Detection and characterization oflipopeptide-specific T cells in guinea pigs sensitized with bacteria of the Mycobacterium tuberculosis complex:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
Gießen, Lahn
VVB Laufersweiler Verlag
2016
|
| Schriftenreihe: | Edition Scientifique
|
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | XIII, 277 Seiten 21 cm x 14.6 cm, 373 g |
| ISBN: | 9783835964426 3835964429 |
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| 100 | 1 | |a Kaufmann, Eva |e Verfasser |0 (DE-588)1103463594 |4 aut | |
| 245 | 1 | 0 | |a Detection and characterization oflipopeptide-specific T cells in guinea pigs sensitized with bacteria of the Mycobacterium tuberculosis complex |c eingereicht von Eva Kaufmann, Heppenheim/Bergstraße |
| 264 | 1 | |a Gießen, Lahn |b VVB Laufersweiler Verlag |c 2016 | |
| 300 | |a XIII, 277 Seiten |c 21 cm x 14.6 cm, 373 g | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 490 | 0 | |a Edition Scientifique | |
| 502 | |b Dissertation |c Justus-Liebig-Universität |d 2016 | ||
| 650 | 7 | |a pigs |2 cabt | |
| 650 | 7 | |a T lymphocytes |2 cabt | |
| 650 | 7 | |a detection |2 cabt | |
| 650 | 7 | |a bacteria |2 cabt | |
| 650 | 7 | |a mycobacterium tuberculosis |2 cabt | |
| 650 | 7 | |a characterization |2 cabt | |
| 653 | |a Doktorarbeit | ||
| 653 | |a Uni | ||
| 653 | |a Wissenschaft | ||
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Datensatz im Suchindex
| _version_ | 1804178465252966400 |
|---|---|
| adam_text | TABLE OF CONTENTS
1 INTRODUCTION AND O
BJECTIVE...................................................................................................
15
2 LITERATURE REVIEW
....................................................................................................................
17
2.1 MYCOBACTERIUM TUBERCULOSIS
COMPLEX....................................................................................
17
2.1.1 TAXONOMY AND GENERAL CHARACTERISTICS OF M. TUBERCULOSIS AND M.
BOVIS
............................
17
2.1.2 TUBERCULOSIS AS
ZOONOSIS.....................................................................................................18
2.1.3 GENETIC EVOLUTION OF THE MYCOBACTERIUM TUBERCULOSIS COMPLEX
.........................................
19
2.1.4 BEYOND THE MYCOBACTERIUM TUBERCULOSIS COMPLEX: NON-TUBERCULOUS
MYCOBACTERIA..............................................................................................
20
2.2 THE MYCOBACTERIAL CELL
ENVELOPE............................................................................................
21
2.3 MYCOBACTERIAL
ANTIGENS...........................................................................................................23
2.3.1
TUBERCULINS...........................................................................................................................23
2.3.1.1 KOCH*S OLD TUBERCULIN AND PURIFIED PROTEIN DERIVATIVES
..................................................
23
2.3.1.2 THE PROTEOME OF PURIFIED PROTEIN DERIVATIVES
..................................................................
24
2.3.2 PROTEIN
ANTIGENS...................................................................................................................25
2.3.3 LIPID
ANTIGENS.......................................................................................................................28
2.3.4 LIPOPROTEIN
ANTIGENS.............................................................................................................30
2.3.4.1 BIOSYNTHESIS OF MYCOBACTERIAL
LIPOPROTEINS.......................................................................30
2.3.4.2 OVERVIEW OF MYCOBACTERIUM TUBERCULOSIS LIPOPROTEINS
.....................................................
31
2.3.4.3 MYCOBACTERIAL LIPOPROTEIN MUTANT
STRAINS...........................................................................33
2.4 HUMAN AND BOVINE
TUBERCULOSIS.............................................................................................
33
2.4.1 THE IMMUNE RESPONSES IN PRIMARY
TUBERCULOSIS..................................................................33
2.4.1.1 INITIAL RECOGNITION AND REPLICATION OF
MYCOBACTERIUM TUBERCULOSIS
....................................
33
2.4.1.2 INNATE IMMUNE RESPONSES WITH PARTICULAR FOCUS ON MYCOBACTERIAL
LIPOPROTEIN
ANTIGENS..........................................................................................................
35
2.4.1.3 ADAPTIVE IMMUNE RESPONSES TO MYCOBACTERIAL LIPOPROTEIN ANTIGENS
.................................
