THE IMMUNOMODULATING ACTIVITY OF LEVAMISOLE AND GAMMA-INTERFERON
ON EXPERIMENTAL MURINE INFECTIONS WITH MYCOBACTERIUM MICROTI AND
MYCOBACTERIUM TUBERCULOSIS AND THE INFLUENCE OF GAMMA-INTERFERON
ON THE BACTERICIDAL ACTIVITY OF ISONIAZID AND RIFAMPICIN
A Thesis submitted for the degree of
Doctor of Philosophy
by
SIEW YAN KHOR
The Royal Postgraduate Medical School
University of London
November, 1985 2
ABSTRACT
Though remarkably successful, chemotherapy of tuberculosis still has
to be given for at least six months and relapses due to endogenous
reactivation remain a problem. Immunomodulating agents which may
complement antituberculous drugs could thus play an important role
in shortening or improving current chemotherapy. This thesis
reports on the effects of two immunomodulating agents, levamisole
and murine gamma-interferon (IFN-X), on models of murine
mycobacterial infections.
Levamisole was investigated in an infection of Mycobacterium microti
and did not have any significant effect on the in vivo growth of
M. microti. The effects of IFN-X were examined in an infection of
M. tuberculosis strain H37Rv. Dosage of 1000 units of IFN-X per mouse every three days with the first dose before infection, caused
a statistically significant but small reduction in bacillary growth
in the organs which occurred mainly in the first day after infec
tion. An increase in dose size, multiple daily dosage before
infection and encapsulation of IFN-X in liposomes did not increase
the effect. IFN-X treatment of mice infected five days previously
did not influence bacillary growth. The administration of IFN-X both before and after infection, did not increase the bactericidal activities of isoniazid or rifampicin. There was no effect of IFN-X on organ counts of mice previously treated for five days with isoniazid. 3
The effects of IFN-Y on the growth of Listeria monocytogenes and
M. microti in peritoneal macrophage monolayers were also examined.
Peritoneal macrophages previously exposed to IFN-Y were activated for listericidal activity. Macrophages incubated with IFN-ft for 48 or 72 hours before infection with M. microti were activated to be bactericidal in the first 15-30 minutes after infection. The addition of IFN-Y after infection with M. microti resulted in an inhibition of growth that occurred after a delay of 24 hours. The addition of IFN-Y did not increase the bactericidal activities of isoniazid or rifampicin. It is unlikely that levamisole or IFN-Y will have a major impact on the treatment of active tuberculosis. 4
ACKNOWLEDGEMENTS
I would like to thank my supervisor, Professor D.A. Mitchison for all his advice, encouragement and helpful discussions throughout
this work. My thanks are also due to Dr D.B. Lowrie and Dr Ann Rees for their interest and advice and Dr A.R. Coates for supplying the recombinant interferon-gamma. I would also like to acknowledge
Mr V.R. Aber for statistical assistance, members of the former MRC
Unit for Laboratory Studies of Tuberculosis for technical advice and the Commonwealth Fellowship Commission in the United Kingdom for the award of the Commonwealth Fellowship.
Finally, I would like to express my appreciation to my family and
Jeremy for all their support and encouragement. 5
CONTENTS PAGE
List of Tables 12
List of Illustrations 14
1. LITERATURE REVIEW AND INTRODUCTION
I. Literature review
1.1 Tuberculosis 18
^.1.1 The mycobacteria 18
1.1.2 Pathogenesis of tuberculosis 21
1.1.3 The immune response to tuberculosis 23
1.1.4 Mycobacterium-macrophage interactions 29
1.1.4.1 Microbicidal mechanisms in macrophages 30
1.1.4.2 Mycobacterial evasion of macrophage killing mechanisms 33
1.1.4.3 Genetic susceptibility 35
1.2 Tuberculosis control 36
1.2.1 BCG Vaccination 37
1.2.2 Case-finding and chemotherapy 39
1.2.2.1 Case-finding 40
1.2.2.2 Chemotherapy 41
1.2.3 Chemoprophylaxis 45
1.3 Immunomodulation and immunomodulating agents 47
1.3.1 Agents derived from microorganisms 48
1.3.2 Agents derived from mammalian cells 50
1.3.3 Synthetic immumomodulating agents 52
1.4 Interferon 54
1.4.1 Description of the interferons 54
1.4.2 Molecules and genes of the interferons 55 6
1.4.3 Mechanisms of action of interferons 56
1.4.4 Immunomodulating effects of the interferons 58
1.4.4.1 Effects on humoral immunity 59
1.4.4.2 Effects on cell surface membrane 59
1.4.4.3 Effects on cell mediated immunity 61
1.5 Levamisole 63
1.5.1 Description of levamisole 63
1.5.2 Immunomodulating effects of levamisole 64
1.5.3 Mechanisms of action of levamisole 66
1.6 Targeting of drugs 67
1.6.1 Description of drug carriers 67
1.6.2 Liposomes 68
1.6.2.1 Types of liposomes 68
1.6.2.2 In vivo interactions of liposomes 69
1.6.2.3 Therapeutic applications of liposomes 70
II. Introductionand objectives of the experimental work 73
1.7 Introduction and objectives 73
2 MATERIALS AND METHODS 78
2.1 Media 78
2.1.1 Bacteriological culture media and supplements 78
2.1.2 Tissue culture media and supplements 79
2.2 Chemicals and reagents 81
2.3 Bacteria 83
2.3.1 Bacterial strains 83
2.3.2 Animal passage and maintenance 83
2.4 Animals 85
2.4.1 Mouse strains 85 7
2.4.2 Random allocation and husbandry of mice 85
2.5 Immunomodulating and chemotherapeutic agents 86
2.5.1 Agents used 86
2.5.2 In vivo administration 86
2.5.2.1 Preparation of liposomes 87
2.5.3 In vitro administration of agents 88
2.6 Preparation of infective inocula 88
2.6.1 Enumeration of total number of bacilli 88
2.6.2 Infective inocula for in vivo experiments 89
2.6.3 Infective inocula for in vitro experiments 89
2.7 Investigation of the T lymphocyte response 91
2.7.1 Proliferative response of splenic T lymphocytes 91
2.7.1.1 Preparation of splenic lymphocytes 91
2.7.1.2 Antigens and mitogens 91
2.7.1.3 Lymphocyte proliferation test 92
2.7.2 Immunoenzymatic staining of splenic T lymphocyte subsets 93
2.7.2.1 Antibodies and sera 93
2.7.2.2 Cytospin preparations 94
2.7.2.3 Preparation of buffers, fixative and substrates 94
2.7.2.4 Immunoenzymatic staining technique 95
2.8 Assessment of growth of mycobacteria in vivo and in vitro 97
2.8.1 Homogenization of organs 97
2.8.2 Enumeration of viable M. tuberculosis and M. microti 98
2.8.3 Enumeration of L. monocytogenes and M. microti in 98
macrophage monolayers
2.9 In vitro techniques used in macrophage microbicidal assays 99
2.9.1 Preparation of monolayers 99
2.9.2 Enumeration of cell suspensions 100 8
2.9.3 Characterization of macrophage monolayers 101
2.9.4 Harvesting of bacteria from the monolayers 102
2.9.5 Analysis of DNA 103
2.10 In vivo models in the assessment of immunomodulating agents 104
2.10.1 Effect of levamisole on M. microti infection in CFLP mice 104
2.10.2 Effect of IFN-X on M. tuberculosis infection in mice 104
2.10.3 Effect of IFN-X in combination with isoniazid and with 105
rifampicin on M. tuberculosis infection in mice
2.11 In vitro assessment of the effects of IFN-Y 106
2.11.1 Effect of saponin on viability of L. monocytogenes 106
and M. microti
2.11.2 Listericidal assay 107
2.11.3 Effect of IFN-X on growth of M. microti in vitro 108
2.11.3.1 Effect of previous exposure of monolayers to IFN-X 108
2.11.3.2 Effect of IFN-Y on the phagocytosis of M. microti 109
2.11.3.3 Effect of IFN-Y added after infection of monolayers 110
with M. microti
2.11.3.4 Effect of IFN-Y in combination with isoniazid and 111
with rifampicin on the growth of M. microti in
macrophages
2.12 Statistical analysis of the data 112
3 THE IMMUNOMODULATING EFFECT OF LEVAMISOLE ON M. MICROTI 113
INFECTION IN MICE
3.1 Preliminary experiments 113
3.1.1 Choice of media for growth of M. microti 113
3.1.2 Course of infection of M. microti in CFLP mice 115
3.2 The effect of levamisole on M. microti infection in CFLP mice 117 9
3.2.1 The effect of levamisole on the growth of M. microti 118
3.2.2 The effect of levamisole on proliferative responses 121
of splenic T lymphocytes
3.2.3 The effect of levamisole on splenic T cell subsets 125
3.3 Discussion 130
4 THE IMMUNOMODULATING EFFECT OF INTERFERON-GAMMA ON 135
M. TUBERCULOSIS INFECTION IN MICE
4.1 Preliminary experiments 136
4.1.1 Sonication of M. tuberculosis 136
4.2 The effect of infective dose size of M. tuberculosis on the 139
growth of bacilli in the lungs and spleens of CFLP mice
4.3 The effect of dose size of IFN-V on the growth of 142
M. tuberculosis in BALB/c mice
4.4 The effect of administration of IFN-^f in liposomes 146
on the growth of M. tuberculosis in mice
4.4.1 The effect of IFN-X administered in liposomes on 146
M. tuberculosis infection in CFLP mice
4.4.2 The effect of the administration of IFN-JT in liposomes 151
on M. tuberculosis infection in BALB/c mice
4.5 The effect of pretreatment of mice with IFN-^ on 156
M. tuberculosis infection in BALB/c mice
4.6, , TUDiscussion . 161
5 THE IMMUNOMODULATING EFFECT OF INTERFERON-GAMMA IN COMBINATION 169
WITH ISONIAZID AND RIFAMPICIN ON MURINE M. TUBERCULOSIS INFECTION
5.1 Assessment of IFN-tf and isoniazid alone and in combination 169
on M. tuberculosis infection in BALB/c mice 10
5.1.1 The effect of IFN-Y and Isoniazid when IFN-Y was 169
administered before and after infection of BALB/c mice
with M. tuberculosis
5.1.2 The effect of IFN-Y and isoniazid when IFN-Y was 175
administered both before and after infection, and only
after infection of BALB/c mice with M. tuberculosis
5.2 The effect of IFN-Y and rifampicin alone and in combination 184
on M. tuberculosis infection in BALB/c mice
5.3 Discussion 188
6 IN VITRO ASSESSMENT OF THE EFFECTS OF INTERFERON-GAMMA 194
6.1 Preliminary experiments 194
6.1.1 Characterization of monolayers 194
6.1.2 The effect of saponin on viability of L. monocytogenes 194
and M. microti
6.2 Listericidal assay 197
6.2.1 The effect of dose size of IFN-V on the listericidal 197
activity of peritoneal macrophages after previous
exposure for 24 hours
6.2.2 The effect of varying the dose size and pretreatment 200
period of IFN-Y on listericidal activity of
peritoneal macrophages
6.3 In vitro assessment of effect of IFN-& on the growth 202
of M. microti
6.3.1 Treatment of macrophage monolayers with IFN-Y before 202
and after infection 11
6.3.1.1 The effect of dose size of IFN-^ added both before and 202
after infection on the growth of M. microti in
macrophages
6.3.1.2 The effect of previous exposure to IFN-Y on the 207
phagocytosis of M. microti
6.3.1.3 The effect of previous exposure of macrophages with 210
IFN-tf on viable intracellular M. microti at intervals
during the phagocytosis period
6.3.2 The effect of IFN-)f added after M. microti infection 215
of peritoneal macrophages
6.3.3 The effect of IFN-Jf and isoniazid alone and in 217
combination on the growth of M. microti in vitro
6.3.4 The effect of IFN-& and rifampicin alone and in 220
combination on the in vitro growth of M. microti
6.4 Discussion 223
7 GENERAL DISCUSSION 229
REFERENCES 240
APPENDIX 1 289 12
LIST OF TABLES PAGE
Viable counts of M. microti on various media under 114 different conditions.
Analysis of variance of levamisole and time on growth 121 of M. microti in CFLP mice
Proliferative responses of splenic T cells of uninfected 122 and infected mice before levamisole treatment
Proliferative responses of splenic T cells of uninfected 123 and infected mice after 2 weeks of levamisole treatment
Proliferative responses of splenic T cells of uninfected 124 and infected mice after 5 weeks of levamisole treatment
Splenic Thy 1.2+ T cells in infected and uninfected mice 127 before and after levamisole treatment
Splenic Lyt 1+ T cells in infected and uninfected mice 128 before and after levamisole treatment
Splenic Lyt 2+ T cells in infected and uninfected mice 129 before and after levamisole treatment
Total counts of M. tuberculosis after sonication 138
Analysis of variance of IFN-tf dose size and time on 143 growth of M. tuberculosis in BALB/c mice
3-way analysis of variance of the effect of IFN-^ dose 150 size, liposomes and time on M. tuberculosis infection in CFLP mice
3-way analysis of variance of the effect of IFN-V dose 155 size, liposomes and time on M. tuberculosis infection in BALB/c mice 13
TABLE 4.5 The effect of IFN-X pretreatment on the uptake of 157
M. tuberculosis in organs of BALB/c mice
TABLE 4.6 Analysis of variance of the effects of IFN-X pretreatment 161
on M. tuberculosis infection in BALB/c mice
TABLE 5.1 3-way analysis of variance of the effects of IFN-X*, 174
time and isoniazid on the growth of M. tuberculosis
in BALB/c mice
TABLE 5.2 3-way analysis of variance of the effects of isoniazid, 179
time and IFN-X given both before and after infection
of BALB/c mice with M. tuberculosis
TABLE 5.3 3-way analysis of variance of the effects of isoniazid, 183
time and IFN-ft given after infection of BALB/c mice
with M. tuberculosis
TABLE 5.4 3-way analysis of variance of the effects of IFN-X, 188
rifampicin and time on M. tuberculosis infection
in BALB/c mice
TABLE 6.1 The effect of saponin on viable counts of 196
L. monocytogenes
TABLE 6.2 The effect of saponin on viable counts of M. microti 197
TABLE 6.3 Analysis of variance of the effects of IFN-X dose size 199
and time on listericidal activity
TABLE 6.4 The effect of previous exposure to IFN-X on viable counts 208
of M. microti in monolayers in 8-chambered slides
TABLE 6.5 The effect of previous exposure to IFN-t# on counts of 210
acid-fast bacilli in macrophage monolayers
TABLE 6.6 3-way analysis of variance of in vitro effects of time, 219
isoniazid and IFN-X on M. microti infection of macrophages 14
TABLE 6.7 3-way analysis of variance of in vitro effects of time, 222
rifampicin and IFN-^ on the growth of M. microti in
macrophages
LIST OF ILLUSTRATIONS
Fig 3.1 Growth of M. microti in lungs and spleens of CFLP mice 116
Fig 3.2 Effect of levamisole on growth of M. microti in lungs of 119
CFLP mice
Fig 3.3 Effect of levamisole on growth of M. microti in spleens 120
of CFLP mice
Fig 3.4 Immunoalkaline phosphatase staining of cytocentrifuged 126
murine splenic T cells with monoclonal Thy 1.2 antibody
Fig 4.1 Effects of sonication on viable counts of mycobacteria 137
Fig 4.2 The effect of infective dose size of IFN-& on growth of 140
M. tuberculosis in lungs of CFLP mice
Fig 4.3 The effect of infective dose size of IFN-V on growth of 141
M. tuberculosis in spleens of CFLP mice
Fig 4.4 Effect of dose size of IFN-X on growth of M. tuberculosis 144
in lungs of BALB/c mice
Fig 4.5 Effect of dose size of IFN-V on growth of M. tuberculosis 145
in spleens of BALB/c mice
Fig 4.6 Viable counts of M. tuberculosis in lungs of CFLP mice 148
after administration of IFN-Jf in liposomes
Fig 4.7 Viable counts of M. tuberculosis in spleens of CFLP mice 149
after administration of IFN-^ in liposomes 15
Fig 4.8 Viable counts of M. tuberculosis in lungs of BALB/c 153
mice after administration of IFN-X in liposomes
Fig 4.9 Viable counts of M. tuberculosis in spleens of BALB/c 154
mice after administration of IFN-fc in liposomes
Fig 4.10 Viable counts of M. tuberculosis in lungs of BALB/c 159
mice after pretreatment with IFN-tf
Fig 4.11 Viable counts of M. tuberculosis in spleens of BALB/c 160
mice after pretreatment with IFN-Y
Fig 5.1 Effect of IFN-V and isoniazid alone and in combination 171
on the growth of M. tuberculosis in lungs of BALB/c mice
when IFN-Y was administered before and after infection
Fig 5.2 Effect of IFN-Y and isoniazid alone and in combination 173
on the growth of M. tuberculosis in spleens of BALB/c mice
when IFN-V was administered before and after infection
Fig 5.3 Effect of IFN-Y and isoniazid alone and in combination 176
on the growth of M. tuberculosis in lungs of BALB/c mice
in the first two days, when IFN-Y was administered before
and after infection
Fig 5.4 Effect of IFN-Y and isoniazid alone and in combination 177
on the growth of M. tuberculosis in spleens of BALB/c mice
in the first two days, when IFN-Y was administered before
and after infection
Fig 5.5 Effect of IFN-X and isoniazid alone and in combination 181
on the growth of M. tuberculosis in lungs of BALB/c mice
when IFN-Y was administered on Days +5 and +7 and
isoniazid from Day 0. 16
Fig 5.6 Effect of IFN-$ and isoniazid alone and in combination 182
on the growth of M. tuberculosis in spleens of BALB/c mice
when IFN-Y was administered on Days +5 and +7 and
isoniazid from Day 0.
Fig 5»7 Effect of IFN-J and rifampicin alone and in combination 185
on the growth of M. tuberculosis in lungs of BALB/c mice
Fig 5.8 Effect of IFN-Y and rifampicin alone and in combination 186
on the growth of M. tuberculosis in spleens of BALB/c mice
Fig 6.1A Wright’s (Diff-Quik) staining of macrophage monolayers 195
established in tissue culture 8-chambered slide for 2 days
Fig 6.IB Non-specific esterase staining of macrophage monolayers 195
established in tissue culture 8-chambered slide for 2 days
Fig 6*2 Effect of dose size of IFN-Y on listericidal activity of 198
peritoneal macrophages after prior exposure for 24 hours
Fig 6.3 Effect of varying the dose size and pretreatment period 201
of IFN-Y on listericidal activity of macrophages
Fig 6.4A Macrophage monolayers after 3 days incubation in BMM 204
Fig 6.4B Macrophage monolayers after 3 days incubation in BMM 204
plus 100 u IFN-Y per ml
Fig 6.5 Effect of dose size of IFN-Y on growth of M. microti 205
in macrophages after prior exposure for 72 hours
Fig 6.6 Effect of dose size of IFN-Y on growth of M. microti 206
in macrophages after prior exposure for 48 hours
Fig 6.7 Distribution of uptake of acid-fast bacilli by 209
macrophages after pretreatment with IFN-Y
Fig 6.8 Effect of previous exposure to IFN-Y on viable 212
intracellular M. microti at intervals during
the phagocytosis period 17
Fig 6.9 Effect of previous exposure to IFN-X on viable 213
intracellular M. microti at intervals during the phago
cytosis period and their subsequent fate 24 hours later
Fig 6.10 Effect of IFN-)£ added after infection of macrophages 216
with M. microti
Fig 6.11 Effect of IFN-% and isoniazid alone and in combination 218
on growth of M. microti in macrophages
Fig 6.12 Effect of IFN-V and rifampicin alone and in combination 221
on growth of M. microti in macrophages 18
CHAPTER 1
LITERATURE REVIEW AND INTRODUCTION
I. LITERATURE REVIEW
1.1 Tuberculosis
Tuberculosis, once known as the great white plague, is an infectious disease of world-wide distribution. Records indicate that it is an ancient disease recognized by early physicians such as Hippocrates and archaeological evidence show that man has been afflicted with tubercu
losis for many thousands of years (Keers, 1978). Over a hundred years ago, the causative organism was identified as Mycobacterium tuberculosis
(Koch, 1882) but despite the advances made in chemotherapy and vaccina tion, there are still 10 million new cases and 3 million deaths every year (World Health Organization, 1982).
1.1.1 The Mycobacteria
The mycobacteria are a group of non-motile, non-sporing, gram-positive rods that are relatively impermeable to basic dyes but once stained are able to resist decolourization with acids or acid-alcohol. Due to this characteristic property, the mycobacteria are often referred to as acid- fast bacilli. The mycobacteria (reviewed by Barksdale and Kim, 1977) comprise pathogenic as well as saprophytic species. The most important human pathogens, M. tuberculosis, M. bovis, M. africanum and M. leprae are strict pathogens usually transmitted from one person to another, but may cause disease in animals. The species which are occasional patho gens have also been called atypical or opportunistic mycobacteria. 19
These species have a saprophytic existence in water or soil, though
M. avium is also a pathogen for birds, pigs and some herbivores. These mycobacteria are less virulent than the strict pathogens for man, and occurrence of disease is thought to be due to host immune deficiency.
Other mycobacteria like M. phlei and M. smegmatis are strict saprophytes and do not cause disease at all.
M. tuberculosis, and probably all mycobacteria are obligate aerobes.
With the exception of M. leprae, M. lepraemurium and M. paratuberculo- sis, all mycobacteria can be grown on a variety of media including simple synthetic media. M. tuberculosis can be grown on very simple media containing only the NH^+ ion as nitrogen source, glucose or glyce rol as carbon source, magnesium, phosphates, iron and trace metals.
Growth is inhibited by the presence of fatty acids on the glass and cotton wool plugs which can be neutralized by the addition of adsorbing agents such as whole blood, serum, albumin, egg yolk or activated charcoal. All mycobacteria grow more slowly than most other bacteria.
The saprophytic species have generation times of 2-4 hours while the pathogenic species like M. tuberculosis have doubling times of 18-20 hours under optimal conditions. M. leprae which is highly adapted for parasitism, has a generation time of 12 days.
M. microti causes tuberculosis in voles, and its serological and patho genic properties suggest that it is very closely related to M. tubercu losis (Wells, 1946). Pathogenic studies have shown that if a large enough dose is used, M. microti will produce progressive and fatal disease in guinea-pigs, rabbits and mice (Corper and Cohn, 1943).
However, it lacks virulence in man and has been used in the form of a live vaccine as an alternative to BCG for vaccination (Medical Research 20
Council, 1956; Sula, 1958). M. microti has a distinctive morphology.
Pleomorphism is seen in vivo, and in culture media, the bacilli are often hooked or curved. It has a slightly slower growth rate than
M. tuberculosis and has been reported to be inhibited by glycerol, particularly in primary isolation (Wells, 1957).
Mycobacteria have cell walls with a unique chemical composition
(Barksdale and Kim, 1977). The mycobacterial cell wall is probably responsible for many of the special characteristics of the genus
including acid-fastness, resistance to host defence mechanisms and adjuvant activity. The cell wall is composed of a basal peptidoglycan structure (murein) covalently linked to an arabinogalactan-mycolate layer to form a complex polymer with a rigid framework which comprises about 60% of the cell wall.
