Jose A. Salas, Gloria Blanco, Alfredo F. Bra%26ntilde;a, Ernestina Fernandez,
Ma Jose Fernandez, Jose Garcia Bernardo, Ana Gonzalez, Felipe Lombo,
Laura Prado, Luis M. Quiros, Cesar Sanchez and Carmen Mendez
1. Introduction ..................................................................................................... 383
3. The aureolic acidgroup........................................................................................ 386
2. Anticancer biosynthetic gene clusters.............................................................. 385
3.1. Genes involved in the biosynthesis of the polyketide moiety. ................... 387
3.2. Genes encoding enzymes modifying the polyketide skeleton.................... 388
3.3. Genes encoding an activated methyl cycle................................................ 388
3.4. Genes encoding sugar biosynthetic enzymes ............................................. 388
3.5. Genes encoding glycosyltransferases.......................................................... 389
3.6. Genes responsible for resistance and secretion................................... 389
4.1. Insertional inactivation.............................................................................. 390
4.3. Combinatorial biosynthesis ............................................................................ 394
4. Generation of novel compounds....................................................................... 390
4.2. Tailoring modification ............................................................................. 392
5. Concluding remarks ............................................................................................... 397
Acknowledgements .............................................................................................. 397
References.................................................................................................................. 397
Cell immobilisation of Taxus media .............................................................................. 401
Summary.................................................................................................................... 401
Chi Wai Tang and Ferda Mavituna
10
3.1.1 Preparation of Collagen Threads........................................................ 372
3.1.2 Ultimate Tensile Strength of Collagen Sponges.................................... 374
4. Conclusion and Future work............................................................................... 379
References.............................................................................................................. 380
2.4 Bioreactor: ............................................................................................... 403
3. Resultsand Discussion......................................................................................403
3.2 Suspension culture: ..................................................................................... 406
3.3 Immobilisation: ........................................................................................... 406
3.4 Bioreactors: ................................................................................................. 406
References............................................................................................................. 406
The role of prebiotics in human gut microbiology.................................................... 411
2. Materials and Methods................................................................................. 402
3.1 Effect ofmedia on callus initiation from explants: .. ..................................... 403
Catherine E . Rycroft, Robert A . Rastall and Glenn R . Gibson
Abstract ...................................................................................................................411
1. The Human Large Intestine.......................................................................... 412
2. Beneficial and Pathogenic Bacteria................................................................. 413
3. The Prebiotic Concept................................................................................... 413
4. Methods for Evaluating Prebiotics............................................................... 413
4.1. In vitro methods.................................................................................... 414
4.2. In vivo methods.................................................................................... 414
4.3. Use of molecular methods..........................................................................414
5. Bifidogenic Factors............................................................................................ 415
6. Oligosaccharides as Prebiotics........................................................................ 415
6.1 Lactulose .....................................................................................................416
6.3. Galacto-oligosaccharides ................................................................................ 418
6.5. Lactosucrose .......................................................................................... 421
6.6. Isomalto-oligosaccharides .....................................................................422
6.2. Inulinandfructo-oligosaccharides........................................................... 416
6.4. Soybean oligosaccharides ..................................................................... 420
6.7. Gluco -oligosaccharides ....................................................................... 423
7. Conclusions................................................................................................... 425
References........................................................................................................... 425
The influence of intestinal microflora on mucosal and systemic immune responses
............................................................................................................................. 429
Stephanie Blum, Dirk Haller, Susana Alvarez, Pablo Perez and Eduardo J .
Schiffrin
1. Introduction ........................................................................................................... 430
3.Adaptive immunity at mucosal sites ............................................................. 431
2.The innate immune system............................................................................ 430
11
1.Introduction.................................................................................................... 401
2.1 Plant Material and Callus induction....................................................... 402
2.2 Callus growth measurement..................................................................... 403
2.3 Suspension Culture and Cell Immobilisation:. ....................................... 403
4. Conclusion.................................................................................................... 406
PART7 – PRE AND PROBIOTICS...............................................................409
6.8. Xylo-oligosaccharides.............................................................................425
Summary............................................................................................................ 429
Abbreviations..................................................................................................... 430
3.1 The mucosal secretory immune system...................................................... 431
3.2 Stimulation of IgA production by the probiotic microorganism L.johnsonii
La1 .................................................................................................................... 432
4. The epithelial compartment.......................................................................... 433
4.1 Intraepithelial lymphocytes....................................................................... 433
4.3 . Intestinal epithelial cells as active partners in mucosal immune defenses
............................................................................................................................ 434
5. Modulation of the mucosal immune response by commensal bacteria.......... 434
........................................................................................................................... 434
populations:.................................................................................................... 436
6. Modulation of the systemic immune response............................................... 438
7. Regulation of the mucosal immune response by luminal factors. New
perspectives ........................................................................................................... 442
8. Conclusion...................................................................................................... 443
References............................................................................................................... 443
INDEX.................................................................................................................... 447
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