37
2.4.1.4 THE PROTECTIVE EFFECT OF IMMUNE RESPONSES ELICITED BY
MYCOBACTERIAL
LIPOPROTEINS........................................................................................................................39
2.4.2 GRANULOMAS AND CASEATION IN HUMAN AND BOVINE TUBERCULOSIS
..........................................
40
2.4.2.1 THE IMMUNOLOGY OF TUBERCULOSIS GRANULOMAS
..................................................................
40
2.4.2.2 CASEATION AND PATHOMORPHOLOGICAL DIFFERENCES BETWEEN M.
TUBERCULOSIS AND
M. BOVIS
INFECTIONS...........................................................................................................
42
2.5 BACILLE
CALMETTE-GUERIN..........................................................................................................
46
2.5.1 ORIGIN AND
CHARACTERISTICS....................................................................................................
46
2.5.2 UTILIZATION AS LIVE VACCINE AND CURRENT MODIFICATIONS
..........................................................
50
2.6 THE GUINEA PIG AS A SMALL ANIMAL MODEL OF TUBERCULOSIS
.......................................................
57
2.6.1 TUBERCULOSIS OF THE GUINEA PIG
............................................................................................
57
2.6.2 USE OF THE GUINEA PIG AS A SMALL ANIMAL MODEL FOR TUBERCULOSIS
RESEARCH
.........................
59
3 MATERIAL AND M
ETHODS..........................................................................................................61
3.1 BACTERIAL
STRAINS......................................................................................................................
61
3.2 MEDIA AND
BUFFERS...................................................................................................................62
3.3
ANTIGENS..................................................................................................................................
62
3.3.1 BACTERIAL CULTURE SUPERNATANT
..............................................................................................
62
3.3.2
TUBERCULINS.........................................................................................................................62
3.3.3 CHLOROFORM-METHANOL EXTRACTS
(CMES)...................................................... 64
3.3.4 LIPOPEPTIDE-ENRICHED SUBTRACTION
(LPPEL)..........................................................................67
3.3.5 ULTRASONICATION OF BACTERIAL
PELLETS......................................................................................
68
3.3.6 HEAT INACTIVATION OF
BACTERIA...............................................................................................
69
3.3.7 DISSOLVING OF CME ANTIGENS IN DIFFERENT
SOLVENTS................................................................69
3.3.8 PHENOL-WATER PARTITIONING OF
CMES.....................................................................................
69
3.3.9 BIOCHEMICAL DEGRADATION OF
ANTIGENS..................................................................................
70
3.3.9.1 MILD SODIUM HYDROXIDE TREATMENT OF
ANTIGENS...................................................................70
3.3.9.2 PROTEINASE K TREATMENT OF
ANTIGENS...................................................................................
71
3.3.9.3 PRONASE E TREATMENT OF
ANTIGENS....................................................................................72
3.3.10 CHROMATOGRAPHIC PURIFICATION OF
ANTIGENS...........................................................................72
3.3.10.1 CHROMATOGRAPHIC PURIFICATION OF PHENOL-PARTITIONED CME
...............................................
72
3.3.10.2 CHROMATOGRAPHIC APPROACH WITH PRESELECTION OF LIPOPHILIC
PEPTIDES
................................
73
3.3.10.3 FURTHER PROCESSING OF THE COLLECTED
FRACTIONS...................................................................73
3.3.11 SYNTHETIC PEPTIDES OF
M. TUBERCULOSIS
LIPOPROTEINS..............................................................74
3.3.12 THE RECOMBINANT PROTEIN AG85A OF M.
TUBERCULOSIS...........................................................74
3.4 ANTIGEN
CHARACTERIZATION.........................................................................................................74
3.4.1 SDS-PAGE
ANALYSIS............................................................................................................74
3.4.1.1 COOMASSIE BLUE
STAINING..................................................................................................
75
3.4.1.2 SILVER
STAINING.....................................................................................................................76
3.4.1.3 WESTERN
BLOTTING.................................................................................................................76
3.4.2 THIN LAYER
CHROMATOGRAPHY.................................................................................................
77
3.4.3 BRADFORD
ASSAY.....................................................................................................................78
3.5
ANIMALS..................................................................................................................................78
3.5.1 DUNKIN-HARTLEY GUINEA
PIGS.................................................................................................
76
3.5.2 INBRED STRAIN 2 GUINEA
PIGS.................................................................................................