A further 25% of the cell is composed of free lipids including Wax D, cord factor and sulpholipids. These lipids can be extracted with organic solvents and have been extensively investigated because of their diverse biological activities. Wax D is a monomer of the rigid frame work containing elements of both the murein and arabinogalactan-mycolate layers, which are thought to be autolysis products of the cell walls.
Cord factor (trehalose mycolate) is highly toxic to mice and destroys mitochondrial membranes. It is also found in the cell walls of M. phlei and M. smegmatis; thus while it may play a role in disease production, it is not the most important virulence factor. Sulpholipids are a group of multiacylated trehalose sulphate derivatives, some of which have been shown to enhance the toxicity of cord factor (Goren et al., 1974) and to be potent inhibitors of phagosome-lysosome fusion (Goren et al., 1976).
The sulpholipids (Goren et al., 1974) together with phospholipids 21 probably account for the binding of neutral red dye in the ‘Neutral Red* virulence test of Middlebrook and colleagues (1947).
At the surface of the cell wall, outside the arabinogalactan-mycolate layer are found various peptides including poly-L-glutamic acid which has been shown to inhibit phagolysosomal fusion in macrophages in vitro
(Hart and Young, 1978). In addition, there are rope-like structures at the surface of the cell wall of some species, but not M. tuberculosis, consisting of species-specific mycosides, which may protect the cell against macrophage lysosomal enzymes.
1.1.2 Pathogenesis of tuberculosis
Mycobacteria give rise to chronic granulomatous diseases in which the host immune response is often responsible for the nature and course of the disease. Even though the tubercle bacillus is highly infectious for most previously uninfected individuals, only a relatively small propor tion will develop clinical disease. This depends on the virulence and infectious dose of the strain, route of infection and also the innate and specific host immunity.
When an infection with M. tuberculosis occurs in a host with no previous experience with mycobacteria it is referred to as a primary infection
(Youmans 1979). In man, a primary infection is usually acquired by the inhalation of infectious droplet nuclei, and thus the lung is usually the first organ involved. The bacilli lodge within the alveolus and are phagocytosed by alveolar macrophages and neutrophils. Neutrophils are short-lived, highly phagocytic cells and are present in the initial stages of the infection when they engulf tubercle bacilli until they are 22 destroyed by the bacilli. Due to their high resistance to destruction, not all the mycobacteria are killed by the macrophages and some may mul tiply within them. As the bacilli multiply, large numbers of monocytes are drawn into the Ghon focus thus established. Dissemination from this focus then occurs by erosion of blood vessels and haematogenous spread throughout the body. Most of the disseminated bacilli will be taken up by the resident mononuclear phagocytes of the organs and will continue to multiply within those cells. In a small proportion of persons with primary tuberculosis this leads to post-primary tuberculous pneumonia, miliary tuberculosis or meningitis which may eventually prove fatal.
However, in the majority, after a period of a few weeks, the number of bacilli dramatically decreases. Many of the macrophages die, the bacilli start to grow extracellularly, and the healing processes begin, brought about by the onset of delayed type hypersensitivity and acquired cellular immunity.
Secondary or adult tuberculosis is the disease that occurs in persons who have previously been infected. It may occur as a result of endo genous reactivation or by reinfection. Secondary disease differs from primary disease in that it is characterized by necrosis and localization of the lesion which are due to tuberculin hypersensitivity and acquired immunity. Spread of this disease can occur by extension to adjacent tissues due to necrosis and in the lung, erosion of a bronchus or a blood vessel can lead to bronchogenic or haematogenous spread, respec tively. The reasons for the breakdown in immunity that may lead to secondary tuberculosis are not known. However, factors like age, hormo nes, immunosuppression and viral infections are known to predispose to the disease. 23
1.1.3 The immune response to tuberculosis
There is evidence that anti tuberculosis immunity is essentially of the cell mediated (CMI) type. Immunity to tuberculosis can be passively transferred by syngeneic lymphocytes but not by serum. This transfer does not occur if the lymphocytes are exposed to anti-0 serum and complement (Lefford, 1975). North (1973) has also shown that experi mental tuberculosis is uncontrolled in T cell depleted mice and that an infusion of thymocytes restores immunity. Undoubtedly, antibodies are produced against a variety of proteins and polysaccharides of the tubercle bacillus (Daniel and Janicki, 1978) but antibodies have been shown to be unimportant in host protection (Reggiardo and Middlebrook,
1974).
Antibacterial cell mediated immunity is based on interactions between macrophages and T lymphocytes. It is now known that the prerequisite for induction of CMI is that the antigen be processed by macrophages
(Bloch and Nordin, 1960; Pearson and Raffel, 1971; Oppenheim and Seeger,
1976) and then presented to T cells on the surface in association with products encoded by the major histocompatibility complex (MHC), the HLA-
D (Bodmer, 1977) or the la (Shreffler and David, 1975) alloantigens in humans or mice respectively. Recently it has also been shown that
Langerhans cells (Stingl et al., 1980) and dendritic cells (Steinman and
Nussenzweig, 1980) also act as antigen-presenting cells and could thus be involved in the CMI response. In mice, it has been shown that Lyt 1
T cells are the cells that recognize antigen in association with the H-2 la determinants presented by the macrophages (Cantor et al., 1976; Farr et al., 1979). Activation of T cells which also seems to require macro phage-derived interleukin 1 (Mizel, 1982), leads to lymphoproliferation. 24
At the peak of lymphoproliferation, histological tubercles begin to appear, the host exhibits delayed hypersenstivity to tuberculin and becomes immune to challenge with tuberculosis (North et al., 1972).
During these events, the draining lymph nodes becomes substantially enlarged. Initially, lymphoproliferation results in actively dividing large lymphocytes or immunoblasts which leave the lymph nodes and remain in the circulation for a few days before disappearing. Subsequently, a population of long-living, non-dividing, recirculating small lymphocytes appears within the circulation and these are the cells that mediate antituberculous immunity (Lefford et al. , 1973). These sensitized lymphocytes were shown to be T cells by North (1973). This sequence of events have been elucidated in animal experiments and it is probable that a similar sequence occurs in man.
The term ’lymphokine' (LK) was first used by Dumonde et al. (1969) to describe non-antibody soluble mediators of cellular immunity generated by lymphocyte activation. It has been proposed that during a CMI response, sensitized T lymphocytes interact with antigen and release LKs which mediate macrophage activation. This hypothesis is supported by in vitro experiments in which macrophages were activated by LKs (Simon and Sheagren, 1971; Nathan et al., 1971). The first LK described and the most intensively investigated is macrophage migration inhibition factor (MIF). Subsequently, it is estimated that up to one hundred factors may be produced and that macrophages and monocytes themselves produce soluble mediators as well (Waksman, 1979). The lymphokines include motility inhibitors like MIF, leukocyte inhibiting factor
(LIF); growth inhibitors like cytotoxic factors (CF), proliferation inhibitory factor (PIF); activators like macrophage activating factor
(MAF) , skin reactive factor (SRF) and other factors like chemotaxins, 25
interferon and transfer factor. In tuberculosis, as the bacilli are
multiplying in the macrophages, the factors that either mobilize or
activate macrophages are likely to be most important. The above func
tions of the lymphokines have been investigated in vitro but their
interactions in vivo are not known.
In addition to lymphokines, macrophages can also be activated as a
result of interactions with microorganisms (Sher et al. , 1975) and microbial products like lipopolysaccharide and other cell wall deriva
tives like muramyl dipeptide (Ellouz et a l ., 1974). In addition to
soluble factors, contact co-operation between macrophages and T cells
forms an autocatalytic system of mutual interactions in T cell mitoge- nesis (Peters and Schimmelpfeng, 1979).
Granulomas begin to appear at sites where tubercle bacilli are located.
The granuloma consists of a focus of mononuclear cells comprising macro phages, lymphocytes and epithelioid cells (Rich, 1951; Lurie, 1964).
Recruitment of mononuclear cells is mediated by the lymphokines produced by the sensitized T cells (Kuhner et al., 1980; Kaufman et al., 1981).
The macrophages and lymphocytes migrate into the lesion from blood while epithelioid cells are derived from macrophages. In granulomas in man, multinucleated giant cells derived from macrophages are present in addi tion to the above cell types. The epithelioid cells are weakly phagocy tic but are active in pinocytosis and extracellular secretion of diges tive enzymes and may play a role in extracellular killing of the bacilli
(Spector, 1976). Giant cells are also weakly phagocytic and their role is unknown. It has been suggested that the formation of giant cells is a means of disposing of altered or effete cells (Spector, 1976). Within 26 the granuloma, the cells are tightly packed together and this allows cell-to-cell interactions (Boros, 1978).
The macrophages in a granuloma differ morphologically and histochemical- ly from normal tissue macrophages, and the term 'activated macrophage' has been coined to describe them (Mackaness, 1962). Activation of macro phages refer to a series of morphologic, functional and biochemical changes (Karnovsky and Lazdins, 1978; Cohn, 1978). Activated macropha ges display a ruffled plasma membrane, an increased capacity for adher ing and spreading on a substratum, an increased capacity for phagocyto sis and microbicidal activity, and increased content of golgi apparatus, lysosomal acid hydrolases, secondary lysosomes (Grogg and Pearce, 1952;
Blanden, 1968; Blanden et al., 1969). In addition, it was later shown that activated macrophages also secrete large quantities of neutral proteinases including plasminogen activator, a specific elastase and a specific collagenase (Gordon, 1976).
One of the most important feature of activated macrophages is the enhancement of microbicidal activity. It has been shown in vitro that activated macrophages have an increased ability to kill intracellular bacteria like L. monocytogenes (Blanden et al., 1969), protozoa like
Leishmania tropica (Titus et a l . , 1984; Oster and Nacy, 1984) and
Toxoplasma gondii (Nathan et al., 1983). Walker and Lowrie (1981), demonstrated that murine peritoneal macrophages exposed to LK killed more than 90 % of ingested M. microti in 24 hours. Previous results had shown that LK-treated macrophages could not kill mycobacteria but inhi bited their multiplication (Patterson and Youmans, 1970; Godal et al.,
1971). 27
In a tuberculous infection, macrophages that have been activated will
not only specifically kill or inhibit the growth of tubercle bacilli but
will also non-specifically inhibit or kill other unrelated organisms
(Mackaness, 1964; Blanden et al., 1969) as well as tumour cells (Hibbs
et al., 1972).
In tuberculosis, hypersensitivity takes the form of a delayed allergic
response termed delayed type hypersensitivity (DTH) which is definded as
an immunological state in which lymphocytes and macrophages show a
sensitivity to tubercle bacilli and their products. There is still
controversy as to the relationship of acquired immunity to DTH. In
experimental tuberculosis, the onset of immunity coincides with the
appearance of DTH, and adoptive transfer experiments have confirmed that
T cells are the mediators of DTH (North, 1973). One hypothesis is that
acquired immunity and DTH are both manifestations of the same cellular
immune response (Mackaness, 1967; Mackaness and Blanden, 1967; Collins
and Mackaness, 1970). The other hypothesis is that DTH and acquired
immunity are merely coincidental events that are unrelated. This view
is supported by demonstrations that each phenomenon can be elicited by
different mycobacterial substances, and thus are independent responses
of the host tissue (Anacker et a l ., 1969; Berthrong, 1970; Youmans,
1975).
The complex system of immunologic responsiveness include feedback
mechanisms by which immunoregulation is achieved. Without feedback,
responses to antigens would be uncontrolled and potentially damaging.
Several types of immunocompetent cells with suppressor function have
been identified in mycobacterial infections. Using a model of mice heavily infected with mycobacteria, Watson and Collins (1980) reported 28 suppression mediated by T cells. Other cell types have also been shown to mediate suppression; adherent cells in spleens of mice heavily infected with BCG have been shown to inhibit cytotoxic T cell generation
(Klimpel and Henny, 1978). Ellner (1978) has shown that a population of adherent cells, presumably monocytes or macrophages, with suppressor activity, can be isolated from the peripheral blood of anergic tubercu losis patients. This suppressor activity was shown to be antigen- specific and to involve complex cell-cell interactions. It has also been reported that B lymphocytes can suppress the cellular immune resp onses of T lymphocytes to mycobacterial antigens (Bona et al., 1976).
It has long been recognized that an occasional tuberculosis patient is tuberculin negative (Adams et al., 1959; Katz et al., 1972). Loss of tuberculin hypersensitivity following chemotherapy or chemoprophylaxis of tuberculosis has also been reported (Robinson et al., 1955; Atuk and
Hunt, 1971). There is evidence that the loss of tuberculin hypersen sitivity can be due to sequestration of sensitized lymphocytes in lymph oid organs which is brought about by their removal from the circulation by 'blocking’ agents; either antigen, specific antibody or specific immune complexes (Schlossman et al., 1971).
It has been postulated that tuberculosis, like leprosy and visceral leishmaniasis is a disease with an immune spectrum based on clinical and immunological grounds. Lenzini et al.(1977) proposed four groups: a) a polar group in which cell mediated immunity is fully active (RR); b) an unreactive polar group in which cell mediated immunity is undetected
(UU); c) an intermediate group towards the reactive pole (RI) and d) an intermediate group towards the unreactive pole (UI). 29
1.1.4 Mycobacteria-macrophage interactions
The pathogenic mycobacteria cause diseases which are characteristically
chronic in nature. There are several prerequisites for establishing
chronic infections. The first is for the infectious agent to be of
sufficiently low toxicity so that the host can survive a prolonged state
of parasitism. Mycobacteria have not been shown to produce exotoxins,
endotoxins or harmful enzymes. Although toxic factors like cord factor
(Middlebrook et a l . , 1947) and other toxic lipids such as sulpholipids
(Goren et al. , 1974) and phthiocerol dimycocerosate (Goren et al.,
1974a) may account for some of the capacity of M. tuberculosis to cause
progressive disease, mycobacteria are considered to be of low toxicity.
There is little or no sign of toxicity when large numbers of live
M. tuberculosis are injected intravenously into mice (Youmans and
Youmans, 1951).
The slow growth rate of pathogenic mycobacteria is another factor in its
success as a chronic pathogen. Another criterion is the ability of the
pathogenic mycobacteria to lie dormant for many years. Although anaero
bic conditions inhibit growth, it has been shown that M. bovis sealed in
ampoules of veal broth incubated at 37°C could survive for as long as 30
years (Corper and Cohn, 1951).
An important prerequisite for chronic infections is an ability of the
infectious agent to avoid host defence mechanisms. Mycobacteria achieve
this by surviving in macrophages. Unlike neutrophils, macrophages are long-lived cells. Hence, persistence in macrophages would ensure survi val in the host for long periods. Intracellular persistence in macro phages could be due to bacterial subversion of the killing mechanisms of 30
macrophages or due to genetically-determined impaired killing mechanisms
of macrophages.
1.1.4.1 Microbicidal mechanisms in macrophages
In macrophages, two main bactericidal mechanisms operate; oxygen-
independent ones such as lysozyme, hydrolases, cationic proteases,
acidity; and oxygen-dependent ones such as superoxide, hydrogen peroxide
and hydroxyl radicals. It is still not certain how macrophages kill mycobacteria. Exposure to peroxide and related oxygen derivatives may occur in the phagosome after ingestion, while exposure to lysosomal enzymes requires phagosome-lysosome fusion. Live M. tuberculosis and
M. microti have been shown to inhibit phagolysosome formation and multi ply within phagosomes (Armstrong and Hart, 1971; Lowrie et al., 1975).
Other successful intracellular parasites have also been found to inhibit phagolysosome fusion (Jones and Hirsch, 1972; Friis, 1972; Weidner,
1975) and it was shown with Toxoplasma gondii that coating the parasites with antibody before phagocytosis led to promotion of fusion and death of the parasites (Jones, 1975). However, the results were less obvious with M. tuberculosis and M. microti. Coating M. tuberculosis with anti bodies promoted phagolysosome fusion with no effect on bacterial growth
(Armstrong and Hart, 1975) and coating M. microti with antibody also led to phagolysosome fusion with occasional stasis (Lowrie et al., 1979).
These results suggest that the lysosomal contents might not be that important in the killing of mycobacteria. However, there is the possi bility that phagolysosomal fusion might be lethal to mycobacteria in the intact animal. 31
Lysozyme is present in large quantities in macrophages and is involved
in the killing of gram-negative organisms. However, many organisms
including mycobacteria are resistant to degradation by lysozyme. It has
been suggested that lysozyme may have tuberculostatic properties (Oshiraa
et al., 1961).
Another possible microbicidal mechanism is the acidity of the macro
phage. It has been known for a long time that phagocytes respond to the
ingestion of particles by acidifying their phagosomes. Early studies
using indicator dyes led Rous (1925, 1925a) to conclude that the intra-
vacuolar pH could be as low as 3.0. Subsequently, Sprick (1956) showed
that phagocytosis of M. tuberculosis and M. smegmatis led to a fall in
pH to 4.7-5.2. Jacques and Bainton (1978) showed that within 10 minutes
of phagocytosis, the pH of neutrophils and monocytes fell to 4.5-5.0.
Recent studies using a pH-sensitive fluorescent probe to measure intra-
vacuolar pH have shown that the pH in newly formed phagosomes in
neutrophils (Segal et al., 1981) and murine macrophages (Geisow et al.,
1981) transiently became alkaline in the first two minutes before the
acidification and that the raised pH is associated with the respiratory
burst. Segal et al. (1981) also postulated that the alkaline pH facili
tates killing and bacteriolysis by granule proteins with alkaline pH
optima following which the pH of the vacuole is reduced to optimize the activities of hydrolases and other proteins of acidic pH optima. The acidity per se may be bactericidal for killing some organisms like
Vibrio cholerae (Looke and Rowley, 1962), but it is more likely that the acidity contributes to other killing mechnisms. It has been shown to enhance hydrogen peroxide toxicity for tubercle bacilli (Jackett et al.,
1978). Acidity also promotes the rate of superoxide reduction to hydrogen peroxide (Stossel, 1974). 32
The oxygen-dependent mechanisms have been established as important bact ericidal mechnisms in neutrophils (Klebanoff and Hamon, 1975). Interme diate products of oxidative phosphorylation like hydrogen peroxide and singlet oxygen have been implicated in bactericidal mechanisms. The first indirect evidence that hydrogen peroxide might be involved in the killing of tubercle bacilli was the isolation of isoniazid resistant mutants of M. tuberculosis which were catalase negative, susceptible to hydrogen peroxide and of low virulence for the guinea-pig (Barnett et a l . , 1953; Cohn et al., 1954; Mitchison, 1954). In the early 1960's it was noticed that a substantial proportion of clinical isolates of
M. tuberculosis in the Indian subcontinent were susceptible to peroxide and isoniazid and of low virulence in the guinea-pig but had the normal content of catalase (Subbaiah e t a l . , 1960; Mitchison et al., 1963).
Subsequently, Walker and Lowrie (1981) showed that the killing of
M. microti was due to macrophage hydrogen peroxide. This was indicted by the protective effect of exogenous catalase and the finding that phagocytosis of M. microti was accompanied by a release of hydrogen peroxide in parallel with the uptake of the bacilli. Another piece of evidence is the correlation of susceptibility of M. tuberculosis strains to hydrogen peroxide and diminished virulence for guinea pigs (Jackett et al., 1981).
Much of the hydrogen peroxide produces by mononuclear phagocytes is derived from superoxide (Rossi et al., 1979). However, in a study with strains of M. tuberculosis of different virulence in guinea-pigs there was no correlation between virulence, resistance to superoxide and content of superoxide dismutase (Jackett, et al., 1978). Thus, supero xide, hydroxyl radical and singlet oxygen are unlikely to have any direct role in tuberculocidal activity in macrophages. 33
Another factor that might be important within a granuloma is reduced oxygen tension. The development of infections with M. tuberculosis is impaired under conditions where oxygen availability is restricted
(Dubos, 1955; Sever and Youmans, 1957; Chandler, 1965). It is unlikely that only a single tuberculocidal mechanism operates in a macrophage.
It may be that the macrophage mycobactericidal mechanisms depend on a combination of acidity, hydrogen peroxide and low oxygen tension.
1.1.4.2 Mycobacterial evasion of macrophage killing mechanisms
Following ingestion, pathogenic mycobacteria have been found to occur in three sites within the host cell; the phagosome, the phagolysosome or the cytoplasm. Live M. tuberculosis and M. microti have been shown to multiply in the phagosome, M. lepraemurium can be found multiplying within the phagolysosomes (Hart et al., 1972) while M. leprae is belie ved to escape from the phagosomes into the cytoplasm (Evans and Levy,
1972). These sites may be interpreted as indicating three ways of avoiding intracellular killing.
At least four substances are known that may, in appropriate conditions, be produced by M. tuberculosis and which will inhibit phagosome-lysosome fusion in cultured mouse peritoneal macrophages. These substances are cyclic AMP (Lowrie et al., 1975; Lowrie et al., 1979); poly-<<-L-glutamic acid (Hart and Young, 1978); sulphatide (Goren et al., 1976); and ammo nia (Gordon et a l . , 1980). It is still not clear which, if any, of these substances is the active material inside the host cell infected with M. tuberculosis. 34
It would seem that M. lepraemurium Is indifferent to the effects of phagolysosomal contents as it may be found multiplying in phagolysosomes of macrophages in vitro (Hart et a l . , 1972) and in vivo (Brown and
Draper, 1976). It has been proposed that the surface structures of
M. lepraemurium are unaffected by lysosomal enzymes and impermeable to bactericidal chemicals due to a protective capsule of a characteristic peptidoglycolipid (Draper and Rees, 1970; Nishiura et al., 1972). Such a capsule is also produced by M. avium (Draper, 1974).
Mouse foot-pad experiments have suggested that M. leprae escapes into the cytoplasm through a phagocytic vacuole (Evans and Levy, 1972). It has also been reported that some rickettsias (Silverman and Wisseman,
1979) and Trypanosoma cruzi (Nogueira, 1974) use the same route. The mechanisms involved are uncertain but once established in the cytoplasm, the pathogen is now removed from killing mechanisms. The lack of cyto toxicity and extremely slow growth rate make M. leprae particularly suited for such intracellular existence.
In addition to the above mechanisms, mycobacteria could also modulate the host response by depressing cellular immune responses. The trigger ing of immune suppression mechanisms is probably a specific response to antigen. Immunopotentiating or adjuvant properties of mycobacteria are well known and studies have identified muramyl dipeptide (Ellouz et al.,
1974) and trehalose mycolates (Goren, 1975) as the mycobacterial cell wall components with adjuvant activity. Mycobacteria have also been reported to contain immunosuppressive substances. Ellner and Daniel
(1979) claimed that mycobacterial arabinomannan is immunosuppressive in vitro, and there are indications that mycobacterial cell wall arabinogalactan is a potent immunosuppressive agent (Kleinhenz et al., 35
1979) . Neta and Salvin (1979) have also shown that mycobacterial cells in Freund's complete adjuvant induced the formation of both suppressor adherent cells and suppressor B lymphocytes.
1.1.4.3 Genetic susceptibility
The heterogeneity of macrophages is well established. Mature macroph ages are more bactericidal than blood monocytes (Gemmell et_al., 1981) and sub-populations of murine peritoneal macrophages vary in their ability to restrict intracellular growth of L. monocytogenes (Harring- ton-Fowler and Wilder, 1982). Thus there are clearly populations of macrophages where intracellular bacteria can survive. In addition there is the possibility of genetic deficiencies adversely affecting the micr obicidal ability of macrophages. It has been shown in mice that geneti cally determined susceptibility to infections occur (Cheers et al.,
1978; Skamene and Kongshavn, 1979). Further studies have identified the macrophage as the cell population that expresses the phenotype of genet ically determined resistance to BCG infection (Stach et al., 1984). It has also been noted that the monocytes and macrophages in patients with chronic granulomatous disease are less efficient at microbicidal func tions (Davies et al., 1968). 36
1.2 Tuberculosis control
The annual risk of tuberculous infection which can used to study the epidemiology of tuberculosis, is estimated by using serial tuberculin conversion rates. In developed countries like the Netherlands, the annual risk of tuberculous infection since 1940 has closely followed an exponential downward trend, the risk decreasing annually by 13.8%. From
1913-1939, the risk was shown to decrease exponentially as well, with an annual decrease in risk of 5.5% (Styblo, et al., 1969). Before 1940, chemotherapy was not available, mass BCG vaccination and radiography was not applied, and could not have contributed towards the decrease.
Hence, the exponential decrease before 1940 appears to have resulted from factors like improvements in socioeconomic conditions and isolation of infectious cases in sanatoria. The steeper decrease after 1940 has been attributed to compulsory pasteurization of milk and the introduc tion of effective chemotherapy (Styblo, et al., 1969).
The situation is rather different in poor developing countries. Similar tuberculin surveys have failed to show any significant reduction in the annual risk of tuberculous infection. In Uganda, the annual risk of infection (at age 10) was 2.8% in 1940, 2.6% in 1950, 2.4% in 1960 and
2.3% in 1970 (Stott et al. , 1973). In Lesotho, the annual risk of infection was 3% in 1957 and was unchanged in 1965 (Styblo, 1980). The current epidemiological trend in most poor developing countries is an annual risk of tuberculous infection of 2-5% about 20-50 times greater than in technically advanced countries (World Health Organization,
1982). 37
Methods of tuberculosis control have consisted of 1) BCG vaccination, 2)
case-finding and chemotherapy, and 3) chemoprophylaxis. The decline in
tuberculosis rates achieved in Eskimos in Alaska, Greenland and Canada
showed that it is possible through intensive antituberculous programmes
to reduce the tuberculosis problem rapidly in a community (Grzybowski
et a l . , 1976). Eskimos had extremely high rates of tuberculosis and in
Alaska and Greenland, the problem was reduced by 90% in 15 years. The
common feature of the control programmes in all three countries was
intensive case-finding followed by thorough chemotherapy. BCG was also
extensively used in Greenland, chemoprophylaxis in Alaska, and both BCG
and chemoprophylaxis in Canada. However, the greatest contribution was
intensive case-finding and treatment which resulted in extremely rapid
decrease of risk of infection.
1.2.1 BCG Vaccination
Despite enormous efforts to develop a non-viable anti-tuberculous
vaccine for clinical use, live BCG still remains the only practical
means of immunization. Previous attempts to immunize with killed whole
mycobacteria have shown that killed vaccines were inferior to viable
vaccines (Bloch and Segal, 1955). However, large doses of phenol-killed
tubercle bacilli were shown to produce an immunity almost comparable to
that seen with live organisms (Acharya et al., 1958). In recent years,
three killed vaccines have been extensively investigated. These are the
mycobacterial ribosome vaccine (Youmans and Youmans, 1969), mycobacte
rial cell wall vaccines (Ribi et al., 1966, 1971) and trypsin-extracted
mycobacterial antigen vaccine (Crowle, 1972). None of these vaccines
were more efficacious in immunization than live BCG and more importan 38
tly, none were suitable for clinical use.
Live BCG was first administered orally to a child in Paris by Weill-
Halle in 1925 with no untoward effects (cited by Collins, 1984) and the use of BCG vaccination spread across Europe and to America. Initially, it was administered orally, then by the sub-cutaneous route which was dropped in favour of the intra-dermal route. On the basis of clinical observations and uncontrolled trials, by 1945, 100 million people had been vaccinated. After World War II, eight major controlled trials have been carried out in Europe and the United States with results ranging from 0-80% protection (Barksdale and Kim, 1977). A trial in American
Indians showed 80% protection (Stein and Aronson, 1953), another trial in British school children gave 78% protection (Medical Research
Council, 1972) while a trial in Georgia showed no protection at all
(Comstock and Palmer, 1966). Several reasons have been put forward to explain the differences, among them the quality of the vaccine and partial protection due to environmental mycobacteria. The Chingleput,
South India trial was started in 1968 in an attempt to assess the influence of environmental mycobacteria on BCG vaccination. After seven and a half years follow-up, the trial had yet to show any protection
(World Health Organization, 1980, 1980a). Whatever the real reasons for the disparate results of the BCG trials, they are likely to be multifac torial and complicated.
The current view of BCG vaccination is that it primarily protects the individual without much effect on disease in the community. The communal effect of BCG vaccination was studied by Styblo and Meijer
(1976). They showed that the decreases in the annual risk of infection 39
in Denmark and Norway, where mass BCG was carried out, were similar to
that of the Netherlands, where mass BCG was never carried out. In
detailed comparisons of the age-related incidence in these countries,
the direct protective effect of BCG on the incidence in the individuals vaccinated could be demonstrated whereas they found no indirect effect within the community as a whole. It was also evident from the
tuberculosis control programmes in Eskimos that mass BCG applied at the
ages of 15-30 did not substantially influence the chain of transmission
(Grzybowski et al., 1976).
Another important factor about BCG vaccination is the duration of immun
ity. It has been shown that the protective effect of BCG decreases with
time from 84% at 0-5 years to 59% at 10-15 years and down to -12% at 15-
20 years (Hart and Sutherland, 1977). This downward trend is signifi
cant at the 1% level.
1.2.2 Case-finding and Chemotherapy
Case-finding and chemotherapy is considered as an entity, as case
finding is a preliminary to treatment and cure. The importance of case
finding and chemotherapy in tuberculosis control is readily seen in the
successful anti-tuberculosis programmes in Eskimos in Alaska, Greenland and Canada where the rapid decrease in risk of infection was attributed
to intensive case-finding and thorough chemotherapy (Grzybowski et al.,
1976). In Alaska, the risk of infection fell some 100 times in 25 years. Case-finding and chemotherapy is now considered the most power ful component of tuberculosis control (World Health Organization, 1982). 40
1.2.2.1 Case-finding
Case-finding may be either passive or active. Passive case-finding depends on patients reporting with symptoms and is essential in any health care programme. Passive case-finding tactics are: 1) detection of chronic cough (greater than 4 weeks duration) in patients attending clinics or hospitals; and 2) re-directing such symptomatic patients for direct smear examination of the sputum. Studies in the developing coun tries have shown this method to be highly effective provided there are adequate facilities for sputum examination. Active case-finding through mass radiography has now largely been abandoned due to the high costs, the rapidity with which new cases develop and the small contribution to the total number of cases detected (Meijer et al. , 1971). It has also been shown that active case-finding by mass tuberculin testing of unvaccinated individuals does not contribute significantly to the number of cases detected (Van Geuns et al., 1975). Active case-finding in high risk groups, such as household contacts of newly diagnosed tuberculous infections should continue to be employed.
In developing countries cases of tuberculosis are diagnosed mainly by direct smear examination of the sputum. In technically advanced coun tries, culture and radiography are used, enabling the detection of smear-negative disease which might account for 50-60% of all newly diagnosed cases of pulmonary tuberculosis. The introduction of culture service in developing countries could significantly improve case-finding and might increase the overall efficiency of the service by facilitating more accurate diagnosis. However, the organization of a culture service is technically complex and relatively expensive and might divert 41
resources from the direct-smear service in the poorer countries.
1.2.2.2 Chemotherapy
Ever since Koch discovered the etiological agent of tuberculosis, there have been attempts to find suitable chemotherapeutic agents. Thiaceta- zone was the first agent with any efficacy to be discovered and was extensively investigated in clinical trials in Germany (Domagk, 1950), but the toxicity associated with the large doses used discouraged wide spread use of the drug (Hinshaw and McDermott, 1950). Streptomycin was
the first chemotherapeutic agent effective for the treatment of tubercu losis in man (Schatz and Waksman, 1944; Feldman and Hinshaw, 1944).
Among the important events in the three decades following the discovery of streptomycin was the discovery that the emergence of drug resistance could be prevented by using a combination of antituberculous drugs
(Medical Research Council Investigation, 1950; Tempel et al., 1951); the introduction of isoniazid, an inexpensive, relatively non-toxic and highly effective antituberculous agent (Robitzek et al., 1952); the demonstration that similar results were obtained in patients treated in
their homes or in a sanatorium with isoniazid and para-aminosalicylic
acid for one year in Madras (Dawson et al., 1966) which brought about ambulatory treatment; the demonstration of the efficacy of intermittent
chemotherapy (Tuberculosis Chemotherapy Centre, Madras, 1964); and the
discovery of the higher sterilizing activity of rifampicin and pyrazin-
amide when combined with isoniazid, which enabled the shortening of
chemotherapy. 42
Standard regimens comprising various combinations of streptomycin, isoniazid, rifampicin, ethambutol, para-aminosalicylic acid and thiacet- azone given for 18-24 months are theoretically capable of producing a long and lasting cure in all patients. However, a review of the bacte rial and non-bacteriological failures of a standard regimen in 1968-1970 in Scotland showed that even in a technically advanced country, there are still practical problems in achieving a lasting cure with the standard regimens (Heffernan et al., 1976). One of the main problems of the standard regimens is non-compliance. In previously untreated patients, it has been estimated that the failure rate attributed to non- compliance is 2% under optimal conditions in technically advanced countries and as high as 50% in poor developing countries (Mitchison,
1980). Two major developments in chemotherapy had been directed at reducing this problem; fully supervised intermittent chemotherapy and short-course chemotherapy.
Short course chemotherapy is based on a combination of appropriate sterilizing drugs and was developed mainly as a consequence of a series of observations from studies on experimental chemotherapy in the mouse and guinea-pig, and in vitro studies (reviewed by Fox and Mitchison,
1975; Grosset, 1978) . Sterilizing activity is taken to mean the killing of the last few persisting organisms in a lesion during effec tive chemotherapy. In experimental tuberculosis, sterilizing activity is measured as the ability to prevent growth of bacilli from the organs of animals after 3-6 months chemotherapy and to prevent subsequent relapse, especially after immunosuppression with steroids. The main facts that emerged from these studies were: 1) rifampicin and 43
pyrazinamide were the two most potent sterilizing drugs, 2) rifampicin
and pyrazinamide were most effective when combined with isoniazid, 3)
the addition of streptomycin or ethambutol did not improve its sterili
zing effect. These conclusions were consistent with findings of short-
course chemotherapy trials conducted on pulmonary tuberculosis patients
in several countries under the auspices of the British Medical Research
Council (reviewed by Fox and Mitchison, 1975).
These findings led Mitchison (1980) to hypothesize the existence of four
special bacterial populations within tuberculous lesions. The first
consists of bacilli growing relatively rapidly and is susceptible to
bactericidal drugs. This is supported by evidence of a very rapid fall
in viable counts of tubercle bacilli in patients in the first few days
after chemotherapy (Jindani et a l . , 1980). Comparisons of different
regimens in these first few days showed that with single drugs, isonia
zid was the most bactericidal drug and that the contribution of the
other antituberculous drugs given in combination with isoniazid is
limited.
The second population consists of bacilli which are slowly metabolizing within the acid environment of the macrophages or in areas of acute
inflammation, and which are killed selectively by pyrazinamide. This is
supported by evidence that pyrazinamide is shown to be weakly bacteri
cidal in vitro at a pH of 5.6 or less (McDermott and Tompsett, 1954;
Dickinson and Mitchison, 1970). The third population of slowly metabo
lizing bacilli with spurts of metabolic activity would be effectively killed by rifampicin which is unique in the speed with which its bacte ricidal activity starts (Dickinson and Mitchison, 1981). 44
The fourth population of dormant bacilli are not known to be killed by any drug. This population has been shown to exist in experimental murine chemotherapeutic models where latent bacilli have been stimulated to grow by the administration of steroids in the post-chemotherapeutic period (McCune et al., 1956; Grumbach, 1975). The bactericidal mecha nisms on the first three populations during chemotherapy probably all
commence at the begining of chemotherapy (Dickinson and Mitchison,
1981). This assumption explains the delayed bactericidal activity that
becomes evident only months after an initial period of treatment with
rifampicin in experimental chemotherapy in mice (Grumbach et al., 1969)
and guinea-pigs (Dickinson and Mitchison, 1976).
Antituberculous drugs can be graded according to their ability to prevent the emergence of drug resistance, for their early bactericidal
activity or for their sterilizing activity (Mitchison, 1985). These
three functions are often unrelated, and the rating for one function
often has no relationship to the rating for another function. Isoniazid has a high activity for preventing the emergence of drug resistance and
for early bactericidal activity; rifampicin has high activity for preve
ntion of drug resistance and sterilizing activity; and pyrazinamide has
a high sterilizing activity but is only moderately effective in preven
ting drug resistance, and has virtually no early bactericidal activity.
In Britain, a standard 9 month daily regimen has been adopted consisting
of rifampicin and isoniazid supplemented with ethambutol for the first
three months. This has been shown to be highly effective with almost no
relapses (British Thoracic and Tuberculosis Association, 1976; British
Thoracic Association, 1980). Later studies have shown that the 45
duration of the regimens could be shortened to 6 months by the adminis
tration of pyrazinamide in addition to isoniazid, rifampicin and either
ethambutol or streptomycin in the initial phase followed by isoniazid
and rifampicin for the duration (Singapore Tuberculosis Service/British
Medical Research Council, 1981; British Thoracic Association, 1982).
There is increasing evidence that there are a variety of highly
effective 6 month regimens (reviewed by Fox, 1985). All these regimens
have in common isoniazid, rifampicin and pyrazinamide initially and
isoniazid and rifampicin in the continuation phase. Some regimens are
daily throughout, some are administered thrice or twice weekly, and some
combine both daily and intermittent regimes. Some regimens include
ethambutol and some include streptomycin.
There have been attempts to reduce the duration of short-course regimens
to less than 6 months in several countries (reviewed by Fox, 1981,
1985). The latest results have confirmed that 6 month regimens had
similar relapse rates as 9 month regimens of 1%. However, 4.5-5 month
regimens had relapse rates of 4%, 4 month regimens of 12% and 3 month
regimens of 16% (Fox, 1985). It still remains remarkable that a large
proportion of patients are 'cured’ by only 3 months treatment.
1.2.3 Chemoprophylaxis
Chemoprophylaxis can be considered as a form of treatment for an
infection that has not yet occurred, is just beginning, or is in an asymptomatic subclinical state (Hoeprich, 1972). Chemoprophylaxis for
tuberculosis only became possible with the availability of a safe, 46
inexpensive oral drug, isoniazid.
In 1955, the United States Public Health Service reported on trials with guinea-pigs which showed that prophylactic isoniazid could convert a massive inoculation of virulent tubercle bacilli into a controlled benign tuberculous infection (Ferebee and Palmer, 1956). This was foll owed by 13 controlled trials in 7 countries involving nearly 100,000 participants (reviewed by Ferebee, 1970). Seven trials were conducted in the United States, one each in Greenland, Tunisia, Japan, Philip pines, Kenya and the Netherlands. While the results of the various trials differ, several general conclusions were reached. Tuberculin testing at the begining and the end of trials in Tunisia, Kenya and
United States indicated that isoniazid reduced the frequency of tubercu lin conversions but did not have much impact on established tuberculin reactivity. The degree of protection also varied from statistically insignificant differences in some of the smaller studies to excellent results in larger studies. Isoniazid produced the largest effects within the treatment year, but there were indications that the effect was still present in the post-treatment period. The results also showed that adverse reactions to isoniazid were uncommon among healthy persons but increased with age and gastrointestinal problems and that bacterial drug resistance was not a problem.
The current recommended duration of chemoprophylaxis is 12 months.
However, a trial conducted by the International Union against Tubercu losis Committee on Prophylaxis (1982) showed that while 52 weeks of isoniazid prevented the most tuberculosis, a 24-week regimen would decrease hepatitis by one-third and increase tuberculosis by 40%. 47
Chemoprophylaxis can prevent the development of tuberculosis in infected
individuals but its impact on the community will be minimal due to the
problems of application on a mass scale, even in technically advanced
countries.
1.3 Immunomodulation and immunodulating agents
There are numerous biological and chemical agents which have been shown to influence the immune system. The response elicted by these agents have been described by terms such as immunomodulation, immunostimula- tion, immunopotentiation, or immunorestoration. There have been reports that immunomodulating agents may synergise with chemotherapeutic agents.
An example of synergism has been the use of levamisole as an adjunct to antituberculous therapy (Yaseen et al., 1980; Singh et al., 1981).
The importance of modulating the immune response by chemical or biolo gical agents has recently been recognized in two areas of medicine: oncology and infectious diseases. In contrast with oncology, experi mental and clinical data referring to infectious diseases have been less abundant. This is not surprising as most bacterial diseases have been effectively controlled by antibiotics and synthetic antibacterial drugs.
However, it is an undeniable fact that even the most effective antibact erial agent needs the additional activity of the host's immune response.
This is seen most clearly in cases of primary immune deficiencies like congenital thymus aplasia and congenital agammaglobulinaemia where infections are difficult or impossible to treat even with the most effective antimicrobial agents. 48
One of the main problems with the use of immunomodulating agents has been the selection of the dose size and timing of doses which has largely been empirical. The difficulties have arisen from the unusual dose-response curve for immunotherapeutic agents which differ from that of the usual chemotherapeutic agents. Both the timing of administration and the dose have been shown to be critical in the effectiveness of BCG against tumours (Hawrylko and Mackaness, 1973). Levamisole has also been shown to have a time and dose-dependent efficacy against a variety of animal tumours (Mantovani and Spreafico, 1975; Fidler and Spitler,
1975). Davies (1983) suggested the existence of phase variations in the modulation of the immune response by various agents including BCG, endotoxin and ubiquinone. He showed that small changes in doses produced profound changes in the response and suggested that the existence of biphasic and multiphasic variations may have been undetected in many instances because the dose intervals chosen are often too large.
The development of safe immunomodulators for clinical use has become a major target for many drug companies. Immunomodulators can be divided into three categories: 1) agents derived from microorganisms, 2) agents derived from mammalian cells and 3) synthetic chemicals.
1.3.1 Agents derived from microorganisms
In the past, bacterial products have been the agents most extensively investigated. These have been shown to have potent immunomodulating activity but the presence of lipopolysaccharide (LPS) has limited the clinical use of preparations of bacterial origin. Mycobacteria and 49
their products have been known for years to possess strong immuno- adjuvant properties. Subsequently, a number of other effects have been reported including stimulation of the reticuloendothelial system (RES) and enhancement of cell-mediated immunity (Howard et al., 1959; Sher et al., 1975). BCG has been one of the first immunomodulating agents to be extensively investigated in experimental tumours (Old et al., 1959;
Mathe et al., 1969; Zbar et al., 1971) and in human cancer (reviewed by
Laucius et al., 1974; Bast et al., 1974).
Other microorganisms shown to have strong immunomodulating properties are Corynebacterium parvum and C. granulomatosum. The ability of C. parvum to strongly activate the RES was recognized over twenty years ago
(Halpern et a l . , 1964; Neveu et a l . , 1964) and has subsequently been shown to have antitumour properties (Scott, 1974; Oettgen et al., 1976), and the ability to enhance resistance to bacterial infections (Cronly-
Dillon, 1974). Killed C. parvum has been used in clinical trials in cancer patients (Israel, 1977) with the attendent problems of crude bacterial products.
Immunomodulating properties have also been attributed to components from other microorganisms including Pseudomonas aeruginosa (Mathe et al.,
1977); Brucella abortus (Glasgow et al., 1979), and Klebsiella pneumonia
(Griscelli et al., 1982).
Glucans, naturally occurring substances in bacteria, yeasts, fungi and higher plants have been known to be powerful RES stimulators and to play important roles in host defences. They have been tested for possible clinical usefulness (Mansell et al., 1976) and have been reported to be 50
effective against infections with some microorganisms (DiLuzio and
Williams, 1978; Williams et al., 1978) and against some tumours (Stewart
et a l ., 1978; DiLuzio et al., 1979). Lentinan is a completely purified
neutral polysaccharide extracted from an edible mushroom and has been
shown to have potent antitumour properties (Shiio and Yugari, 1980) and
the ability to enhance host resistance to M. tuberculosis (Usuda et al.,
1981; Kanai and Kondo, 1981).
Ubiquinones (Coenzyme Q), important participants in the oxidation-
reduction reactions of the mitochondrial respiratory chain, have been
reported to induce host-specific resistance to a number of bacterial and
viral infections (Block et al., 1978). Another microbial component is
bestatin, a metabolite dipeptide of Streptomyces olivoreticuli which has
been shown to stimulate both humoral and cellular immunity in vitro and
in vivo (Blomgren, 1980).
1.3.2 Agents derived from mammalian cells
Among the immunomodulating agents derived from mammalian cells are
thymic hormones, dialysable leukocyte extracts (transfer factor),
tuftsin and interferons which will be described separately in another section.
Many factors with thymic hormone-like activity have been isolated and described including thymosin fraction 5 (Hooper et al., 1975), thymopo ietin (Goldstein, 1975), serum thymic factor (Bach and Carnaud, 1976) and thymus humoral factor (Kook et al., 1975). These factors have been shown to promote T cell differentiation (Goldstein et a l ., 1978; 51
Trainin, 1974) and to promote antitumour effects in thymectomized mice
(Bach, 1977). Thymosin fraction 5 was the first well-defined thymic hormone preparation to be used clinically in patients with primary
immunodeficiency diseases (Wara et al., 1975; Barrett et al. , 1980) and advanced cancers (Chretien et al., 1978; Cohen et al., 1979).
Transfer factor (TF) prepared by a dialysis method (Lawrence, 1955) and shown to transfer delayed hypersensitivity from an immune to a non- immune person, is in fact a crude dialysable leukoycte extract (DLE) which has now been shown to contain several hundred chemical moieties
(Wilson and Fudenberg, 1983). Consequently, DLE is the current designation for such preparations and the term TF is now reserved for the components with antigen-specific activity. Crude DLE has been shown to contain non-specific immunomodulatory activity in addition to TF activity (Wilson et a l . , 1980). DLE has received widespread clinical use and the results obtained in some viral, fungal and other diseases have been striking (reviewed by Arala-Chaves et al., 1978).
Tuftsin is a hormone-like tetrapeptide which has been isolated from the
Fc portion of IgG. It has been shown to be the physiological stimulator of motility, phagocytosis and pinocytosis for all macrophages and blood neutrophils (Najjar, 1974); and has also been shown to possess anti tumour properties (Nishioka, 1979). 52
1.3.3 Synthetic immunomodulating agents
The first synthetic immunomodulating agent to be extensively investi gated was levamisole, which will be described in a later part of the literature review (section 1.5).