79
3.5.3 NUMBERS OF GUINEA PIGS USED IN THE PRESENT
STUDY............................................... 80
3.6 ANIMAL
EXPERIMENTS.................................................................................................................80
3.6.1
ANESTHESIA...........................................................................................................................
80
3.6.2 SENSITIZATION
PROCEDURES......................................................................................................81
3.6.2.1
BCG.................................................................................................................................81
3.6.2.2
IAN5...................................................................................................................................
81
3.6.3 BLOOD SAMPLE COLLECTION BY
CARDIOCENTESIS..........................................................................61
3.6.4
EUTHANASIA...........................................................................................................................62
3.7 SERUM PROCESSING AND CYTOLOGICAL TECHNIQUES
.....................................................................
83
3.7.1 SERUM
PROCESSING................................................................................................................63
3.7.2 PBMC
PURIFICATION................................................................................................................83
3.7.3 FREEZING OF PBMCS AND CELL
LINES.......................................................................................
84
3.7.4 IRRADIATION OF PBMCS
.................................................
.........................................................
84
3.6 CYTOLOGICAL
ANALYSES................................................................................................................64
3.8.1 EX VIVO LYMPHOCYTE PROLIFERATION ASSAY (CFSE ASSAY)
.......................................................
84
3.8.2 LYMPHOCYTE RESTIMULATION
ASSAY.........................................................................................
85
3.8.2.1 ANTIGEN-SPECIFIC EXPANSION OF
LYMPHOCYTES......................................................................86
3.8.2.2 SORTING OF EXPANDED
LYMPHOCYTES.....................................................................................
66
3.8.2.3 RESTIMULATION OF SORTED
LYMPHOCYTES................................................................................
67
3.8.3 PHENOTYPICAL CHARACTERIZATION OF
LYMPHOCYTES....................................................................67
3.8.4 FLOW
CYTOMETRY.....................................................................................................................68
3.8.5 TLR2
ASSAY..........................................................................................................................93
3.9 TNFA
ELISA............................................................................................................................93
3.10 ESTABLISHMENT AND CHARACTERIZATION OF GUINEA PIG T CELL
LINES.................................................94
3.10.1 PRODUCTION OF RECOMBINANT GUINEA PIG LNTERLEUKIN-2
...........................................................
94
3.10.2 PRIMARY T CELL CULTURE
.........................................................................................................
95
3.10.3 LIMITING
DILUTION....................................................................................................................95
3.10.4 MAINTENANCE, TRANSFER AND STIMULATION OF T CELL LINES
.........................................................
96
3.10.5 SPECIFICITY ASSAYS FOR GUINEA PIG T CELL
LINES.......................................................................96
3.10.6 FREEZING OF GUINEA PIG T CELL
LINES.......................................................................................
96
3.11
STATISTICS.................................................................................................................................96
4
RESULTS......................................................................................................................................98
4.1 EX VIVO LYMPHOCYTE PROLIFERATIVE RESPONSES TO TUBERCULINS AND
CMES....................................98
4.1.1 LYMPHOCYTE PROLIFERATION IN RESPONSE TO TUBERCULINS
..........................................................
96
4.1.2 LYMPHOCYTE PROLIFERATION IN RESPONSE TO CHLOROFORM-METHANOL
EXTRACTS
.............................
105
4.1.3 PHENOTYPICAL CHARACTERIZATION OF RESPONDING LYMPHOCYTES
................................................
114
4.2 SPECIFICATION OF THE STIMULATORY
ANTIGENS.................................................................................
116
4.2.1 BIOCHEMICAL DEGRADATION OF
ANTIGENS..................................................................................
116
4.2.2 THE STIMULATORY POTENTIAL OF A LIPOPEPTIDE-ENRICHED SUBFRACTION
(LPPEL)
...........................
123
4.3 ANTIGEN SPECIFICITY OF THE PROLIFERATING
LYMPHOCYTES................................................................127
4.3.1 ENRICHMENT OF EXPANDED LYMPHOCYTES BY MAGNETIC SORTING
...............................................
127
4.3.2 ASSESSMENT OF THE ANTIGEN SPECIFICITY OF THE SORTED LYMPHOCYTES
......................................
131
4.4 ANALYSIS OF THE STIMULATORY
ANTIGENS......................................................................................135
4.4.1 CHARACTERISTICS OF DIFFERENT CME
PREPARATIONS....................................................................135
4.4.2 STRAIN SPECIFICITY OF THE STIMULATORY
ANTIGENS.......................................................................142
4.4.3 DISTRIBUTION OF THE STIMULATORY ANTIGENS WITHIN THE BACTERIAL CELL
.........................................