Another synthetic molecule that has been extensively examined is N- acetylmuramyl-L-alanyl-D-isoglutamine or muramyl dipeptide (MDP). MDP is a small molecular weight subunit of the mycobacterial cell wall peptidoglycan which can be used in place of whole mycobacteria for immunoadjuvant activity (Ellouz et al., 1974). However, the pyrogeni- city of MDP has been a major obstacle for clinical applications
(Dinarello et al . , 1978; Rotta et a l . , 1979). Subsequently, several hundred compounds containing the muramyl moiety have been synthesized and tested and some have been found to be superior to MDP as immuno- modulators. The N-butyl-ester of MDP has been shown to be apyrogenic and to enhance non-specific resistance to Klebsiella infection (Chedid et al., 1982). Lipophilic derivatives like 6-0-acyl esters of MDP have been shown to retain immunostimulating properties (Matsumoto et al.,
1981). Other lipophilic derivatives containing the mycolyl group at the end of the peptide chain have been shown to stimulate strongly adjuvant activity, delayed hypersensitivity and non-specific antibacterial resistance (Parant et al., 1980).
Several compounds containing nucleotides have also been shown to possess immunomodulating properties. The polynucleotides, polyinosinic-polycy- tidylic (poly I:C) and polyadenylic-polyuridylic (poly A:U) have been reported to be potent antiviral (Richmond and Hamilton, 1969) and 53
antitumour (Levy et a l . , 1968) agents as well as interferon inducers
(Field et al., 1967). Another important compound is methisoprinol or
isoprinosine (ISO), which appears to be effective in a wide variety of
viral diseases (Waldman and Ganguly, 1977; Laude et al., 1980). ISO
increases cell mediated immune functions in vitro (Bradshaw and Summer,
1977; Wybran et al ., 1978) and increases T cell levels in patients
(Friedman et al., 1980). NPT 15392 is a hypoxanthine analogue that has
recently been shown to possess neutrophil, T cell and NK cell stimula
tory properties. Its structure is related to inosine and its action to
methisoprinol. It augments human T cell proliferation and suppressor
cell induction (Hadden et a l . , 1982) and also modulates a variety of
cytocidal functions (Florentin et al., 1982).
Pyran (maleic divinyl ether copolymer) was first used as a chemothera
peutic antiviral agent against Friend virus-induced leukemia and the
protection was attributed to interferon induction (Merigan, 1967; Hirsch
et a l . , 1972). The problem with these early copolymers was extensive
toxicity which was related to the high molecular weight polymers. This
led to the development of copolymers based on the same structure but of
smaller molecular weights called maleic vinyl ethers (MVE). MVE-2 has
been shown to be a potent immunostimulator of low toxicity and has been
tested in clinical trials (Carrano et al., 1984).
Recently, a compound belonging to the 2-cyanaziridines group, azimexon, has been shown to have immunomodulating properties which include incre ased cellular immunity, NK cell activity, increased granulopoiesis and antitumour activity (Bicker, 1984). 54
1.4 Interferon
Interferon (IFN) was discovered by Isaacs and Lindenmann (1957) when they observed anti-viral activity in the supernatant fluids of chick allantoic membrane cultures inoculated with influenza virus. It is now known that IFN is a complex group of small proteins and glycoproteins produced by animal cells in response to a wide variety of stimuli.
Although IFNs were first recognized for their anti-viral activity, recent interest in them has centered on the discovery of a wide range of biological effects on the immune system. IFNs have been shown to regul ate cellular proliferation or differentiation as well as cellular morph ology and expression of cell surface antigens. In addition, IFNs appear to be potent immunoregulatory agents affecting both cell mediated and humoral immunity.
1.4.1 Description of the interferons
To date, three different types of IFNs have been described, IFN-alpha
(IFN-*<-or leukocyte IFN), IFN-beta (IFN-p or fibroblast IFN) and IFN- gamma (IFN-V or immune IFN). IFN-«* is mainly produced by B cells, natural killer (NK) cells and macrophages; and IFN-|3 mainly by fibro blasts after induction by a variety of agents. Among the inducing agents are viruses; double-stranded RNA; intracellular organisms like brucella, listeria, rickettsia, mycoplasma and chlamydia; microbial products like LPS; organic polymers such as pyran co-polymers; and several low molecular weight substances like cycloheximide, kanamycin and tilorone (Epstein, 1979). T lymphocytes are the main cells which produce IFN-K after stimulation by mitogens or specific antigens. 55
However, B cells and NK cells also produce IFN-Y after mitogenic stimulation (Epstein et al., 1974; Kirchner et al., 1979).
The IFNs differ antigenically, physically and chemically from each other. Antigenic differences between all three IFNs can be detected by polyclonal and more recently, by monoclonal antibodies (Secher & Burke,
1980; Nyari et a l . , 1981; Hochkeppel, 1982). Human IFN-o( and |2> have molecular weights ranging from 18,000 to 25,000 daltons but native human
IFN-X has a molecular weight of 50,000 daltons. Recent studies however, have suggested that it may be composed of dimers of smaller subunits of
20,000 and 25,000 daltons (Yip et al. , 1982). IFN-Y and IFN-[iare glycoproteins whereas most IFN-0C moieties do not appear to have carbohy drate components.
The antiviral effects of IFNs are relatively species specific but there are exceptions. Subtypes of human leukocyte IFN may have significant antiviral activity on bovine or mouse cells (Week et al., 1981). It has been shown that each IFN has a characteristic species activity profile.
Human IFN-0( has a high degree of antiviral activity in bovine and porc ine cultures whereas human IFN-{S is hardly active and human IFN-X is totally without activity in these cells (Rager-Zisman and Bloom, 1985).
1.4.2 Molecules and genes of the interferons
IFN-o( has been purified to homogeneity (Rubinstein et al., 1978; Berg and Heron, 1980) and human IFN-o( has been shown to consist of at least
13 different proteins (Berg and Heron, 1982). Recently, several groups have succeeded in cloning at least 12 distinct but almost homologous 56
IFN-<* proteins in Escherichia coli (Goeddel et al., 1980; Streuli et^ a l . , 1981; Rehberg et a l . , 1982). Only one human I F N - g e n e has been definitely isolated (Derynck et al., 1980) and has led to the production of a polypeptide with human IFN-{3 activity in E. coli (Derynck et al.,
1980a). However, it has been observed that there are at least five translationally active human IFN-^3 mRNAs (Sagar et al., 1982). There is evidence that only one IFN- gene consisting of 146 amino acids exists
(Gray and Goeddel, 1982). Recently, the human IFN-^ gene (Gray et al.,
1982), followed by the murine IFN-tf gene (Gray and Goeddel, 1983) were cloned in E. coli and monkey cells.
1.4.3 Mechanisms of action of the interferons
It is now known that IFNs exert their action by binding to specific cell-surface receptors probably consisting of gangliosides and, or glycoproteins (Friedman, 1967; Aguet, 1980). IFN-^and IFN-appear to share a class of receptors whereas IFN-# binds to a different receptor
(Branca and Baglioni, 1981). Binding assays using radiolabelled IFN-if have demonstrated that there are about 2400 and 12,000 high-affinity binding sites per human fibroblast and mouse macrophage respectively
(Celada et al., 1984). Anderson et al.(1983) showed that labelled human
IFN- bound to cells is rapidly internalised and degraded at 37°C.
Binding of IFNs initiates a series of metabolic modifications which involve the de novo synthesis of RNA and polypeptides. Two-dimensional gel electrophoresis has shown that all IFNs induce several common poly peptides but that IFN-JT induces in addition 12 distinct polypeptides
(Weil et al., 1983). 57
Among the proteins synthesized are 2'5 '-oligoadenylate synthetase and
protein kinase. 2'5'-oligoadenylate synthetase catalyzes the synthesis
of small molecules of 2'5' adenylate oligomers which activate endogenous
ribonucleases (Baglioni et a l . , 1978). The ribonucleases can degrade
both viral and probably host cell mRNAs which could lead to diminished
synthesis of viral specific proteins. Protein kinase is activated by
the presence of double-stranded RNA and mediates through phosphorylation
the inactivation of a eukaryotic protein synthesis initiation factor
eIF-2 (Lebleu et a l . , 1976). These two enzymatic activities could be
responsible for the restriction of viral replication. However it is
still not firmly established that either is responsible for anti-viral
activity. Both need double-stranded RNA for full activation and this
suggests that there are likely to be other mechanisms of action for the non-viral effects of IFNs.
There has been some evidence of the possible mechanisms of action of the non-viral effects of IFNs. Recently, Hamilton et al. (1985) demonstrat ed that murine peritoneal macrophages treated with recombinant IFN-Y had
2+ a five fold increase in Ca , phospholipid-dependent protein kinase act ivity (Protein kinase C). The kinetics of the elevation of kinase acti vity was identical to that required for induction of other activities by
IFN-Y, suggesting that protein kinase C may have direct functional con sequence in macrophage activation. Similarly, Weiel et al. (1985) used a model of depression of transferrin receptors on murine peritoneal macrophages after exposure to IFN- Another enzyme induced by IFN-^ is indoleamine-2,3-dioxygenase which catalyzes the breakdown of tryptophan. Since eukaryotic cells and many intracellular parasites require tryptophan for growth, this enzyme may play a role in inhibition of cell growth or intracellular parasite (Pfefferkorn, 1984). Nagata et al. (1984) have shown that in a macro phage cell line, cyclic AMP may mediate the effects of IFN-*. They selected mutant macrophage cell lines which were susceptible to anti viral effects but resistant to growth inhibiting effects of IFN-JT and showed that the mutant cell lines were either lacking IFN-JT receptors or were mutants for adenylate cyclase. 1.4.4 Immunomodulating effects of the interferons IFN preparations especially those containing IFN-f have long been known to modulate the immune response (Gresser et a l ., 1972; Virelizier et a l . , 1977; De Maeyer and De Maeyer-Guignard, 1982). All three types of IFNs have been shown to affect antibody production, cell-mediated immunity and other functions of the immune system. However, as most of these earlier studies used only crude or partially purified IFNs or mixtures of IFNs it had been difficult to separate the immunoregulatory effects of IFNs from those of other biologically active molecules. Recently, with the cloning of the IFN genes in mouse and man, it will now be possible to assess the actual effects of each of these molecules on the immune response. 59 1.4.4.1 Effects on humoral immunity Various types of IFNs have either suppressive or enhancing effects on humoral B-cell responses both in vivo and in vitro, depending on the relative timing of exposure to IFN and to the antigen. IFN-V in parti cular potentiates the effects of immunoglobulin secretion when added late during an immune response in vitro (Sonnenfeld et al., 1978). Recent reports with recombinant IFN-V confirm the effects observed previously with partially pure preparations. Nakamura et al. (1984) described an enhancement of antibody formation when antigen and IFN-V were administered together to mice. In another study, Leibson et al. (1984) showed that cloned murine IFN-^T could act synergistically with other helper factors as a T-cell Replacing Factor (TRF) in stimulating B-cell antibody response in vitro. IFN-V has also been reported to act as one of several B-cell maturation factors and induces surface phenotype changes and immunoglobulin secretion in resting B cells (Sidman et al., 1984). 1.4.4.2 Effects on cell surface membrane The cell surface membrane plays a vital role in direct contacts between cells, recognition of molecular structures and initiation of cell activ ation and differentiation (Loor, 1979). All these interactions are known to be mediated through surface molecules on the cell surface memb rane. IFNs induce a variety of alterations in and on the cell surface (Friedman, 1979). Knight and Korant (1977) described an increase in net negative surface charge on murine L cells and Chang et al. (1978) reported an increase in bouyant density of the plasma membranes in IFN 60 treated murine cells. Other studies have shown that IFN-(S treatment can alter the structure and organization of the cytoskeleton of a variety of cells (Pfeffer et al., 1980; Pfeffer et al., 1980a). All three types of IFNs enhance the expression of Class I Major Histo compatibility (MHC) antigens, although IFN-J seems to be the most effi cient (Sonnenfeld et al., 1981; Fellous et al., 1979; Wallach et al., 1982). There have been conflicting reports on the effect of IFN-** and IFN- £ on the expression of MHC Class II antigens. However, IFN-if has been shown to cause a substantial increase in these antigens on lymphoid cells, myelo-monocytic cells, mast cells, fibroblasts, tumour cell lines and melanoma cells (Wong et al., 1983; King and Jones, 1983; Virelizier et a l . , 1984). Walker et al. (1984) reported that IFN-^ may induce la antigens on mouse macrophages via a discrete secondary factor. Other changes consistently seen on human and murine cells following IFN treatment are the expression of Fc-IgG receptors (Fridman et al., 1980; Itoh et al., 1980; Guyre et a l ., 1981). However, these effects are quantitatively not as large as the increases in MHC Class I antigens and are furthermore only seen in certain subpopulations of cells. The biological significance of the IFN-induced surface alterations is not yet established. However, the Class II MHC antigens are important in the recognition processes for cell interactions or cytotoxic acti vity. Zlotnik et a l . (1983) has shown that IFN-T treatment renders macrophage cell lines capable of antigen-presentation. Pober et al. (1983) reported that human T lymphocytes recognized and lysed endothe lial and fibroblast cells with IFN-)f induced la antigens. Becker (1985) 61 showed that the IFN-V enhancement of monocyte antigen-induced and autologous proliferative responses was a consequence of the increased density of monocyte HLA-DR antigens induced on the accessory cells. 1.4.4.3 Effects on cell mediated immunity IFN-tf appears to play a more important role in immunoregulatory function than IFN-c* or IFN-£J. It is one of the principle lymphokines regulating the activation of macrophages. It had been previously postulated that IFN-X and MAF are the same molecules (Schultz and Chirigos, 1978; Kleinschmidt and Schultz, 1982). Studies with recombinant I F N - h a v e yet to resolve the controversy. There have been reports of antigenic and functional similarities of MAF and IFN-fr (Schultz and Kleinschmidt, 1983; Schreiber et al . , 1983; Svedersky et al., 1984). Other studies suggest that IFN-3T and MAF are distinct molecules. It has been shown that culture supernatants from human T cell lines contain a soluble factor with MAF activity that is not abrogated by treatment with anti human IFN-V antibody (Andrew et al., 1984; Kleinerman et al., 1984). It has recently been hypothesized that the intact IFN-2T molecule may contain distinct domains for antiviral activity and for MAF activity (Peters et al., 1985). This hypothesis is supported by the development of monoclonal antibodies specific for various epitopes of the recombi nant murine IFN-# molecule that differentially inhibit the antiviral and MAF activities (Schreiber et al., 1985). The availability of recombinant IFN-if and the development of monoclonal antibodies to IFN-^fhave enabled the unambiguous identification of IFN-k* as the active factor in lymphokines activating macrophages to produce 62 and for killing a variety of intracellular organisms in both the human and the murine system. Rothermel et al. (1983) showed that it was IFN- X present in human concanavalin A (Con A) induced lymphokine that activated monocytes to inhibit chlamydial replication. Similarly, Murray et al. (1983) reported that IFN-^ is the main macrophage activa ting molecule present in human lymphokines and that recombinant IFN-& enhances dependent and independent anti-leishmanial activity of monocytes. Nathan et al. (1983) identified IFN-if as the lymphokine that activates human macrophage oxidative metabolism and anti toxoplasmic activity. In the murine system, Murray et al. (1985) has shown that IFN-8 activates mouse peritoneal macrophages both in vitro and in vivo for increased oxidative metabolism and anti-protozoal activity. There is also evidence that IFN-tf can activate cells other than macro phages to kill intracellular organisms. Pfefferkorn (1984) reported that IFN-# blocks the growth of Toxoplasma gondii in human fibroblasts by inducing host cells to degrade tryptophan. Wiseman and Waddell (1983) showed that factors in human lymphokines with the properties of IFN-y’ have antirickettsial activity on Rickettsia prowazekii infected endothelial cells, fibroblasts and macrophages. There have been similar reports of the effects of IFN-& in murine cells. IFN- X has been shown to inhibit the growth of R. prowazekii in mouse fibroblasts (Turco and Winkler, 1983). Turco et a l . (1984) reported that crude lymphokines and recombinant IFN-tf, but not a crude preparation of IFN-<* and IFN-J2, inhibited the growth of Coxiella burnetii in mouse fibroblasts. In addition, IFN-|f has been shown to regulate natural killer cell activity (Rager-Zisman and Bloom, 1985); stimulate production of 63 interleukin-1 production (Palladino et a l . , 1983); and enhance the growth of interleukin-2 (IL-2) dependent murine cytotoxic T cell clones in the absence of IL-2 (Peters et al., 1985). 1.5 Levamisole 1.5.1 Description of levamisole Levamisole is the levo isomer of tetramisole stereospecifically synthesized in 1966 , and available as an antihelminth for clinical use in 1968. It is a stable, white crystalline powder of molecular weight 240.75 and is very soluble in water and aqueous acidic solutions. In neutral buffers and especially alkaline solutions, the solubility drops and hydrolysis to 3-oxo-3(2-mercaptoethyl)-5-phenyl imidazoline (OMPI) occurs. Recent studies suggest that OMPI is the active compound (Van Ginckel and DeBrabander, 1979). Levamisole is active against most nematodes but has no direct toxic effect on bacteria, viruses and fungi (Thienpont et a l . , 1966) nor on normal and tumour cells at concentra tions up to 100 pg ml ^"(Sampson and Lui, 1976; Pabst and Crawford, 1974). Pharmacologically, levamisole stimulates parasympathetic and sympathetic ganglia (Van Nueten, 1972). Pharmacokinetic studies in animals (Graziani and De Martin, 1977) have shown that levamisole is rapidly absorbed from the gastrointestinal tract and well distributed in the tissues with the highest tissue levels in the liver and kidneys. Plasma half lives for the unchanged drug vary from 1-4 hours and drugs were rapidly removed from the tissues within 2-4 days. It is a drug of low toxicity with therapeutic doses in the range of 2-3 mg kg 64 1.5.2 Immunomodulating effects of levamisole The first evidence of the immunomodulating properties of levamisole was obtained in 1971 when it was observed that levamisole increased the immunity of Brucella vaccinated mice (Renoux and Renoux, 1971). Since then, there have been a multitude of studies on levamisole, some con tradicting and some supporting the immunotherapeutic potential of levamisole. The effects of levamisole on the immune system has been investigated in isolated cells, experimental animal infections, normal volunteers and patients. These studies showed that levamisole influenced cell-mediated immune reactions. In vitro and in vivo studies showed that levamisole was able to restore effector mechanisms of cell mediated immunity. The effects are most pronounced in compromised hosts with subnormal T cell or phagocyte functions (Bensa et al., 1976; Ellegaard and Boesen, 1976; Bruley-Rosse t et a l . , 1976; Rosenthal et al., 1976). Levamisole does not usually increase an adequate immune response except at doses greatly exceeding therapeutic concentrations. The effector phagocytic functions which may be restored by levamisole include chemotaxis (Rabson et al., 1978); phagocytosis (Molin and Stendahl, 1977); random migration (Anderson et al.,1976); adherence and antibody and complement receptor activity (Schmidt and Douglas, 1976; Schreiber e t a l . , 1975) of polymorphonuclear and mononuclear phagocytes. Effector lymphocyte functions which may be restored include spontaneous, mitogen or antigen-specific proliferation (Chan and Simons, 1975; Chan 65 et a 1 ., 1976; Lewinski et al., 1977); lymphocyte counts (Moncada- Gonzalez et a l . , 1976); antibody plaque formation (Renoux and Renoux, 1974) and migration inhibition and lymphokine production (Golding £t_ al., 1976). Levamisole does not stimulate B-cells directly. It has been shown not to increase B-cell mitogenic proliferation (Hadden £t_ al. , 1975) nor antibody production (Flannery et al., 1975). There is in vivo evidence that levamisole is capable of inducing maturation of T cells. It induces thymic antigens in nude mice (Renoux and Renoux, 1977) and stimulates lymphocyte proliferation in nude and thymectomized mice (Merluzzi et al., 1976). Further evidence is provided by the findings that levamisole restores cell-mediated immune functions in children with primary immune deficiencies (Griscelli et al., 1978). Levamisole has been shown to restore delayed skin sensitivity in anergic subjects. Such observations have been made in cancer patients (Lewinski et al., 1977; Tripodi et al., 1973); aged healthy persons (Kondo et al., 1978) ; pulmonary tuberculosis patients (Singh et al., 1981); and leprosy patients (Cardama et al., 1973). The activity of levamisole in restoring cell-mediated functions indicates a potential use in diseases where hypofunction of T cell, polymorphonuclear cells or macrophages occurs. Levamisole has been tested in large numbers of diseases with suspected immune imbalance and some have responded well, others partially or not at all. Responses to levamisole has been mainly seen in chronic or recurrent diseases (reviewed by Symoens and Rosenthal, 1977), some cancers (reviewed by Amery and Verhaegen, 1978) and primary immune deficiencies (Griscelli et al, 1978). 66 1.5.3 Mechanisms of action of levamisole The mechanisms of action of levamisole are rather unclear at present. There have been several proposals. One possibility is that levamisole increases cyclic GMP or reduces cyclic AMP (Hadden et al., 1975; Hogan and Hill, 1978) in lymphocytes and phagocytes which would promote proliferative and secretory functions as well as receptor reactivity. Levamisole contains an imidazole ring and it has.been suggested that this moiety is responsible for immune regulation by altering the levels of cyclic nucleotides. In vitro studies have shown that levamisole and imidazole exerted virtually identical effects, however, none of the in vivo effects of levamisole could be reproduced by imidazole (Renoux and Renoux, 1977a). Another possible mechanism could be through the activity of the thiol OMPI metabolite which appears after the hydrolysis of levamisole. Thiols like all other antioxidants could be important for maintenance of the cellular redox potential. Levamisole and OMPI have been shown to restore cell functions by the inhibition of peroxidase formation (Ander son et a l . , 1981). Levamisole also shares with all other antioxidants the ability to stimulate DNA synthesis (Van Wauwe and Goosens, 1979). Another aspect is the indirect thymomimetic activity of a molecule mediated by a serum factor which is produced by responders but not non responders to levamisole. The serum factor has been shown to mimic thymic hormone; to be neither a complement factor nor a levamisole meta bolite; and animals that do not respond to levamisole respond to serum 67 factor (Symoens et al., 1979). Different mechanisms might operate depending on the conditions of the experiment, whether it was performed in vivo or in vitro or whether the immune response of the host was normal or impaired. 1.6 Targeting of drugs An important feature of successful drugs is the ability to act predomin antly on the target cell thus minimizing the effects on the rest of the organism. Antibacterial agents are a good example. Most of them owe their unique selectivity to their ability to interfere with some meta bolic pathway peculiar to the target bacteria and not shared by the human host. The use of carriers has been proposed to improve the selec tivity of drugs. Carriers could deliver the drug directly to the target organ, tissue or cells. They might also favourably alter the pharmaco kinetics of the drug like clearance and metabolism enabling the reduc tion of therapeutic dosages to acceptable toxicity levels. It is also conceivable that carriers might prolong the intervals between admins- tration of the drug. 1.6.1 Description of drug carriers A number of drug carriers have been investigated and they can be broadly divided into two categories, natural products and synthetic systems. Natural carriers are extracted from animals or plants and either selec tively bind to receptors on potential targets, for example antibodies; or migrate to specific targets, for example, neutrophils attracted to 68 inflammation sites. Most of the studies with natural carriers involved the use of antibodies in the treatment of experimental cancers. Poly clonal antibodies raised against the whole target cells or surface anti gens and coupled with a wide variety of cytotoxic drugs have been quite selective in experimental systems in killing cells in vivo and in vitro, but were not as successful as expected in clinical use (Everall et al., 1977; Ghose et a l ., 1977). The recent advent of monoclonal antibodies has given new impetus to antibody-mediated targetting (Thorpe et al., 1982). Synthetic carriers include polymers, albumin beads, acrylic microsphe res, magnetic particles and liposomes. Of all the synthetic carriers, liposomes have been most extensively investigated. 1.6.2 Liposomes ■1.6.2.1 Types of liposomes A liposome is a minute spherical vesicle composed of phospholipid bilayers that enclose a volume. There are four main types of vesicles. Multilamellar vesicles (MLV), originally described by Bangham et al. (1965), are composed of many concentric lamellar membranes and range in diameter from 0.1 to 10 jum. Small unilamellar vesicles (SUV) with one membrane and one cavity of diameter 25-50 nm, were initially described by Papahadjopoulos and Miller (1967). Large unilamellar vesicles (LUV) are similar to the SUV except for a larger diameter of 0.2-1.0 jam (Papa- hadjopoulos et a l ., 1975; Deamer and Bangham, 1976). Reverse phase evaporation vesicles (REV) are large unilamellar liposomes of diameter 69 0.2-0.8 Aim formed by evaporation of the organic phase from an emulsion of phospholipids and aqueous buffer (Szoka and Papahadjopoulos, 1978). 1.6.2.2 In vivo interactions of liposomes All liposomes can entrap hydrophilic substances in the aqueous compart ments and lipophilic substances in the phosholipid membranes of the vesicles. The use of liposomes as carriers is based on their inter action with cells. It has now become apparent that liposomes can associate with cells in a variety of ways. Endocytosis has been shown to occur (Weissman et al., 1975; Poste and Papahad jopoulos, 1976), and is followed by lysosomal fusion leading to disruption and release of the entrapped agents which then either act within the lysosomal vacuole or diffuse into other cell compartments. Liposomes have also been shown to fuse with the plasma membrane of the cell and introduce their contents directly into the cytosol (Poste and Papahad jopoulos, 1976). Another mechanism that has been proposed is that liposomes adsorb onto the cellular membrane without subsequent interiorization, with the release of agents by diffusion from the liposomes (Poste, 1980). It is quite likely that more than one mechanism occurs simultaneously and that parameters like cell type, liposomal lipid composition, size, surface charge and other experimental conditions will favour one mechanism over another. After intravenous injection, liposomes are rapidly cleared from the plasma and found mainly in mononuclear phagocytes of the reticuloendo thelial system in the liver, spleen and circulating monocytes (Finkel- stein and Weissmann, 1981). This natural fate of the liposomes has 70 created difficulties when the drugs are to be targeted to other cell types like solid tumours. However, this passive localization of lipo somes can be exploited in diseases where the mononuclear phagocyte plays an active and important role in host defence like with intracellular parasites. The blockade of RE phagocytic activity produced by an intra venous injection of liposomes is transient and reversed fully within 24 hours (Abra and Hunt, 1982; Ellens et al., 1982). 1.6.2.3 Therapeutic applications of liposomes Among the substances that have been encapsulated in liposomes are enzymes (Gregoriadis and Buckland, 1973; Tyrell et al., 1976); anti tumour drugs (Kaye and Richardson, 1979; Mayhew et.. al. ,1978) , chelating agents (Rahman et al.,1973; Young et al.,1979); steroids (Shaw et al., 1976; De Silva et a l ., 1979); antibiotics (Desiderio and Gordon-Camp- bell, 1983; Lopez-Berestein et al., 1983); lymphokines (Fidler, 1980; Fidler et al., 1982) and immunomodulators (Fidler et al., 1981; Philips et al., 1985). The feasibility of exploiting passive targeting of liposomes as a means of activating macrophage-mediated host defence mechanisms has been inve stigated in several laboratories. Fidler, Poste and their colleagues have investigated the in vitro and in vivo effects of lymphokines encap sulated in liposomes (Poste et al., 1979; Fidler, 1980; Fogler et al., 1980). They demonstrated that liposomes containing MAF activated macrophages to be tumoricidal in experimental murine metastatic cancer. Fidler et al. (1980) showed that the formulation of the liposomes could influence the in vivo distribution after intravenous administration. 71 They demonstrated that the proportion of liposomes trapped in the lungs of mice could be increased by using liposomes containing phosphatidyl choline (PC) and phosphatidylserine (PS) in a molar ratio of PC:PS = 7:3. However, even though more liposomes of this formulation are delivered to the lungs, the majority of them are still to be found in the liver and spleen (Poste et al., 1982). Reed et al. (1984) also showed that lymphokine encapsulated in liposomes enhanced the in vivo inhibition of the proliferation of L. donovani chagasi in mice. Liposomes containing immunomodulating agents have been shown to be highly effective in stimulating macrophage-mediated host resistance. Fidler et a l . (1981) demonstrated the eradication of spontaneous meta- stases and the activation of alveolar macrophages by liposomes con taining MDP. Fraser-Smith et al. (1983) reported the protective effect of an analog of MDP encapsulated in liposomes against an experimental Candida albicans infection. In addition to their role in host defence, mononuclear phagocytes are also important sites of replication for many parasites. A number of viruses, bacteria, fungi and protozoa multiply within these cells. Such intracellular infections are often difficult to eradicate and liposomal encapsulation of therapeutic agents could resolve the problem. It could provide a more efficient drug delivery system directly to the target cells, enabling a reduction of dosages thereby reducing potential toxi city effects. This has been shown in experimental therapy of leishma niasis with antimonials (Alving et al.,1978; New et al., 1978). These studies showed that the doses of liposome-encapsulated drug required to inhibit parasitic growth and cure infected animals are about 1/100 to 72 1/800 of those required of the free drug. Lopez-Berestein et al. (1983) demonstrated that encapsulation of amphotericin B in liposomes improved the therapeutic index in a model of experimental murine candidiasis. In a study on chemotherapy of murine cryptococcosis, Graybill et al. (1982) showed that liposome-associated amphotericin B had only 1/17 the toxi city of the free drug. The enhanced effects of liposomal amphotericin B in cryptococcosis was mainly due to the ability to deliver larger doses without increased toxicity. When the liposomal dose was reduced to a dose that could be safely administered as a free drug, liposomes did not convey any therapeutic advantages. Experimental murine histoplasmosis was also more effectively treated with the higher amphotericin B concen tration that could be achieved with liposomes (Taylor et al., 1982). 73 II. INTRODUCTION AND OBJECTIVES OF THE EXPERIMENTAL WORK 1.7 Introduction and objectives After taking into consideration the impact of current tuberculosis control measures on the epidemiology of tuberculosis, there is universal agreement that the most powerful component in tuberculosis control is case-finding and chemotherapy. In addition to their direct and imme diate effect of reducing suffering and mortality, case-finding and chemotherapy also eliminate sources of infection. BCG vaccination can prevent tuberculosis in uninfected individuals but does not contribute significantly to the reduction in overall risk of infection in the community. Isoniazid prophylaxis can prevent the development of tuberc ulosis in infected individuals but is also unlikely to have much impact on the community as it is cannot be easily implemented on a mass scale. At present there seems little prospect for any improved vaccines and BCG remains the only vaccine. It is possible that with the application of genetic engineering a more efficacious vaccine will be engineered (Young et a l . , 1985). However, it is likely to be decades before any new vaccine has an impact on the disease as it will have to be tested for safety and then properly evaluated for its ability to produce long lasting protection in lengthy controlled trials. Furthermore, there is an inevitable time lag between vaccination in childhood and its efficacy in preventing adult disease 30-60 years later. Immunological research may be helpful in two ways: 1) improving case finding by new diagnostic techniques, and 2) improving chemotherapy by 74 using immunomodulating agents. Considering first new diagnostic methods, development of better serological tests could lead to improved diagnosis of childhood tuberculosis, non-pulmonary tuberculosis and smear-negative cases of active pulmonary tuberculosis. The isolation of pure antigens or more specific tuberculins would also be of value for skin testing and other in vitro immunological tests. Turning to therapy, it is important to learn whether chemotherapy might be improved by simultaneous treatment with immunotherapeutic agents. The second question is the subject of the present thesis. Two immunomodulating agents, levamisole and interferon-gamma, were examined for their effects on experimental murine tuberculosis; and for interferon, for possible synergism between the immunomodulating agent and the chemotherapeutic drugs, isoniazid and rifampicin. Levamisole has been shown to have immunotherapeutic potential and has been tested in a large number of diseases with suspected immune imbalance. There have been claims of the clinical benefits of levami sole as an adjunct to chemotherapy on tuberculosis patients. In a study with 73 cases of advanced pulmonary tuberculosis (Yaseen et al., 1980) it was reported that the addition of levamisole to a standard antituber culous regimen improved the speed of sputum conversion and the radiolo gical findings. They also reported increases in T cell counts and restoration of dinitrochlorobenzene (DNCB) sensitization. A trial of levamisole as an adjunct in the treatment of 50 newly diagnosed pulmo nary tuberculosis patients with mild immunodepression (Singh et al., 1981) showed improved radiological clearing in the levamisole-treated group but no difference in sputum clearance rate. In the levamisole- treated group, 48% reacted to DNCB compared to 18% in the control group. 75 In another study of 100 pulmonary tuberculosis patients, Singh et al. (1983) reported that levamisole given in conjunction with antituber culous drugs caused a significant difference in sputum conversion time, subjective improvement, weight gain and radiological improvement. However, another study on the effect of levamisole in combination with chemotherapy on 15 patients with miliary tuberculosis (Singh et al., 1983a) showed only a restoration of Mantoux and DNCB reactivity, with no clinical improvement. A clinical trial on the effect of levamisole on the response to chemo therapy of pulmonary tuberculosis patients in Kenya and Zambia was being organized under the auspices of the Medical Research Council at the start of the work described here. The double-blind trial consisted of three regimens: chemotherapy with streptomycin, isoniazid, rifampicin and pyrazinamide daily for two months followed by daily thiacetazone and isoniazid for 4 months; chemotherapy supplemented with levamisole once a week for one month; and chemotherapy with levamisole for two months. Levamisole was given at a dose of 150 mg for patients weighing 50 kg or more and 100 mg for patients less than 50 kg in weight. The existence of this major clinical study was an important reason for investigating the efficacy of levamisole in experimental murine tuberculosis. The effects of levamisole were assessed on an infection of M. microti in mice. As M. microti is non-pathogenic for humans, all manipulations and assays could be carried out with comparative ease. In addition to moni toring the effect of levamisole on the growth of M. microti, the effects on T lymphocyte function and subsets were also examined to monitor the effects on the immune system. The work with levamisole was not carried 76 to completion in that no experiments were done with levamisole in combi nation with antituberculous drugs. The reasons for this was because levamisole appeared to have no effect on the M. microti model and in part because recombinant interferon-gamma became available and was given priority for further study. Recent evidence has implicated interferon-gamma as the important factor in lymphokine preparations which activates macrophages for antitumour and antimicrobial action (Pace et a l . , 1983; Rothermel et al. , 1983; Murray et al., 1983; Nathan et al., 1983). Recombinant interferon-gamma was examined for its effects on the growth of M. tuberculosis in the lungs and spleens of mice. The model of an acute infection with the virulent strain H37Rv of M. tuberculosis was chosen as it was considered to be a more relevant model than an infection with M. microti. Lipo somes have been shown to be effective in delivering drugs or immunomodu- lating agents in both in vivo and in vitro systems. Fidler (1980) had shown that MAF encapsulated in negatively-charged MLV liposomes composed of PC and PS (PC:PS =7:3 mole ratio) was effective in activating alveolar macrophages for tumouricidal activity in murine lungs. It was decided to administer interferon-gamma in liposomes formulated in a similar way on M. tuberculosis infection in mice. The effects of interferon-gamma in combination with isoniazid and with rifampicin were examined with M. tuberculosis infection in mice. Ison iazid was chosen because it is an important component of all chemothera peutic regimens, and for its high bactericidal activity while rifampicin was chosen for its high sterilizing activity. 77 The mononuclear phagocyte plays an important role in both the afferent and the efferent arms of the immune response to tuberculosis. Tubercle bacilli multiply in macrophages in susceptible hosts and it has been demonstrated that macrophages can be activated in vitro by lymphokines to kill M. microti (Walker and Lowrie, 1981). The effects of IFN-Y on the growth of L. monocytogenes and M. microti in murine peritoneal macrophages were examined. The listericidal assay was chosen because L. monocytogenes is an intracellular pathogen with a rapid growth rate and this facilitated the development of the microbicidal assays. The choice of M. microti enabled the in vitro assays to be performed with comparative ease. In addition, the in vitro activity of IFN-Y in combination with isoniazid and with rifampicin on M. microti infection in macrophages was also investigated. 78 CHAPTER 2 MATERIALS AND METHODS 2.1 Media 2.1.1 Bacteriological Culture Media and supplements All media were obtained from Difco Laboratories (P.0. Box 14B, Central avenue, West Molesey, Surrey, UK) unless specified otherwise. Middlebrook 7H11 Oleic acid-albumin Agar was reconstituted from the dehydrated product in distilled water containing 0.5% glycerol. After sterilization, the media was supplemented with 10% Oleic Albumin Dext rose Complex (OADC). Selective 7H11 Agar (Mitchison et a l ., 1973) was prepared by the addition of antibiotics to the reconstituted Middlebrook 7H11 medium above. The following antibiotics at their respective final concentra tions were added after autoclaving : 1. Polymyxin B , 200 units ml ^ 2. Carbenicillin , 100 }ig ml ^ 3. Trimethoprim , 20 jig ml ^ 4. Amphotericin B , 10 jig ml ^ Dubos Oleic Agar Base was prepared by reconstituting the dehydrated medium with distilled water and adding 10% OADC after sterilization. Middlebrook 7H11 agar, Selective 7H11 agar and Dubos Oleic agar supple mented with 5% lysed horse blood were prepared as described above and 79 supplemented with 5% lysed horse blood after sterilization. Dubos Broth (2X) was prepared double strengthed from Dubos Broth Base (Difco Laboratories). It was used either in this form or with the addition of 10% ADC. Middlebrook 7H9 Broth was reconstituted from the dehydrated medium with distilled water. It was supplemented with 10% ADC. Middlebrook ADC Enrichment and Middlebrook OADC Enrichment were either obtained from Difco Laboratories or were prepared in the laboratory from the ingredients. Tryptic Soy Agar Blood Base (TSA) and Tryptic Soy Broth (TSB) were prepared from the dehydrated products. Horse blood was obtained from Tissue Culture Services Ltd. (2 Perth Estate, Slough, Berkshire SL1 4XX, UK) and hemolysed by repeated freez ing and thawing. 2.1.2 Tissue Culture Media and supplements All tissue culture media and supplements were obtained from Gibco Europe Ltd. (Unit 4, Cowley Mill Trading Estate, Uxbridge UB8 2YG) unless specified otherwise. Tissue culture media RPMI 1640 and RPMI 1640 containing 25 mM HEPES and L-Glutamine. 80 Medium 199 containing Earle’s Salts, 25 mM HEPES Buffer and L-Glutamine. Hanks’ Balanced Salts Solution without Phenol Red (HBSS). L-glutamine was obtained as a lyophilised powder. Foetal Calf Serum and Horse Serum (Mycoplasma-free). Both sera were heat-inactivated at 56°C for 30 minutes before being used. Antibiotic-antimycotic was obtained as a lyophilised powder containing penicillin (10,000 units ml ^), streptomycin (10,000 meg ml and amphotericin B(25 meg ml ^). Bovine Embryo Extract (50% in Earle’s Salt Solution) was obtained from Flow Laboratories (Woodcock Hill, Harefield Road, Hertfordshire, WD3 1PQ, UK). HEPES Buffer was obtained as a 1 Molar solution from Flow Laboratories. Heparin sodium B.P. (1000 units ml ^) was obtained from Paines and Bryne Ltd. (Greenford, England). Liver Fraction L was obtained from the United States Biochemical Corpor ation, Cleveland, Ohio, USA. HBSS-HEPES was prepared by the addition of 25 mmolar HEPES buffer to HBSS. 81 Basic Maintenance Medium (BMM) was used for the culture of macrophage monolayers. BMM contained: Medium 199 75% (v/v) Horse serum 20% (v/v) Bovine Embryo Extract 4% (v/v) Liver Fraction L (1 mg ml ^) 1% (v/v) 2.2 Chemicals and reagents Chemicals: Ammonium chloride, bovine serum albumin (Fraction V powder), deoxyribo nucleic acid, N, N-dime thylf ormamide , formaldehyde, Hoechst No. 33258 (bisBenzimide) , 2-mercaptoethanol, <*-naphthyl butyrate, pararosaniline HC1, trypan blue, saponin ,sodium nitrite, were obtained from Sigma Chemical Company Ltd. (Fancy Road, Poole, Dorset BH17 7NH, UK). 2-( 4 * tert.-Butylphenyl)-5-(4"-biphenylyl)-l,3,4-oxadiazole [Butyl-PBD], gelatine powder, hydrogen peroxide (3% w/v), potassium dihydrogen ortho phosphate, di-sodium hydrogen orthophosphate, tris(hydroxymethyl)methyl- amine were obtained from BDH Chemicals Ltd. (Broom Road, Poole BH12 4NN, UK) . Dulbecco’s phosphate buffered saline tablets were from Oxoid Ltd. (Wade Road, Basingstoke, Hants. RG24 OPW, UK). Ficoll 400 was obtained from Pharmacia (Great Britain) Ltd. (Prince Regent Road, Hounslow, Middlesex TW3 1NE, UK). 82 Hypaque sodium (sodium diatrizoate) was from Sterling Research Labora tories (Onslow Street, Guildford, Surrey, UK). Toluene, Hydrochloric acid, methanol and acetone (analytical grade) were from May and Baker Ltd. (Liverpool Road, Barton Moss, Manchester M30 7RT, UK). Immunological reagents: Concanavalin A was obtained as a powder from Sigma Chemical Company. Phy t ohaemagglutinin (Reagent grade) was obtained from Wellcome Diagnos tics (Temple Hill, Dartford DAI 5AH, UK) as a lyophilised pellet of 45 mg which was then reconstituted in 5 ml sterile water. Tuberculin Purified Protein Derivative (PPD) was obtained from Evans Medical Ltd. (Greenford, Middlesex, UK) and used a concentration of 2 mg ml ^« 3 [methyl- H] Thymidine was obtained as an aqueous solution containing 25 Ci/mmol from Amersham International pic (Lincoln Place, Aylesbury, Buckinghamshire HP20 2TP, UK). Histochemical reagents: 3,31 Diaminobenzidine tetrahydrochloride, Fast Red TR, levamisole hydro chloride, Naphthol AS-MX were obtained from Sigma Chemical Company. Liposomal reagents: L-o<-"phosphatidylcholine (Type V-E) from frozen egg yolk in chloroform- 83 methanol solution and L-(X-phosphatidyl-L-serine from bovine brain in chloroform-methanol solution were obtained from Sigma Chemical Company. Staining reagents: Cold AFB stain LMR 22, Methylene blue, Haematoxylin (Harris), Methyl Green, Methylene blue, D.P.X mounting medium and Apathy's mounting medium were all obtained from Raymond A. Lamb (6 Sunbeam Road, London NW10 6JL). Diff-Quik was obtained from American Hospital Supply, UK Ltd.(Didcot, Oxfordshire OX 117 NP, UK). 2.3 Bacteria 2.3.1 Bacterial Strains The bacteria used were the virulent strain, H37Rv of M. tuberculosis obtained from Trudeau Mycobacterial Culture Collection (TMC 102), Trudeau Institute, Saranac Lake, New York; M. microti strain 0V 254, pathogenic for voles, obtained originally from Dr.R.J.W. Rees and L. monocytogenes strain NCTC 9373. 2.3.2. Animal passage and maintenance Prior to their use in the in vivo experiments, both M. tuberculosis and M. microti were passaged through animals. L. monocytogenes was also mouse passaged. This was to counter the risk of attenuation following serial transfer on laboratory media. 84 M. tuberculosis: M. tuberculosis H37Rv was recovered from the spleen of a guinea-pig that had been infected intravenously with the bacilli. A portion of the spleen was homogenised in 2% Bovine albumin serum in 0.85% sodium chloride (2% BSA) and plated on Middlebrook 7H11 agar plates. After two weeks incubation at 37°C , the growth was scraped into Middlebrook 7H9 broth containing 10% Albumin Dextrose Complex (ADC) and reincubated for 7 days at 37°C. The M. tuberculosis suspension was dispensed aseptic- ally into 4 ml amounts in sterile Bijou bottles and frozen at -70°C. This suspension was used as the infecting inoculum in all subsequent experiments. M. microti: M. microti was injected intravenously into the tail-vein of CFLP mice and the mice were sacrificed after 12 days and the spleens removed. The spleens were homogenized aseptically in 2% BSA and 0.1 ml of the spleen homogenate was inoculated into 100 ml quantities of Dubos broth supplemented with 10% ADC and incubated at 37°C for 4 weeks. This broth was used as the inoculum for the next serial passage and the process was repeated. After the third passage, the broths were pooled and frozen in 4 ml aliquots in sterile Bijou bottles at -70°C. This was used as the inoculum for all subsequent experimental M. microti infections in mice. L. monocytogenes: L. monocytogenes was passaged through BALB/c mice. The mice were injected intraperitoneally with a broth culture of L. monocytogenes. After two days the mice were sacrificed and the spleens removed asepti cally. The spleens were homogenized in 2% BSA and the homogenate plated 85 onto Tryptic Soy Agar (TSA) plates. Colonies from the plates were used to inoculate Tryptic Soy Broth (TSB) and incubated at 37°C for 8 hours. The culture obtained was frozen at -70°C in 1 ml amounts in sterile ampoules (Nunc cryotubes, Nunc UK Ltd., 16 Salter Street, Stafford ST16 2JU, UK). This culture was used to seed other broths for all subsequent in vitro experiments. M. microti for in vitro experiments: A culture of M. microti was serially transferred at weekly intervals in Dubos broth medium. This was used as the infecting inoculum in the in vitro experiments with the macrophage monolayers. 