145
4.5 APPROACHES TO IDENTIFY INDIVIDUAL MYCOBACTERIAL LIPOPEPTIDE ANTIGENS
..................................
147
4.5.1 CHROMATOGRAPHIC PURIFICATION OF MYCOBACTERIAL LIPOPEPTIDE ANTIGENS
.................................
147
4.5.1.1 SOLUBILITY OF CME ANTIGENS IN DIFFERENT
SOLVENTS................................................................147
4.5.1.2 CHROMATOGRAPHIC APPROACHES AND INITIAL CHARACTERIZATION OF
PURIFIED FRACTIONS
................
150
4.5.1.2.1 CHROMATOGRAPHIC PURIFICATION ATTEMPT WITH INITIAL PHENOL
PARTITIONING
..........................
150
4.5.1.2.2 CHROMATOGRAPHIC PURIFICATION ATTEMPT WITH PRESELECTION OF
LIPOPHILIC PEPTIDES
............
154
4.5.1.3 LYMPHOCYTE STIMULATION BY THE CHROMATOGRAPHICALLY PURIFIED
FRACTIONS
............................
155
4.5.2 EX VIVO LYMPHOCYTE PROLIFERATION IN RESPONSE TO SYNTHETIC
N-TERMINAL PEPTIDES
OF M. TUBERCULOSIS
LIPOPROTEINS.............................................................................................
156
4.6 DEVELOPMENT AND CHARACTERIZATION OF GUINEA PIG T CELL
LINES.................................................165
5
DISCUSSION.................................................................................................................................170
5.1 GUINEA PIGS MOUNT PROMINENT LIPID-REACTIVE T CELL RESPONSES UPON
SENSITIZATION
WITH BACTERIA OF THE MYCOBACTERIUM TUBERCULOSIS
COMPLEX.....................................................170
5.2 THE GUINEA PIG MODEL PROVIDES IMMUNOLOGICAL EVIDENCE FOR THE
STIMULATORY
IMPORTANCE OF LIPOPEPTIDES IN MYCOBACTERIAL LIPID
PREPARATIONS...............................................174
5.3 BCG SENSITIZATION INDUCES LYMPHOCYTE RESPONSES AGAINST LIPID
ANTIGENS
EXPRESSED BY SEVERAL TUBERCULOUS BUT ONLY RARELY BY NON-TUBERCULOUS
MYCOBACTERIA
............
179
5.4 SYNTHETIC PEPTIDES REVEAL POTENTIAL STIMULATION BY DEFINED M.
TUBERCULOSIS
LIPOPEPTIDE
ANTIGENS................................................................................................................187
5.5
CONCLUSIONS.............................................................................................................................
194
6
SUMMARY...................................................................................................................................
196
7 ZUSAM
MENFASSUNG.................................................................................................................199
8
REFERENCES...............................................................................................................................
202
9 A
NNEX........................................................................................................................................
240
ANNEX 1 DEFINED PROTEINS IN PURIFIED PROTEIN DERIVATIVES
.........................................................
240
ANNEX 2 MULTIPLE CARDIOCENTESES IN GUINEA
PIGS.......................................................................243
ANNEX 3 MEDIA, BUFFERS, CHEMICALS, CONSUMABLES AND OTHER MATERIAL,
ANTIBODIES...................249
ANNEX 4 INSTRUMENTS AND
DEVICES.............................................................................................
255
ANNEX 5 N-TERMINAL AMINO ACID SEQUENCES OF M. TUBERCULOSIS LIPOPROTEINS
............................
258
ANNEX 6 NUMBERS OF DUNKIN-HARTLEY GUINEA PIGS USED IN THE PRESENT STUDY
..........................
264
10
ABBREVIATIONS...........................................................................................................................
267
11 LIST OF
FIGURES............................................................................................................................
271
12 LIST OF
TABLES.............................................................................................................................