2.4 Animals 2.4.1 Mouse strains Two different strains of mice were used. Specific-pathogen free CFLP mice were obtained from Interfauna UK (Abbots Ripton Road, Wyton, Huntingdon, UK) and specific-pathogen free BALB/c mice were obtained from the Imperial Cancer Research Fund (ICRF). 2.4.2 Random allocation and husbandry of mice In the in vivo animal models, the mice were allocated into the different treatment groups by selection based on a table of random permutations of 20 numbers (Fisher and Yates, 1963). 86 The mice were kept in cages and fed on a pelleted food diet (Labsure PRD, Labsure Company, Poole, Dorset, UK) and water ad libitum. The cages of mice infected with M. tuberculosis were kept within a negative- pressure isolator (Vickers Medical Isolator, Vickers Instruments, Haxby Rd., York Y03 7SD, UK). 2.5 Immunomodulating and Chemotherapeutic Agents 2.5.1 Agents Used Levamisole hydrochloride was kindly presented by Janssen Pharmaceutical Ltd. (Janssen House, Marlow, Bucks SL7 1ET, UK). Recombinant murine gamma interferon derived from Escherichia coli was generously given by Genentech Inc. San Francisco, United States of America. The lot number was 1551/43 with a specific activity of 7.2 x 106 U/mg. Isonicotinic acid hydrazide (Isoniazid) was obtained from Sigma Chemical Company, Poole, Dorset, United Kingdom. Rifampicin (Rimactane A.S Batch No. 1935410) was a gift from Ciba Laboratories, Horsham, West Sussex, UK. 2.5.2 In vivo administration Levamisole was dissolved in sterile distilled water and the required dose was adminstered to the mice in 0.2 ml aliquots by oral gavage with 87 the aid of a syringe and a blunted needle. Recombinant gamma interferon was diluted in Phosphate Buffered Saline (PBS) containing 1 mg ml ^ of homologous mouse serum immediately before use. The required doses of interferon was intravenously injected through the tail vein in 0.1 ml aliquots. IFN-# was also administered intravenously in liposomes (see section 2.5.2.1). Isoniazid was dissolved in sterile distilled water and administered to the mice by oral gavage in 0.2 ml amounts. Rifampicin was suspended in 0.2% Methyl Cellulose containing 0.05% Tween 80. The suspending fluid was sterilized by autoclaving at 115°C for 10 minutes. The required dose of rifampicin was administered in 0.2 ml aliquots by oral gavage. 2.5.2.1- Preparation of liposomes IFN-8 was administered intravenously in multilamellar vesicles (MLV) composed of phosphatidylcholine (PC) and phosphatidylserine (PS) in a PC:PS ratio of 7:3 moles. The required dose of IFN-)f was delivered in 2.5 jumole of lipid contained in a final volume of 0.2 ml per mouse. The liposomes were prepared by modifications of procedures described elsewhere (Fidler, 1980). The required amounts of PC and PS were dispensed into a 50 ml round bottomed flask, thoroughly mixed and evaporated to dryness with the aid of a vacuum rotary evaporator (Corning Type 349/2, Corning Ltd., Staffordshire ST15 0BG, England) at 88 room temperature in a high vacuum. The lipids were left under vacuum for 30 minutes to ensure the complete removal of all the chloroform and methanol. The lipid film was then hydrated with the IFN-^ dilution or diluent for 30 minutes at room temperature before being vortexed for 5 minutes. The resultant liposomal preparation was then injected in the tail-vein in 0.2 ml aliquots. Liposomes were prepared and used within 2 hours. No attempt was made to separate free IFN-& from liposomal IFN-& because of the uncertainty about the stability of recombinant IFN-^. 2.5.3 In vitro administration of agents Recombinant gamma interferon was diluted in BMM just prior to being used. Isoniazid was dissolved in pyrogen-free water to obtain a stock solution of 100 ^g ml K The final dilution of the drug (1 pg ml was made in BMM. Rifampicin was dissolved in N/100 Hydrochloric acid and immediately diluted in pyrogen-free water to a stock solution of 500 pg ml The final dilution of 10 jig ml ^ was made in BMM. 2.6 Preparation of infective inocula 2.6.1 Enumeration of total number of bacilli The total number of bacilli in a suspension was determined by using a Helber counting chamber (Thoma, Webber, England). The Helber chamber is 1/50 mm deep and has a ruled area of large squares enclosing small 89 squares whose sides measure 1/5 and 1/20 mm. Dilutions of the suspen sion were made in saline or HBSS and the final count was calculated from the dilution factor and the number of organisms counted per small square by the following formula: Number of bacilli per ml = Dilution Factor x 2n x 10^, where n is the mean number of organisms per small square. 2.6.2 Infective inocula for in vivo experiments M.microti : The frozen vials of bacilli were quickly thawed in a 37°C waterbath. The bacilli were pooled into Universal containers and sonicated with a sonicating probe (Rinco Ultrasonics UK Ltd., P.O.Box 217, London W5 1BL) at a setting of 70 amplitude % for 15 seconds and with the probe 1 cm below the surface. The mice were injected with 0.2 ml of this inoculum in disposable 2 ml syringes (Monoject, Sherwood Medical, United Kingdom) with 16 x 0.5 mm sterile disposable needles (Monoject, UK). M. tuberculosis : The frozen vials of bacilli were rapidly thawed in a 37°C waterbath. The bacilli were pooled into sterile Universal contai ners and diluted 1:1 by the addition of an equal volume of sterile 0.1% gelatine saline. The suspension was then sonicated (see above) and immediately used for infecting the animals as detailed above. 2.6.3 Infective inocula for in vitro experiments L. monocytogenes : The infective inocula were prepared fresh for each experiment. A frozen vial of passaged bacilli was rapidly thawed in a 90 37°C waterbath and 100 pi of the thawed suspension was inoculated into 15 ml of TSB and incubated at 37°C for 8 hours. The resultant log phase culture was used to infect the monolayers. The suspension was centrifuged at 1000 g (MSE Chilspin 2, MSE Scientific Instrument, Manor Royal, Crawley, West Sussex) for 15 minutes after which the supernatant was decanted and the bacilli resuspended in HBSS-HEPES . Dilutions were then made and the total count was determined in a Helber chamber (section 2.6.1). The inoculum was diluted to a concentration lOOx that of the final concentration needed. 100 of the suspension was opson ized by incubating with 25 ul of sterile normal mouse serum at 37°C for 30 minutes. At the end of the opsonization period, the required concen tration of the infective inoculum was obtained by diluting lOOx with Medium 199. M. microti : A culture of M. microti serially transferred in Dubos broth was used for the in vitro experiments. A 6 day culture of the strain was centrifuged for 15 minutes at 1000 g (MSE Chilspin 2). The supernatant was decanted and the pellet gently resuspended in HBSS- HEPES. The centrifugation step was repeated and the bacteria resuspended in HBSS-HEPES. The bacterial suspension was then sonicated and the total count determined in a Helber chamber. The required infective inoculum was then obtained by diluting in Medium 199 contain ing 10% heat-inactivated FCS. 91 2.7 Investigation of the T lymphocyte response 2.7.1 Proliferative response of splenic T lymphocytes 2.7.1.1 Preparation of splenic lymphocytes Spleens from three mice were aseptically removed and gently teased apart with forceps over a wire mesh in a sterile 20 mm plastic petri dish filled with media (RPMI 1640 with 20% Foetal Calf Serum). The resulting suspension was layered gently over Ficoll-Hypaque (65 g Ficoll and 100 g Hypaque in 1000 ml water) in a universal container. The universal was centrifuged at 900 g at 4°C and the band of cells at the interface was collected. These cells were washed twice with media by centrifugation at 150 g at 4°C and the cell pellet was resuspended in the final medium of RPMI 1640 supplemented with 20% Foetal calf serum, 2mM L-glutamine, 5 x 10 2-mercaptoethanol (2-ME) and 1% Antibiotic-antimycotic mixture (see section 2.1.2). The lymphocytes were diluted in 0.2% Trypan blue and enumerated in an improved' Neubauer haemocytometer (section 2.9.2) 2.7.1.2 Antigens and mitogens The mitogens used were phytohaeraagglutinin (PHA) and concanavalin A (Con A). PHA was used at a dilution of 1/20 (stock solution of 9 mg ml ^) and Con A at a concentration of 10 ;ug ml The specific antigen used was Tuberculin Purified Protein Derivative (PPD) at the concentrations r —A _ O — O — 1 ™ 1 1x10 , 1x10 , 1x10 , 1x10 , and 1x10 ;ug ml 92 2.7«1.3 Lymphocyte Proliferation test The proliferative responses of the T lymphocytes were determined by a micro method using terasaki plates (O'Brien et al., 1979). The splenic lymphocytes were suspended at various cell concentrations in the final medium of RPMI 1640 supplemented with 20% Foetal calf serum, 2mM L-glut- amine, 5x10 2-ME and 1% Antibiotic-antimycotic mixture. Cell concen trations of 1x10^ and 3x10^ per well were used for the PHA and Con A 5 6 6 assays and 5x10 , 1x10 and 3x10 for the PPD assay. 2 pi of the antigen/mitogen in various concentrations were first distri buted to the microterasaki plates (Nunc UK Ltd.) followed by 20 pi aliquots of the cell suspension dilutions. These aliquots were dispensed by the use of a programmable microdispenser (Microlab P, Hamilton Company, USA). The plates were then inverted and the cells cultured on hanging menisci in humidified chambers at 37°C with 5% CO2 • Each assay was set up in triplicates. Cells stimulated with PHA and Con A were incubated for 3 days and the 3 PPD assays for 6 days. Before harvest, 0.5 Ci of [ H] thymidine were added to each well. Plates were then reincubated for 4 hours after which the cells were harvested using a precut filter block onto filter discs and were washed sequentially for 30 seconds in PBS, 5% Trichloro acetic acid, and methanol. The discs were removed into plastic insertion vials and allowed to dry. They were then dissolved in 1 ml of scintillating fluid (Toluene 3 containing 0.5% butyl PBD. Incorporation of [ H] thymidine was counted 93 in a liquid scintillation counter (SL 4000, Intertechnique, France) and the results were expressed as mean counts per minute (cpm) + s.e. for triplicate cultures. 2.7.2 Immunoenzymatic staining of splenic T lymphocyte subsets Splenic T lymphocytes were isolated and cytospin preparations made for immunoperoxidase and immunoalkaline phosphatase staining. 2.7.2.1 Antibodies and sera Monoclonal antibodies: 1. Mouse anti-mouse Thy 1.2 2. Mouse anti-mouse Lyt-1 3. Mouse anti-mouse Lyt-2 The monoclonal antibodies were obtained as 0.5 mg purified immunoglo bulin in 0.5 ml buffered saline from Becton Dickinson, Laboratory Impex Ltd. (Impex House, Lion Road, Twickenham, Middlesex TW1 4JF, UK). Other antibodies: Peroxidase-conjugated rabbit anti-mouse IgG (Sigma Chemical Company) Alkaline phosphatase-conjugated rabbit anti-mouse IgG (Sigma) Serum: Normal mouse serum (Sigma) 94 2.7.2.2 Cytospin preparations Splenic lymphocytes were prepared as described in section 2.7.1.1. The lymphocyte suspension was counted (see section 2.9.2) and resuspended at a concentration of 1x10^ cells ml ^ . 100 }il of the cell suspension together with 25 ^pl of FCS was spun down on clean glass slides in a cytocentrifuge (Shandon Cytospin 2, Shandon Southern Products Ltd., Astmoor, Cheshire, UK) at 500 rpm for 5 minutes. The cell spots were allowed to dry at room temperature overnight, indi vidually wrapped in aluminium foil and stored at -20°C until stained. It had been shown by Moir et al., (1983) that slides treated in such a manner retained antigenic activity even after several months of storage. 2.7.2.3 Preparation of buffers, fixative and substrates Buffers: 5x Concentrated buffer consisted of 2g Na^HPO^ and 10 g KH^PO^ in 60 mis of distilled water. Tris HC1 buffer (pH 7.6) and tris HCl buffer (pH 8.2) were prepared by dissolving Tris base (0.05 M) and Tris base (0.1 M) in distilled water respectively and adjusting the pH with concentrated HCl. Tris-buffered saline (TBS) was prepared by adding Tris HCl buffer (pH 7.6, 0.05 M) to normal saline (0.15M). TBS was used to dilute all reagents unless stated otherwise. 95 Fixative: Buffered formol acetone (BFA) was made fresh each time. It consisted of: 8 ml of 5x Concentrated buffer 138 ml of distilled water 33.2 ml of formalin 60 ml of acetone Substrates: Peroxidase substrate. Immediately before staining, 3,3' diaminobenzi- dine tetrahydrochloride was dissolved in TBS at a final concentration of 0.6 mg ml ^ and hydrogen peroxide (10 volumes, 3% w/v) added to give a final concentration of 0.01 %. Alkaline phosphatase substrate. 2 mg Naphthol AS-MX was dissolved in 200 >il N,N-*dimethylformamide in a glass universal and 9.8 ml of Tris HC1 buffer (pH 8.2, 0.1M) was added. Immediately before staining, Fast Red TR was dissolved in this solution at a final concentration of 1 mg ml ^ together with levamisole (final concentration 1 mM). The substrate was filtered through Whatman No.l filter paper directly onto the cell spots. 2.7.2.4 Immunoenzymatic staining technique The slides were removed from the freezer and allowed to thaw at room temperature. The cell spots were stained by an indirect immunoperoxi- dase technique and an indirect immunoalkaline phosphatase procedure (Moir et al., 1983). The cell spots were fixed for 30 seconds in BFA , rinsed in distilled water and then in TBS. They were then stained by either technique. 96 Indirect Immunoperoxidase procedure: The slides were flooded with the optimal dilution of the monoclonal antibody and incubated for 60 minutes at room temperature in a humidified chamber to prevent drying of the cell spots. The slide was then washed with TBS and incubated at room temperature for 30 minutes with peroxidase-conjugated rabbit anti-mouse IgG (1/50 dilution) with normal mouse serum at a final concentration of 1/20 (to block cross reactivity against mouse IgG). The slide was again washed in TBS and the reaction developed with diaminobenzidine-H^O^ substrate. The devel opment of the reaction was monitored with a light microscope (x200 magnification) and the reaction, was stopped by washing in tap water. The slide was then counterstained very briefly with dilute haematoxylin (1/6 dilution) and mounted in DPX. Indirect immunoalkaline phosphatase procedure: The technique was essentially similar to the procedure for the immuno peroxidase staining as described above except that the second incubation was carried out with alkaline phosphatase-conjugated rabbit anti-mouse IgG and the reaction was developed with alkaline phosphatase substrate. The substrate was filtered onto the slide and the reaction monitored microscopically. The reaction was then stopped by rinsing in tap water. The slide was briefly counterstained with dilute haematoxylin and mounted in Apathy’s mountant. 97 2.8 Assessment of growth of mycobacteria in vivo and in vitro 2.8.1 Homogenization of organs The mouse was killed by cervical dislocation, immersed briefly in 2% Clearsol and pinned ventral aspect upwards on a cork dissecting board. The skin was opened up and deflected to either side. The abdominal musculature was swabbed with 70% alcohol and allowed to dry. The abdominal wall was then incised with sterile scissors right to the top to enable easy removal of the lungs. The spleen and lungs were removed aseptically with sterile instruments into labelled sterile precision- bore homogeniser tubes made of hard glass with a diameter of 16 mm. The organs were ground with a motor-driven homogeniser using PTFE grinders and glass-homogeniser tubes. The PTFE grinder and the homo geniser tubes were separately autoclaved and assembled together just before homogenization. All the organs were ground in a final volume of 5 ml of a diluent, 2% bovine serum albumin in 0.85 % sodium chloride (BSA). The diluent served the purpose of protecting the bacilli from the toxic substances released by grinding or autolysis of the tissues (Pierce et al., 1953). For the same reason, all further dilutions were made in 0.1% BSA. During homogenization, the tube was pushed up and pulled down the revolving PTFE grinder about 10 times for spleens and about 20 times for lungs to ensure complete disruption of the tissues. Homogenates were kept at 4°C until dilutions were made to determine the number of viable mycobacteria. 98 2.8.2 Enumeration of viable M. tuberculosis and M. microti The numbers of viable bacilli present in the infective inocula and the homogenates of organs were determined by plating appropriate dilutions for surface plate counts on suitable media. The culture media used was Middlebrook 7H11 Oleic acid-albumin agar or Selective 7H11 agar for M. tuberculosis and Dubos Oleic Base Agar for M. microti. All media were incubated at 37°C for 24 hours before use to check for sterility and to ensure that the surface was sufficiently dry. Appropriate ten-fold dilutions of the homogenates were made in 0.1% BSA and 100 pi aliquots were pipetted on 1/3 segments of the agar plate in duplicate. The inocula were allowed to dry into the agar and the plates were then packed in polythene bags and incubated at 37°C. 7H11 and Selective 7H11 plates were incubated for 3-4 weeks before the number of colonies were counted. Plates of M. tuberculosis were exposed to formalin vapour overnight before being enumerated. Dubos Oleic Base Agar plates were sealed by parafilm before being packed into polythene bags and were enumerated after 4 weeks incubation at 37°C. 2.8.3 Enumeration of L. monocytogenes and M. microti in macrophage monolayers The number of viable bacteria per macrophage monolayer was determined either by scraping the macrophages from the tissue culture wells or by lysing the monolayer with 1% saponin and then performing a viable count. 99 After the monolayers had been harvested from the tissue culture wells by either method, the cell lysates were placed Into sterile tubes and submitted to a brief sonication step to ensure that all the cells were lysed and that the bacilli were not clumped. The determination of the viable count was accomplished by a method similar to the one described above. L. monocytogenes was grown on TSA plates and M. microti on Dubos Oleic Base agar. The TSA plates were enumerated after overnight incubation at 37°C. 2.9 In vitro techniques used in the macrophage microbicidal assays 2.9.1 Preparation of monolayers The mice were sacrificed by cervical dislocation, pinned ventral aspect upwards on a cork board and the abdominal region liberally swabbed with 70% alcohol. The skin was incised and deflected to the left side of the body. 3 ml of ice-cold RPMI 1640 containing 10 units of heparin per ml were injected slowly into the peritoneal cavity with a 21 gauge needle. The lavage fluid was carefully injected by first going obliquely through the abdominal wall musculature before penetrating the peritoneum. This ensured that there was no leakage. The peritoneal cavity was gently massaged for a few minutes and then the lavage fluid was removed by drawing back into the syringe. The peritoneal cells from several mice were pooled, kept on ice and were centrifuged at 4°C at 150 g (MSE Chilspin 2). The cells were resuspended in Medium 199 containing 10% heat-inactivated FCS. The cell suspension was then enumerated (see section 2.9.2), diluted to the required concentration and aliquoted in 100 500 pi or 200 ;ul amounts Into either 16 mm tissue culture multidishes (Linbro Space Saver, Flow Laboratories) or Lab-Tek 8-Chambered Tissue culture slides (Miles Scientific Laboratories, Stoke Poges, Slough SL 4LV, UK) respectively. The cell suspension was incubated at 37°C in a CO2 incubator (5% CO^) for 2-3 hours to enable macrophages to adhere. At the end of this time, the plates were gently agitated, the superna tants removed and the monolayers were washed twice with 500 ;ul amounts of warm Medium 199 containing 5% FCS to ensure the removal of all non adherent cells. The monolayers were then overlaid with 500 ;ul amounts of BMM and reincubated. 2.9.2 Enumeration of cell suspensions The enumeration of cells (macrophages, lymphocytes) in a suspension was achieved by adding 25 til of the cell suspension to 225 til of tris- ammonium chloride and 250 til of 0.2% Trypan Blue (1/20 dilution). The tris-ammonium chloride lyses any erythrocytes present and the trypan blue distinguishes dead from living cells by dye exclusion. After thorough mixing, the dilution was transferred to an improved Neubauer haemacytoraeter chamber and counted microscopically by phase contrast. The improved Neubauer chamber consists of 25 large squares, each divided 3 into 16 smaller squares. The large square has a volume of 0.004 mm , and the number of cells are calculated from the formula: 4 No. of cells per ml= Dilution factor x n x 10 , where n is the number of cells in 25 large squares 101 2.9.3 Characterization of macrophage monolayers Characterization of the monolayers were performed on those set up in the 8-Chamber tissue culture slides. At required intervals, some monolayers were differentially stained by a modified Wright's stain (Diff-Quik) and for non-specific esterase (Koski et al., 1976). Diff-Quik Staining: Reagents : Diff-Quik solutions I and II and fixative Procedure : The supernatant was aspirated and the monolayers washed by warm HBSS-HEPES. The chamber component was removed and the slide was immersed in fixative for 15 seconds followed by 15 seconds in Diff-Quik I and 30 seconds in Diff-Quik II. It was then thoroughly washed, air dried, mounted in DPX and examined under a light microscope. Non-specific esterase staining: Reagents: 1. Fixative (pH6.6) containing 20 mg Na2HP0^, 100 mg KI^PO^, 45 ml acetone, 25 ml formaldehyde (30%) and 30 ml distilled water. 2. Pararosaniline solution containing 1 g pararosaniline HC1 in 25 ml 2N HC1. 3. 4% Sodium nitrite (freshly prepared) 4. M/15 Sorenson's phosphate buffer (pH 6.3) 5. c^-Naphthyl butyrate solution containing 1 g o<-Naphthyl butyrate in 50 ml dimethyl formamide. 6. 0.5% methyl green counterstain. Procedure: The monolayers were washed with warm HBSS-HEPES, the chamber component 102 discarded and the slide was immersed in fixative for 30 seconds, rinsed thoroughly in distilled water and air-dried for 30 minutes. Hexazotiza- tion of pararosaniline was carried out by adding 1 ml of filtered para- rosaniline solution to 1 ml of sodium nitrite and leaving the mixture for 1 minute. The reaction mixture was then prepared by adding in sequence 44.5 ml M/15 phosphate buffer, 0.25 ml hexazotized pararos aniline and 3 ml cX-naphthyl butyrate solution. After immersion of the slide in this mixture for 45 minutes at 37°C, it was rinsed thoroughly in distilled water, counterstained briefly in methyl green and rinsed in distilled water. It was then air-dried, mounted in DPX and examined mi croscopi cally. 2.9.4 Harvesting of bacteria from the monolayers Two methods were used to harvest bacteria from the adherent macrophage monolayers : 1. HBSS-HEPES was added to the tissue culture vessel and the monolayer was physically scraped off the well with the aid of a specially designed autoclavable PTFE-tipped rod. This procedure was repeated to ensure that all the cells were scraped off. The process was also monitored with an inverted microscope. This method allowed the DNA content and hence the number of cells in the monolayer to be determined. 2. 1% saponin was added to the culture chamber which was then reincu bated for 20 minutes at 37°C. At the end of this time, the cells were easily removed by gently scraping with the blunted ends of sterile 1 ml serological pipettes. HBSS-HEPES was then added and the process repeated. The complete removal of all the cells was confirmed by 103 observation under an inverted microscope. 2.9.5 Analysis of DNA The analysis of DNA content of cell lysates was determined for some of the samples and used as a means of quantitating of the number of cells in the monolayer. DNA analysis was carried out by a simple and rapid assay based on the enhancement of fluorescence seen when bisbenzimid- azole (Hoechst Compound 33258) binds to DNA (Labarca and Paigen, 1980). The assay can detect as little as 10 ng of DNA. The number of macro phages can be established from the conversion factor that 1x10^ murine peritoneal macrophages has 7.5 jug of DNA (Lowrie et al., 1979a). Reagents: 1. 0.05M phosphate buffer pH 7.4 made 2M in NaCl 2. 0.10M phosphate buffer pH 7.4 made 4M in NaCl 3. Hoechst compound 33258, stock solution in DMSO, used at 5 ;ig ml ^ with 0.05M phosphate buffer 4. DNA standards prepared in 5 mM NaOH Procedure: The monolayers were scraped into 500 jul of HBSS-HEPES and 500 jul of 0.10M phosphate buffer and 250 jul of H33258 (5 jug ml ^) were added. Standards were set up with 0-20 jug ml ^ of DNA in HBSS-HEPES in exactly the same way. The mixture was vortexed and left for 3 hours for penetration into the cells. Fluorescence was read with a Perkin Elmer 1000M Fluorimeter with an excitation filter of 365 nm and an emmission wavelenght of 456 nm. Results were read off a DNA standard curve. 104 2.10 In vivo models In the assessment of immunomodulating agents 2.10.1 Effect of levamisole on M. microti infection in CFLP mice The model of an infection of M. microti in CFLP mice was used to assess the effects of levamisole in vivo. The effects of levamisole on the infection of M. microti was monitored in two ways : firstly, the effect on the growth of M. microti in the lungs and spleens of the infected animals by performing viable counts (see section 2.8.2); and secondly, the effect on the splenic T lymphocytes of uninfected and infected mice as measured by the proliferative response of T lymphocytes (section 2.7.1) and immunoenzymatic staining of subsets of T lymphocytes (see section 2.7.2). 2.10.2 Effect of interferon-gamma on M. tuberculosis infection in mice The model of an acute infection of M. tuberculosis H37Rv was used to assess the effects of the IFN-Y on the growth of M. tuberculosis in the whole animal. In all the experiments, mice were infected with animal- passaged M. tuberculosis (section 2.6.2). The mice were infected by intravenous injection through a tail vein by the use of a specially designed mouse restrainer. The size of the infective dose was decided by a preliminary experiment in which diffe rent dilutions of the passaged H37Rv were used to infect mice and the outcome of the infection was then monitored by performing viable counts of the bacilli in the lungs and the spleens (see sections 2.8.1 and 2 .8 .2). 105 The effects of size of dose of IFN-tf was investigated. This was done by randomly allocating mice into different groups receiving different doses of IFN-Jf. Several schedules of administration of IFN-& were also tested to determine the optimal schedule. The feasibility of using MLV lipo somes as drug delivery vehicles was also investigated. Different dilu tions of IFN-fr were encapsulated within liposomes (see section 2.5.2.1) and were intravenously administered to the mice. Assessment of the effect of IFN-'fc was achieved by performing viable counts of M. tuberculosis in the lungs and the spleens of the animals at selected time points after infection. The number of viable bacilli present in the inoculum was determined in each experiment by performing the viable count at infection. The uptake of bacilli by the organs in each experiment was determined by performing viable counts of the lungs and spleens of 4-6 mice an hour after intravenous infection. 2.10.3 Effect of interferon-gamma in combination with isoniazid and with rifampicin on M. tuberculosis infection in mice A series of experiments were performed to determine the effect of IFN-Jf in combination with antibacterial agents of tuberculosis. A model of infection with M. tuberculosis was used. In the first set of experiments, mice were randomly allocated into groups that received either IFN-tf, isoniazid, isoniazid and IFN-V or were untreated. IFN-ft was given at a dose of 2000 units per mouse while isoniazid was given at a daily dose of 25 mg kg In one experiment, IFN-J was given both before and after infection, and isoniazid was 106 started at Day+1. In another experiment, IFN-# was either given both before and after infection or only 5 days after infection. The effects of each treatment were assessed by determining the viable counts of M. tuberculosis in lungs and spleens. In another experiment, mice were allocated into groups which received either IFN-tf , rifampicin, IFN-Jf and rifampicin or were untreated. Two doses of IFN-if at 2000 units per mouse were given at Days -2 and +1. Rifampicin was given at 25 rag kg ^ per day with the first dose on Day 0. The efficacy of each treatment group was ascertained by the growth of M. tuberculosis in the lungs and the spleens. 2.11 In vitro assessment of the effects of interferon-gamma The in-vi t ro assessment of the effects of interferon-gamma were all performed on murine resident peritoneal macrophages. Monolayers were established in 16 mm tissue culture multi-well plates and 8-chamber tissue culture slides (see section 2.9.1). In all of the experiments, triplicate wells were used and the experiments were performed at least twice unless stated otherwise. 2.11.1 Effect of saponin on the viable count of L. monocytogenes and M. microti In some experiments, saponin was used to lyse the macrophage monolayers and it was thus important to determine if saponin had any effect on the viable counts obtained. 107 To investigate the effect of saponin on L. monocytogenes and M. microti, experiments were performed to compare the viable counts of bacterial suspensions left in room temperature in distilled water, 1% saponin or 2.5% saponin for at least two hours. The suspensions were then briefly sonicated (M. microti only) and dilutions made in distilled water and viable counts performed (see sections 2.8.2 and 2.8.3). 2.11.2 Listericidal assay The listericidal assay used was based on the method of Harrington-Fowler e t a l . (1981) with several modifications. One major modification was that no antibiotics were used at any stage of the assay. The macrophage monolayers were prepared in 16 mm Linbro wells (section 2.9.1). In this assay, the monolayers were pre-incubated with various concentrations of IFN-X in 3MM or with BMM for either 24 or 48 hours prior to infection. Treated and untreated (control) monolayers were infected with L. monocytogenes at a bacteria:macrophage ratio of 1:10. The infective inoculum was prepared as described in section 2.6.3. The wells were washed twice with warm HBSS-HEPES and the inoculum in a volume of 500 jjl was added to each well and the plates incubated at 37°C to allow phagocytosis to occur. After 40 minutes, the supernatant was aspirated and the monolayers washed five times with warm HBSS-HEPES to remove non-attached bacteria. The last rinse was cultured to give an indication of the level of viable bacteria In the supernatant at the begining of the experiment. The replicate monolayers which were to represent the number of cell-associated bacteria after phagocytosis (T^) were then lysed while the rest of the monolayers were reincubated with 108 Medium 199 containing 10% heat-inactivated FCS. These monolayers were incubated at 37°C with 5% CO^ for a further four hours, with the media being changed every half hour. At the end of four hours, all the monolayers (T^) were lysed with 1% saponin (see section 2.9.4) and the lysates diluted and plated on TSA (section 2.8.3) to determine the viable count. The experiments were done with duplicate monolayers. 2.11.3 Effect of interferon-gamma on growth of M. microti in vitro In these experiments, macrophage monolayers were either pre-incubated with IFN-X before and after infection with M. microti or were infected and then treated with IFN- . 2.11.3.1 Effect of previous exposure of monolayers to interferon-gamma In these series of experiments, monolayers were previously exposed to various concentrations of IFN-K for either 48 or 72 hours. Monolayers in 16 mm Linbro wells were infected with M. microti at a macrophage: bacteria ratio of 1:1. The infective inocula were prepared as in section 2.6.3. The monolayers were washed twice with warm HBSS-HEPES and infected with the required number of bacilli in a volume of 500 pi per well. The wells were then incubated at 37°C with 5% CO2 to allow phagocytosis to occur. Phagocytosis periods of 15, 30, 60, 90 and 120 minutes were used in the different experiments. After the phagocytosis period, the wells were washed four times with warm HBSS-HEPES to remove non-attached bacteria. This was checked with an inverted phase-contrast microscope and the last rinses were cultured 109 to determine the level of viable bacteria in the supernatant at the end of the phagocytosis period. The monolayers representing (immediately after phagocytosis) were then lysed and the other monolayers were reincubated at 37°C with 5% CO^ for a further 24, 48 or 72 hours with either BMM and IFN-S or BMM alone. The medium was changed every day by removing the old medium and adding fresh BMM with and without IFN-^. In these series of experiments, both the scraping method and the saponin method were used to disrupt the monolayers and release the bacteria (section 2.9.4). The viable counts of the M. microti were determined as described in section 2.8.3. The number of cells per monolayer was determined by assaying the DNA content of the cell lysates for those experiments where the scraping method was used (section 2.9.5). 2.11.3.2 Effect of interferon-gamma on the phagocytosis of M. microti Some experiments were performed to investigate the effect of previous exposure of monolayers to interferon-gamma on the uptake of bacilli. The monolayers were set up in 8-chambered tissue culture slides. After infection with a bacteriarmacrophage ratio of 10:1 and a phagocytic period of 2 hours, some of the wells were assessed for the viable count and the others were stained for acid-fast bacilli. Viable mycobacterial counts were obtained by lysing the macrophages with 1% saponin and plating dilutions of the lysates on Dubos Oleic Base agar plates (see section 2.8.3). Acid-fast bacillary counts were obtained by staining the monolayers with a cold Ziehl-Nielsen method. The monolayers were washed once with HBSS- 110 HEPES and fixed with methanol for 5 minutes. The chambers were then removed and they were rinsed with HBSS-HEPES, air-dried and stained for 5 minutes with Cold AFB stain LMR 22. This was followed by decolouriza- tion with acid-alcohol and counterstaining with 2% methylene blue. The slides were then mounted with D.P.X. Mounting Medium and enumerated under oil-immersion. Macrophages were enumerated in a random way by utilizing the vernier scales on the microscope stage to scan fields at regular intervals enabling all sections of the monolayer to be examined. 300 macrophages were counted per monolayer and scored as the number of macrophages containing no acid-fast bacilli, the number with 1-2 bacilli, the number with 3-5 bacilli and the number with more than 5 bacilli. For the acid-fast counts, the experiments were done with 6 replicates and for the viable counts, triplicate wells were used. 2.11.3.3 Effect of interferon-gamma added after infection of monolayers with M. microti In this series of experiments, monolayers were first established for 48 hours in 16 mm Linbro wells and then infected with M. microti at a bacteria:macrophage ratio of 1:1 for 120 minutes. At the end of the phagocytosis period, the monolayers were thoroughly washed 4 times with HBSS-HEPES to remove all extracellular bacteria. The Tq monolayers were then lysed to determine the number of bacilli phagocytosed and the remaining monolayers were reincubated with various doses of IFN-JT at 37°C with 5% CC^* At various time points after infection (24, 48 and 72 hours) the monolayers were assessed for the number of viable mycobact eria (see section 2.8.3). Media was removed and fresh BMM with and without IFN-^ was added daily. In some experiments, the medium that was Ill removed was pooled and cultured for the number of viable bacteria. 2.11.3.4 Effect of interferon-gamma in combination with isoniazid and with rifampicin on the growth of M. microti in macrophages A series of experiments were carried out to investigate the effect of interferon-gamma in combination with either isoniazid or rifampicin on the growth of M. microti in macrophage monolayers. In these experiments, the monolayers were established in 16 mm Linbro wells for 48 hours before infection. A macrophage:bacteria ratio of 1:1 was used. Phagocytosis was allowed to proceed for 90 minutes and the wells were thoroughly washed four times to remove non-attached bacteria and the last rinses were cultured to give an indication of the number of viable bacteria in the supernatant before the start of the experiment. The Tq monolayers were then scraped off the wells to determine the number of M. microti taken up by the macrophages (section 2.8.3) and the remainder of the monolayers were reincubated at 37°C in 5% CO^* The dose of IFN-K used was 100 units ml ^. Isoniazid was used at 1 jig ml ^ and rifampicin at 10 jjg ml The media containing the antibact erial agents were prepared as described in section 2.5.3, and were replaced every 24 hours with fresh media. In these experiments, the media that was removed was pooled and cultured to give an estimate of the viable bacteria in the supernatants at all the time points. At 24, 48 and 72 hours after infection, the monolayers were lysed with saponin and the number of viable mycobacteria was determined (section 2.8.3). 112 2.12 Statistical analysis of the data Standard error of the mean (s.e.m.) was used to measure the precision of the means of the different experimental groups. The analysis of variance (ANOVA) is a technique for performing multiple simultaneous comparisons. The analysis of variance was used to evaluate the results obtained in both the in vivo and the in vitro experiments. Two-way and three-way analyses were used, depending of the number of variables in the experiments. Analysis of variance was carried out by log^ transformation of the data which were then entered into a statistical package computer programme (Mini tab) for the two-way analysis or GLIM (General Linear Interactive Modelling) for the 3-way analysis. The variance ratio F was calculated and referred to F ratio tables (Fisher and Yates, 1963) for the prob ability of it being significant. 113 CHAPTER 3 THE IMMUNOMODULATING EFFECT OF LEVAMISOLE ON M. MICROTI INFECTION IN MICE This Chapter reports the effects of levamisole on a M. microti infection in mice. The immunomodulation by levamisole is assessed by monitoring the growth of the bacillus in organs of the mice and the effect on splenic T lymphocyte subsets and proliferation. 3.1 Preliminary experiments 3.1.1 Choice of media for growth of M. microti A variety of media and conditions were tested to select the best media and conditions for performing viable counts with M. microti. The media tested were: 1) Middlebrook 7H11 agar with and without 0.5% glycerol 2) Middlebrook 7H11 agar with and without 5% lysed horse blood 3) Dubos Oleic Base agar with and without 5% lysed horse blood 4) Selective 7H11 agar with and without 5% lysed horse blood The media were prepared as described in section 2.1.1 and dried for 24 hours before use. A culture of M. microti was incubated at 37°C for 6 days in Dubos broth. The suspension was sonicated (section 2.6.2) and 10-fold dilutions in saline were made. 100 jil of the dilutions were plated in duplicate on all the different media (see section 2.8.2). The media were then incubated under a variety of conditions. The conditions 114 tested Included sealing with parafilm, incubation in air or with 5% CC^ at 37°C. Before incubation, all the plates that were incubated in air were bagged in polyethylene envelopes. The plates were examined at weekly intervals and counts were made when the colonies were large enough. The results are shown in Table 3.1. TABLE 3.1 VIABLE COUNTS OF M. MICROTI ON VARIOUS MEDIA UNDER DIFFERENT CONDITIONS Media L o g ^ c^u *** microti per ml* for condition: tested Air Air 5% C02i sealed unsealed 7H11 agar 7.95 (5w) 6.82 (8w) 6.78 (8w) 7H11 agar + 5% blood 8.34 (3w) 8.32 (5w) CT Selective 7H11 agar 8.20 (4w) 8.04 (5w) 8.00 (6w) Selective 7H11 agar + 5% blood 8.26 (4w) 8.18 (5w) CT 7Hllg agar 7.60 (5w) 6.20 (8w) 6.35 (8w) 7Hllg agar + 5% blood 8.18 (4w) 8.26 (5w) 8.26 (6w) Selective 7Hllg agar 8.15 (4w) 6.83 (8w) 6.53 (8w) Selective 7Hllg agar + 5% blood 8.23 (4w) 8.20 (5w) 8.20 (6w) Dubos oleic agar 8.23 (3w) 8.32 (4w) 8.28 (5w) Dubos oleic agar + 5% blood CT 8.32 (4w) 8.32 (4w) * Figures in parentheses indicates the length of incubation in weeks 7H11 agar refers to Middlebrook 7H11 agar prepared without glycerol 7Hllg refers to Middlebrook 7H11 agar prepared with 0.5% glycerol CT = contaminated plates 115 The results showed that Dubos Oleic base agar with and without 5% horse blood supported the growth of M. microti equally well and that counts could be made within 4 weeks on plates sealed with parafilm. The results of the viable counts obtained using Middlebrook 7H11 agar or Selective Middlebrook 7H11 agar with or without glycerol were variable. Some plates gave very low counts even after prolonged incubation. The low counts observed on 7H11 and selective 7H11 agar were abrogated by the addition of 5% lysed horse blood. The addition of lysed horse blood also hastened the growth rate on 7H11 plates as did incubation with 5% CC>2; however both these conditions increased the contamination rate. 0.5% glycerol had no effect on the growth of this strain. After taking into consideration all the above results, it was decided to perform viable counts of M. microti with Dubos Oleic base agar and to seal culture plates with parafilm and incubate for 4 weeks at 37°C. 3.1.2 Course of infection of M. microti in CFLP mice The course of infection of M. microti was investigated in CFLP mice. A volume of 0.2 ml of the inoculum of M. microti that had been mouse- passaged (see sections 2.3.2 and 2.6.2) was injected intravenously into each of 54 female CFLP mice weighing 18-20 g at the begining of the experiment. From viable counts set up at infection, the infective dose was 1.4 x 10^ cfu of M. microti. The infection was monitored over 106 days by performing viable counts of the lungs and spleens; 5 mice were sacrificed at each time point. The viable count results (Figure 3.1) showed that the mouse-passaged M. microti was capable of multiplying in CFLP mice. In the lung, there was an initial decrease in the counts . irt i te ug (•) ad h sles A o 5 mice. of (A) spleens the and ) • ( microti in lungs the M. i 31 rwho . irt n ug ad pen o FP ie Mice Growth were infected microti with mice.M.CFLPof of spleens and 3.1in lungs Fig 1.4 x 10® viable organisms per mouse on Day 0. Each point represents the mean ± s.e cfu the means.e ± Each point represents 10® x mouse 0. Day viableon per organisms 1.4 Mean log. viable mycobacteria per organ asatr infection after Days 117 followed by a period of exponential growth for 4-5 weeks after which there was a period of a very gradual increase till Day 106. The mice sacrificed on Day 106 were apparently healthy but upon post-mortem, the lungs were full of necrotic lesions. The counts in the spleen gradually increased until Day 48 and then began to decrease. Overall, the results suggest the emergence of appreciable immunity in the 4th and 5th weeks of infection which substantially reduced bacillary growth. The outcome was eventually fatal after approximately four months (results not shown). From these results it was decided to infect CFLP mice with this infec tive dose size and allow the infection to proceed for 10 days, when in vivo growth was most rapid in the lungs and spleen, before commencing levamisole treatment. 3.2 The effect of levamisole on M. microti infection in CFLP mice 150 female CFLP mice, 20-22 g at the begining of the experiment, were randomly allocated into six groups. Three groups were infected with mouse-passaged M. microti (see section 2.6.3) by intravenous tail vein inoculation. One group consisted of the untreated controls, another received 2.5 mg kg ^ of levamisole twice a week and the third group received 25 mg kg ^ twice weekly. The remaining three groups of mice were kept as uninfected controls, one group being the uninfected untreated controls and the other two groups receiving 2.5 and 25 mg kg ^ of levamisole twice weekly respectively. Levamisole was administered by oral gavage twice weekly for 5 weeks with the first dose on Day +10. The effect of levamisole on the growth of M. microti was monitored by 118 performing organ counts on Days +3, +7, +15, +22, +29, +36 and +43. The effect on splenic T lymphocytes was assessed by the proliferative res ponse to PPD, PHA and Con A before treatment started, half-way through and at the end of treatment. The subsets of splenic T-cells were also determined at the same time points by immunoenzymatic staining. In the immunological tests, comparison was also made with the uninfected controls. 3.2.1 The effect of levamisole on the growth of M. microti 4 mice were sacrificed for each time point. Viable counts set up at infection showed that the infective dose was 2.4 x 10^ cfu per mouse. The results of the viable counts in the lungs and spleens are shown in Figure 3.2 and Figure 3.3 respectively. For clarity, the standard errors are not depicted in the Figures but full details are shown in Tables 3A.1 and 3A.2 in Appendix 1. It was evident from the Figures that there was not much difference between the growth of M. microti in the untreated and the levamisole treated groups in both lungs and spleens. This was confirmed by analysis of variance of the results (Table 3.2) which showed that only time (Days) had a highly significant effect on the growth of M. microti in both lungs and spleens. i. . Efc flvmsl ngot fM mcoi inlungslevamisole ongrowthofM. microti Effect Fig. 3.2 fCL ie Mc ee infected2.4xwere Mice with CFLPmice.of e os nDy0ad rae ih0m/g •, . gk (♦), 2.5mg/kg (•), treatedand0on0perDay with mouse mg/kg r2 gk () eaioe wc ekyfo a 1. Each twicefrom levamisoleDay +10.weekly (A) or25 mg/kg point represents the mean logio cfu per lung of 4 the lunglogio cfuofmice.pointperrepresentsmean Mean log ^viable mycobacteria per lung 4 3 5 6 7 8 0 r I i I I1 7 5 2 9 36 29 22 15 7 3 I I I I I I I I as fe infection after Days ---- 119 i i I I 1 ---- 106 i viable bacilli viable U 1 --- 1 H3 * A Control 25mg/kg 2.5mg/kg 2.5mg/kg of CFLP mice. Mice were infected with 2.Ainfectedwere with x Mice CFLPmice.of Fig. 3.3 Effect of levamisole on growth of M. microti in spleenslevamisoleongrowthinof M. microti Effect 3.3Fig. e os nDy0ad rae ih0m/g •, . gk (♦), 2.5mg/kg (•), treated0and0on with mg/kgDay permouse r2 gk () eaioe wc ekyfo a.1. Each from twice levamisoleweeklyDay.+10. (A) 25ormg/kg point represents the mean logio cfu per spleem theperlogiocfuAof mice.point represents mean Mean log ^viable mycobacteria per spleen 3 1 2 2 3 43 36 29 22 15 7 3 0 i i i i i i i I I I II II II I L L asatr infection after Days LLLLLL ----- 120 1 ----- 106 L=levamisole viable bacilliviable 1 ----- LL 1 121 TABLE 3.2 ANALYSIS OF VARIANCE OF EFFECT OF LEVAMISOLE AND TIME ON GROWTH OF M. MICROTI IN CFLP MICE Source of LUNG SPLEEN variation DF MS F MS F LMS 2 0.010 0.1 0.046 0.9 *** Days 4 13.450 140 0.772 8.9 LMS x Days 8 0.188 1.9 0.059 0.6 Replicate Error 45 0.096 0.090 LMS = levamisole *** pCO.OOl 3.2.2 The effect of levamisole on proliferative responses of splenic T lymphocytes The proliferative responses of splenic T lymphocytes from uninfected and infected mice that were treated with 0, 2.5 or 25 mg kg ^ levamisole were tested before treatment (Day +9), after 2 weeks treatment (Day +24) and after 5 weeks treatment (Day +45). Different cell concentrations were investigated but invariably, the concentration of 1 x 10^ cells per ml gave the lowest background counts and hence only proliferative responses of this cell concentration were reported. The results of the proliferative responses on Day +9 to PHA, Con A and PPD together with their respective controls are shown in Table 3.3. 122 TABLE 3.3 PROLIFERATIVE RESPONSES OF SPLENIC T CELLS OF UNINFECTED AND INFECTED MICE BEFORE LEVAMISOLE TREATMENT T cells Uptake of [3H] Thymidine + s.e. (cpm) cultured with: Uninfected mice Infected mice No mitogen 394 + 68 1689 + 490 PHA 65331 + 9010 118086 + 19480 Con A 12190 + 3600 31715 + 9800 No antigen 50 ± 26 544 + 304 * PPD 337 + 200 51103 + 9333 PPD 0.001 pg ml The above results showed that 9 days after Infection, there was an increase in proliferative response to PHA, Con A as well as a substantial increase in antigen-specific response to PPD when compared with the uninfected controls. The proliferative responses to PHA and Con A after two weeks of levamisole treatment are shown in Table 3.4. 