273
13
ACKNOWLEDGEMENTS.................................................................................................................274
14
ERKLAERUNG...................................................................................................................................277
|
| any_adam_object | 1 |
| author | Kaufmann, Eva |
| author_GND | (DE-588)1103463594 |
| author_facet | Kaufmann, Eva |
| author_role | aut |
| author_sort | Kaufmann, Eva |
| author_variant | e k ek |
| building | Verbundindex |
| bvnumber | BV044906444 |
| ctrlnum | (OCoLC)1031428493 (DE-599)DNB1102946915 |
| dewey-full | 630 |
| dewey-hundreds | 600 - Technology (Applied sciences) |
| dewey-ones | 630 - Agriculture and related technologies |
| dewey-raw | 630 |
| dewey-search | 630 |
| dewey-sort | 3630 |
| dewey-tens | 630 - Agriculture and related technologies |
| discipline | Agrar-/Forst-/Ernährungs-/Haushaltswissenschaft / Gartenbau |
| format | Thesis Book |
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| illustrated | Not Illustrated |
| indexdate | 2024-07-10T08:04:24Z |
| institution | BVB |
| institution_GND | (DE-588)1065553803 |
| isbn | 9783835964426 3835964429 |
| language | English |
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| physical | XIII, 277 Seiten 21 cm x 14.6 cm, 373 g |
| publishDate | 2016 |
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| publisher | VVB Laufersweiler Verlag |
| record_format | marc |
| series2 | Edition Scientifique |
| spelling | Kaufmann, Eva Verfasser (DE-588)1103463594 aut Detection and characterization oflipopeptide-specific T cells in guinea pigs sensitized with bacteria of the Mycobacterium tuberculosis complex eingereicht von Eva Kaufmann, Heppenheim/Bergstraße Gießen, Lahn VVB Laufersweiler Verlag 2016 XIII, 277 Seiten 21 cm x 14.6 cm, 373 g txt rdacontent n rdamedia nc rdacarrier Edition Scientifique Dissertation Justus-Liebig-Universität 2016 pigs cabt T lymphocytes cabt detection cabt bacteria cabt mycobacterium tuberculosis cabt characterization cabt Doktorarbeit Uni Wissenschaft (DE-588)4113937-9 Hochschulschrift gnd-content VVB LAUFERSWEILER Verlag (DE-588)1065553803 pbl DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030300100&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Kaufmann, Eva Detection and characterization oflipopeptide-specific T cells in guinea pigs sensitized with bacteria of the Mycobacterium tuberculosis complex pigs cabt T lymphocytes cabt detection cabt bacteria cabt mycobacterium tuberculosis cabt characterization cabt |
| subject_GND | (DE-588)4113937-9 |
| title | Detection and characterization oflipopeptide-specific T cells in guinea pigs sensitized with bacteria of the Mycobacterium tuberculosis complex |
| title_auth | Detection and characterization oflipopeptide-specific T cells in guinea pigs sensitized with bacteria of the Mycobacterium tuberculosis complex |
| title_exact_search | Detection and characterization oflipopeptide-specific T cells in guinea pigs sensitized with bacteria of the Mycobacterium tuberculosis complex |
| title_full | Detection and characterization oflipopeptide-specific T cells in guinea pigs sensitized with bacteria of the Mycobacterium tuberculosis complex eingereicht von Eva Kaufmann, Heppenheim/Bergstraße |
| title_fullStr | Detection and characterization oflipopeptide-specific T cells in guinea pigs sensitized with bacteria of the Mycobacterium tuberculosis complex eingereicht von Eva Kaufmann, Heppenheim/Bergstraße |
| title_full_unstemmed | Detection and characterization oflipopeptide-specific T cells in guinea pigs sensitized with bacteria of the Mycobacterium tuberculosis complex eingereicht von Eva Kaufmann, Heppenheim/Bergstraße |
| title_short | Detection and characterization oflipopeptide-specific T cells in guinea pigs sensitized with bacteria of the Mycobacterium tuberculosis complex |
| title_sort | detection and characterization oflipopeptide specific t cells in guinea pigs sensitized with bacteria of the mycobacterium tuberculosis complex |
| topic | pigs cabt T lymphocytes cabt detection cabt bacteria cabt mycobacterium tuberculosis cabt characterization cabt |
| topic_facet | pigs T lymphocytes detection bacteria mycobacterium tuberculosis characterization Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030300100&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT kaufmanneva detectionandcharacterizationoflipopeptidespecifictcellsinguineapigssensitizedwithbacteriaofthemycobacteriumtuberculosiscomplex AT vvblaufersweilerverlag detectionandcharacterizationoflipopeptidespecifictcellsinguineapigssensitizedwithbacteriaofthemycobacteriumtuberculosiscomplex |