123 TABLE 3.4 PROLIFERATIVE RESPONSES OF SPLENIC T CELLS OF UNINFECTED AND INFECTED MICE AFTER TWO WEEKS OF LEVAMISOLE TREATMENT 3 T cells from Uptake of [ H]-Thymidine + s.e. (cpm) group: Control ^ PHA CON A Uninfected 0 mgkg LMS 1061 + 241 73809 + 18330 16106 + 1341 2.5 mgkg 1 LMS 18962 + 5542 100693 + 6100 28004 + 2771 25 mgkg 1 LMS 21796 + 4492 137111 + 8320 53260 + 7203 Infected 0 mgkg 1 LMS 244 + 36 124227 + 15620 23416 + 6842 2.5 mgkg 1 LMS 1238 + 222 69186 + 6790 10809 + 3800 25 mgkg 1 LMS 1725 + 329 36038 + 2520 34437 + 5900 LMS = levamisole These results showed several evident trends. In the uninfected controls, treatment with levamisole increased the background prolife ration of control cultures by 18-20 fold with a concomitant increase in mitogen-stimulated cultures. However, in the infected cultures, the background proliferation of unstimulated cultures from treated mice were just a little greater than those from untreated mice and considerably less than in the uninfected mice. The response to PHA of the infected and untreated mice was much greater than those of treated mice. Hence, it appeared that levamisole elevated the PHA response in uninfected mice but decreased the response in the infected mice. The response to Con A in the uninfected mice was also different to the response in the infected mice. Like the PHA response, levamisole in uninfected mice 124 elevated the Con A response; but in infected mice the lower dose of levamisole decreased, while the higher dose increased the response. The PPD response was very variable (results not shown) with very high back ground counts for the unstimulated cultures. The proliferative responses to PHA and Con A after 5 weeks of levamisole treatment are shown in Table 3.5. TABLE 3.5 PROLIFERATIVE RESPONSES OF SPLENIC T CELLS OF UNINFECTED AND INFECTED MICE AFTER FIVE WEEKS OF LEVAMISOLE TREATMENT T cells from Uptake of [^H]-Thymidine + s.e. (cpm) group : Control PHA CON A Uninfected -1 0 mgkg LMS 942 + 90 64879 + 3371 5219 + 2131 -1 2.5 mgkg LMS 7864 + 1124 108035 + 7076 9353 + 890 -1 25 mgkg LMS 5222 + 1247 104348 + 5721 16170 + 3600 Infected , -1 0 mgkg LMS 92 + 51 61755 + 6195 3376 + 790 -1 2.5 mgkg LMS 1296 + 412 46050 + 8642 6883 + 1645 -1 25 mgkg LMS 1754 + 153 57137 + 12018 7998 + 3500 LMS = levamisole The proliferative response to PHA after 5 weeks levamisole still showed a similar trend. Levamisole increased the PHA response in uninfected mice but decreased the response in infected mice. The background pro liferation of unstimulated cultures were still high but not as high as 125 after 2 weeks levamisole treatment. The response to Con A had decreased compared to the earlier time points but levamisole still elevated the response in the uninfected mice and also the infected mice. PPD res ponses were again very variable with very high background counts (results not shown). 3.2.3 The effect of levamisole on splenic T cell subsets Preliminary results had shown that the optimal dilution of the mono clonal antibodies was 1 in 500. The monoclonals were stored at 4°C and diluted just prior to use. Both immunoenzymatic staining methods gave similar results but the immunoalkaline phosphatase method facilitated enumeration as the cells were stained red against a bluish background (Figure 3.4). The monoclonal antibodies enabled the following determinations to be made: a) total T cell count using the anti-Thy 1,2 antibody; b) helper T cell phenotype count using the anti-Lyt 1 antibody; and 3) suppressor /cytotoxic T cell phenotype using the anti-Lyt 2 antibody. The splenic T cell subsets were determined on Day +9 (pre-treatment), Day +24 (after 2 weeks treatment) and on Day +45 (after 5 weeks treatment). In all the results, the number of T cells stained by a particular antibody was calculated as a % of the total number of spleen cells counted. Differential staining (Diff-Quik staining) of the spleen cell suspensions had shown that they comprise 85% lymphocytes, 10% macrophages and 5% polymorphonuclear cells. 126 Fig. 3.4 Immunoalkaline phosphatase staining of cytocentrifuged murine splenic T cells with monoclonal Thy 1.2 antibody. Thy 1.2+ T cells are stained red (Naphthol AS-MX/Fast Red substrate, light haematoxylin counterstain). Magnification x400. 127 The results of the total number of T cells stained by the anti-Thy 1,2 antibody are shown in Table 3.6. TABLE 3.6 SPLENIC THY 1,2+ T CELLS IN INFECTED AND UNINFECTED MICE BEFORE AND AFTER LEVAMISOLE TREATMENT Spleen cells Thy 1,2+ T cells (% total spleen cells + s.e.) on: from: Day 9 Day 24 Day 45 Uninfected 0 mgkg 1 26.2 + 2.3 26.5 + 1.6 28.0 + 2 2.5 mgkg 1 3 2 . 2 + 3 34.0 + 2.2 25 mgkg 1 18.4 + 4.3 38.7 + 2.3 Infected 0 mgkg”1 25.A + 2.2 2 1 . 3 + 2 25.4 + 1.9 2.5 mgkg 1 2 1 . 0 + 1 23.9 + 3.1 25 mgkg 1 20.4 + 1 27.4 + 3.7 These findings showed that after 2 weeks treatment with 2.5 mg kg 1 levamisole there was an increase in the number of total T cells in the uninfected mice but had no effect on the number of T cells in the infected mice. The 25 mg kg 1 dose caused a decrease in T cells in the infected mice and a slight decrease in the uninfected mice. After 5 weeks of levamisole, the T cell numbers in uninfected mice were still elevated but there were hardly any differences between the treated and untreated infected mice. 128 Table 3.7 shows the results of the cells staining with anti-Lyt 1 antibodies. TABLE 3.7 SPLENIC LYT 1+ T CELLS IN UNINFECTED AND INFECTED MICE BEFORE ANJ) AFTER LEVAMISOLE TREATMENT Spleen cells Lyt 1+ T cells (% total spleen cells + s.e.) on: from Day 9 Day24 Day 45 Uninfected 0 mgkg 20.1 + 1.5 18.4 + 1.6 16.3 + 1 2.5 mgkg 18.4 +1 . 5 22.6 + 1.6 25 mgkg 10.3 + 1 . 2 32.0 + 3.4 Infected 0 mgkg 17.3 + 1.7 17.0 + 1.2 15.9 + 1 - 5 2.5 mgkg 18.2 + 2.1 18.4 + 1 . 9 25 mgkg 12.5 + 0.9 15.9 + 1.4 These results showed that in the uninfected mice, treatment with 2.5 mg kg ^ did not change the number of helper (Lyt 1+) T cells but the higher dose caused a decrease after 2 weeks and an increase after 5 weeks of treatment. In the infected mice, there was not much difference in the number of helper T cells between treated and untreated mice. The number of cells stained by the Lyt 2+ antibodies are shown in Table 3.8. 129 TABLE 3.8 SPLENIC LYT 2+ T CELLS IN INFECTED AND UNINFECTED MICE BEFORE AND AFTER LEVAMISOLE TREATMENT Spleen cells Lyt 2+ cells as % total spleen cells + s.e. on: from: Day 9 Day 24 Day 45 Uninfected 0 mgkg 1 8.9 + 1.4 7.4 + 1 7.1 + 0.7 2.5 mgkg 1 4.1 + 0.5 10.2 + 0.3 25 mgkg 1 6.1 + 0.7 15.7 + 1.1 Infected 0 mgkg 1 5.2 + 0.7 3.5 + 0.5 2.5 mgkg 1 9.9 + 1.0 5.4 + 1 . 5 25 mgkg 1 5.6 + 0.6 3.2 + 0.8 Approximately 10% of the total spleen cells have the Lyt 2+ phenotype in both the uninfected and infected untreated mice at the begining of the infection. As the infection progresses, the number of Lyt 2+ cells decreases slightly in the infected mice and the 2.5 mg kg ^ levamisole dose seemed to cause a slight elevation of the number of cells. In the uninfected mice, treatment with levamisole seemed to increase the number of Lyt 2+ cells on Day 45 after an initial decrease on Day 24. 130 3.3 Discussion M. microti has been reported to grow slower than M. tuberculosis and to be inhibited by glycerin in primary culture (Wells, 1946). It had also been observed (Leach and Wells, 1956) that the addition of 5% whole blood and adequate retention of moisture improved the recovery of freeze-dried M. microti. Later, a method for obtaining viable counts of M. microti in 21 days was reported (Sharp, 1973) using oleic-albumin agar containing 5% defibrinated horse blood and a tight seal. It was decided to perform some preliminary experiments with a variety of media and conditions to determine the best system. Those experiments showed that reproducible results could be obtained in 4 weeks by using Dubos Oleic Base agar supplemented with 10 % OADC and sealing the plates with parafilm before enclosing in polyethylene bags. Under these circum stances, growth was only slightly slower than that of M. tuberculosis. The standard oral dose of levamisole is 150 mg twice weekly in adult humans. The choice of 2.5 mg kg ^ levamisole twice weekly was based on the clinical therapeutic dose given in a clinical study of the effect of levamisole on the response to chemotherapy of pulmonary tuberculosis in Kenya and Zambia (Kenyan/Zambian/British Medical Research Council Collaborative Study, in preparation). The 25 mg kg ^ dose was selected to represent a higher dose. At these doses, levamisole did not have any effect on the growth of M. microti in the lungs or spleens. This was evident from the growth curves and confirmed statistically. Shepherd et al. (1977) also reported that levamisole had no effect on the mouse footpad growth curve. In a study of the effect of levamisole 131 on experimental murine infection with M. tuberculosis (Chumak and Kostromin, 1981) the authors reported that levamisole (12 mg kg \twice weekly) did not increase the survival rate nor improved the extent of disease when given on its own. However, when levamisole was given in conjunction with isoniazid, there was an increase in survival rate and increased lymphoid proliferation in the spleen and thymus. In another study, there was a similar finding that levamisole at 2.5 and 25 mg kg ^ (thrice weekly) in mice and guinea-pigs respectively, was effective in decreasing the extent of disease in combination with isoniazid but not on its own (Alexandrova and Zabolotnykh, 1981). However, in both these reports, the actual growth of M. tuberculosis was not monitored. It has been reported that responses to levamisole are not always predictable due to the presence of responders and non-responders in every animal species (Symoens et al., 1979). In mice, the responsive ness to levamisole seems to be related to their ability to produce a serum factor (Renoux, 1978). In a series of experiments Renoux and colleagues (1979) showed that host factors like genetics, sex and age of mice can lead to differences in response to levamisole. A recent report showed that the fat composition of a mouse diet modified the effects of levamisole on growth and spread of a murine tumour (Boeryd and Hallgren, 1985). That could be one of the reasons for the conflicting reports that have appeared in the literature about the immunotherapeutic potential of levamisole. Although there have been a few reports in the literature about the effects of levamisole in conjunction with chemotherapy on clinical cases of pulmonary tuberculosis, the evidence is not very convincing (see 132 section 1.7). Preliminary results of a trial of levamisole in combina tion with chemotherapy on tuberculosis patients in Zambia and Kenya have shown no clinical benefit of levamisole (Kenyan/Zambian/British Medical Research Council Collaborative Study, in preparation). Gatner and Anderson (1982) examined immune parameters, sputum bacterio logy and radiological evidence in seven newly diagnosed pulmonary tuber culosis patients given a single oral dose of levamisole (150mg) once a week in addition to standard antituberculous therapy for 3 months. The humoral parameters (serum immunoglobulin, salivary IgA, C-reative prot ein, ^-antitrypsin levels) were initially elevated but returned to normal. Among the CMI parameters, neutrophil chemotactic responses increased within 2 hours after ingestion but was not sustained; lympho cyte proliferation to PHA and PPD was also augmented 2 hours after ingestion and was sustained. No evidence of improved sputum conversion time or significant radiological improvement was observed. It was noted that while immune function parameters were restored during levamisole therapy, a gradual return of these indices to normal generally occurred during standard therapy. The proliferative responses to PHA and Con A after levamisole treatment were surprisingly different in uninfected and infected mice. Levamisole elevated the PHA response in the uninfected mice but had the reverse effect in the infected mice both after 2 weeks and 5 weeks treatment. The effects on Con A response were not so clearly defined but generally, levamisole increased the response to Con A regardless of infection. The other noticeable effect of levamisole was the elevation of the back ground proliferation of control cultures especially after 2 weeks. The 133 response to PPD was not clear due to the presence of a very high and variable background proliferation on Days 24 and 45. The reasons for the variable background proliferation counts of control cultures in the PPD assay were not known. The mi croteraski method uses only a small number of cells. In the PPD assay, there are relatively fewer antigen- reactive cells compared to reative cells in mitogenic assays and the microterasaki method may have been too insensitive. The response to PHA correlated with the increase in total splenic T cells seen in uninfected but not infected mice. Generally, the total T cell and T cell subsets were not very different after levamisole treatment in infected mice although there were some slight differences in the uninfected mice. The significance of these observations is unknown. It would seem that infection with M. microti radically affects the proliferative response of splenic T cell to PHA. These findings confirm the earlier observations that the effect of levamisole to a large extent depended on host factors and that infection with M. microti altered the host and consequently altered the proliferative responses. Ultimately though, despite the differences seen in the immunological tests, levamisole did not affect the in vivo growth of M. microti. Levamisole has been shown to augment the responsiveness of T cells to mitogens and antigens. Levamisole has been shown to be weakly mitogenic for mouse spleen cells (Merluzzi et al., 1975) and Renoux (1978) reported an increase in DNA synthesis of murine splenocytes after in vivo treatment with levamisole. This was consistent with the findings reported in this Chapter of increased background proliferative responses of unstimulated splenocytes. 134 In an In vitro study Chan e t a l . (1976) showed that preincubation of lymphocytes from tuberculosis patients with levamisole induced an enhanced proliferative response to PHA. Gatner (1981) subsequently reported that a single 150 mg dose of levamisole augmented in vitro PHA and PPD-induced lymphocyte proliferation of tuberculin-positive control subjects. As levamisole did not have any significant effect on the growth of M. microti in organs of mice, no attempts were made to investigate the effect of levamisole in combination with antituberculous drugs. At the time this decision was taken, preliminary results of the clincal trials in Kenya and Zambia were begining to show a lack of clinical effect of levamisole, and recombinant IFN-)( also became available. 135 CHAPTER 4 THE IMMUNOMODULATING EFFECT OF INTERFERON-GAMMA ON M. TUBERCULOSIS INFECTION IN MICE The immunomodulating effect of Interferon-gamma was investigated in an acute infection of M. tuberculosis H37Rv in mice. The experiments were performed with two strains of mice. The initial experiments were performed on CFLP mice, an outbred strain, but most of the results reported in this chapter were experiments done on BALB/c mice. The reason for the switch from the outbred strain to an inbred strain was due to preliminary results which had shown that the effects of IFN-V were not very large. In addition, it was noted that there were diffe rences in the growth rate of M. tuberculosis in untreated controls between some experiments with the CFLP mice. It is well known that the cleanliness of mice affects the host resistance and even though the CFLP mice were obtained from a specific-pathogen free colony, there was uncertainty about their cleanliness. Thus, when a source of BALB/c mice from a reputable specific-pathogen free colony became available, most of the later experiments were performed on these mice. As each experiment had its own control, there was no problem with correlation of results from both sets of mice. The untreated controls showed that both strains reacted very similarly to infection with M. tuberculosis. A range of dose sizes were tested as well as the efficacy of liposomal carriage of IFN-J^. The schedule of administration of IFN-tf was also investigated. The results of some preliminary experiments on the in vivo model are also reported. 136 4.1 Preliminary experiments 4.1.1 Sonicatlon of M. tuberculosis The hydrophobic nature of the mycobacterial species means that they readily clump. The introduction of surfactants like Tween 80 into culture media (Dubos, 1945) has helped to resolve the problem but it has been shown that suspensions of mycobacteria grown in liquid media with Tween 80 are still mainly in small clumps, each containing 4-20 organisms (Fenner, 1949). Ultrasonication has been used as a means of dispersing clumps in mycobacterial suspensions (Blanden et al., 1969). Some experiments were carried out to determine the optimal conditions for obtaining a dispersed suspension with a sonicating probe (Rinco Ultrasonics UK Ltd.). 12 ml samples of a suspension of H37Rv in 0.1% gelatine saline were placed in universals and the probe was used at an amplitude of 70% with the probe 1 cm below the surface of the suspension. In one experiment, viable counts were performed after sonication periods of 0, 5, 10, 15, 30, 45 and 60 seconds in non-continuous bursts of 5 seconds. In another experiment, total counts were carried out after the same sonication periods. In addition to enumeration, the total counts were also scored as the number of single bacilli, the number of clumps of 2, 2-5 and >5 bacilli. The results of different lengths of sonication on the viable counts are shown in Figure 4.1. It can be seen that the viable counts increased dramatically after 5 seconds of sonication to its maximum after 30 seconds. Even 60 seconds ahpitrpeet the tuberculosisrepresentspointnumberatofcfuof M.Each aiu tm onsatr sonication. timepointsaftervarious Fig. 4.1 Effect of sonicationofofmycobacteria. Effectoncounts viable Fig. 4.1 Viable mycobacteria (x10 )/ ml Time in seconds 137 138 did not seem to affect the viability of the mycobacteria. The results of different sonication lengths on the total counts are shown in Table 4.1. TABLE 4.1 TOTAL COUNTS OF M. TUBERCULOSIS AFTER SONICATION Sonication Total count % Bacteria in aggregates of : time (secs) (orgs /ml) one two 2-5 >5 0 1.0 x 107 46 6 27 21 5 3.6 x 107 65 10 18 7 10 3.8 x 107 73 17 7 3 15 4.4 x 107 80 13 6 1 30 5.2 x 107 81 10 8 1 45 6.2 x 107 80 14 6 0 60 6.1 X 107 83 14 3 0 Before sonication, 27 % of the bacilli were aggregated as clumps of 2-5 and 21% as clumps of >5 bacilli. These aggregations dropped consider ably after sonication but even after 60 minutes sonication, there were still 3.6% of the bacilli in small clumps of 2-5. It was decided that a sonication period of 15 seconds at a setting of 70% amplitudes and with the probe 1 cm below the surface of the suspen sion was to be adopted in all the experiments. 139 4.2 The effect of infective dose size of M. tuberculosis on the growth of bacilli In the lungs and spleens of CFLP mice In this experiment, different infective doses of M. tuberculosis were used to determine an optimal inoculum size. Different dilutions of the animal-passaged H37Rv inoculum stored at -70°C were injected into CFLP mice. 72 mice were divided randomly into 3 groups. Viable counts performed at infection showed that one group received 5 x 10^ cfu per 5 4 mouse, another 5 x 10 cfu and the last group 5 x 10 cfu. The growth of the bacilli in the mice was monitored by performing viable counts of the bacilli in the lungs and the spleens at days 1, 8, 15, 22, 29 and 43 after infection. Four mice were sacrificed at each time point. The results are shown in Figures 4.2 and 4.3. The details of the results are shown in Table 4A.1 in Appendix 1. The mice that received 5 x 10^ cfu per mouse had enlarged spleens and necrotic lesions in the lungs by day 15 and began to die after day 20. None of the mice in the 4 other two groups died and the mice in the group that received 5 x 10 cfu per mouse were still apparently healthy at day 43. However upon post-mortem, the lungs showed evidence of necrotic lesions. From these results, it was decided that an infective dose size mid-way between 5 x 10^ and 5 x 10^ cfu per mouse was to be used in all subsequent experments. This infective dose size was achieved by diluting the original frozen inoculum 1 in 2. 1 40 1 cn . c 3 i- Fig. 4.2 The effect of infective dose size on the growth of M. tuberculosis in lungs of CFLP mice. Mice were infected with 5 x 106 (#), 5 x 105 (A) or 5 x 10^ ( ♦ ) cfu per mouse. Each point represents the mean + s.e. of 4 mice. 141 c n i i i i i 01 8 15 22 29 43 Days after infection Fig. 4.3 The effect of infective dose size on the growth of M. tuberculosis in spleens of CFLP mice. Mice were infected with 5 x 106 (#), 5 x 105 (A) or 5 x 10^ (♦) cfu per mouse. Each point represents the mean + s.e. of 4 mice. 142 4.3 The effect of dose size of interferon-gamma on the growth of M. tuberculosis In BALB/c mice 65 male BALB/c mice, 18-20 g in weight, were randomly allocated into 4 groups. 20 mice were used as controls and the other groups of 15 mice each received either 200, 1000 or 5000 Units of interferon-gamma per mouse. The control group was given PBS. Treatment was started 2 days prior to infection (Day-2) and each group received a dose every third day, that is, on Days +1, +4 and +7. 5 mice were sacrificed per treatment group for each time point. In this experiment, the viable count of the M. tuberculosis was assessed in the lungs and spleens on Days +3, +5 and +9 after infection. Viable counts performed at infection showed that the inoculum per mouse was 1.26 x 10^ cfu. The results of the mean viable counts in the lungs are shown in Figure 4.4. For clarity, the s.e. of the means are not depicted but are shown in Table 4A.2 in Appendix 1. The mean viable count of M. tuberculosis in the lungs an hour after infection was 4.78 x 10** cfu. Over the next 3-5 days, there was a fall in viable counts in all the groups followed by an increase after Day+5. On Day+9, the mean viable count was 1.3 x 10^ cfu in the untreated controls and consider ably lower in the groups treated with IFN-V . There was hardly any difference between the groups given 1000 or 5000 units IFN-V but both these groups had a lower cfu than the group given 200 units. 143 The corresponding results of the spleen counts are shown in Figure 4.5 with the full details in Table 4A.3 (Appendix 1). The mean viable count an hour after infection was 1.43 x 10^ cfu. There was hardly any diffe rence in the growth curves of the untreated controls and the group that received 200 units of IFN-S. The growth curves of the groups that received 1000 or 5000 units were very similar and there was an obvious inhibition of bacillary growth. The viable count results of all the groups were then examined statistic ally by analysis of variance (AN0VA). The results of the AN0VA of both lungs and spleens are shown in Table 4.2. TABLE 4.2 ANALYSIS OF VARIANCE OF EFFECTS OF INTERFERON-GAMMA DOSE SIZE AND TIME ON GROWTH OF M. TUBERCULOSIS IN BALB/C MICE Source of Lung Spleen variation DF MS F MS F ** IFN-8 3 0.291 2.6 0.104 10.8 *** *** Days 2 2.244 20.0 6.960 718 Days x IFN-# 6 0.068 0.4 0.004 0.4 Replicate error 48 0.112 0.009 *** p < 0.001 ** p<0.01 The IFN-y dose effect was highly significant in the spleens (p<0.01) but did not attain statistical significance at the 5% level in the lungs. 144 U) c 3 L. o CL 0 u .2il 0) ■*-> u (0 JD O 200u U 5000u E o 10OOu Si to Days after infection Fig. 4.4 Effect of dose size of interferon gamma on the growth of M. tuberculosis in lungs of BALB/c mice. Mice were infected with 1.3 x 106 cfu M. tuberculosis per mouse on Day 0 and treated with 0 u (•), 200 u (O ), 1000 u (♦) or 5000 u (A ) IFN-Jf per mouse on Days -2, +1, +4 and +7. Each point represents the mean + s.e. cfu per lung of 5 mice. Mean log^viable mycobacteria per spleen + s.e. cfu per spleen of spleen5 permice. cfus.e. + os nDy 2 1 + n 7 Ec on ersns therepresentspoint Each mean andon +4 Days -2,+1, +7. mouse with 0 u (•), 200 u 200 (•), 0u with treated tuberculosisonand0per cfumouseDay M.1.3106 x with fM tbruoi i pen fBL/ ie Mc ee infectedwere Mice spleensBALB/cof in tuberculosismice. ofM. i. . Efc fds sizetheinterferon-gammaofgrowthdoseonof Effect Fig. 4.5 as fe infection after Days (O ), ), (O 00u( o 00u A IFN-Yper (♦ ) (A) or50001000uu 145 146 This was probably due to the higher mean square of the replicate error in the lungs compared to the spleen. As there was no difference between dosage with 1000 or 5000 units of IFN-* , most of the subsequent experi ments were performed with doses of 1000 or 2000 Units IFN-V per mouse given at 3 day intervals, with the first dose given 2 days prior to infection. 4.4 The effect of the administration of interferon-gamma in liposomes on the growth of M. tuberculosis in mice Two sets of experiments were performed on the effect of the administra tion of IFN-y in liposomes on the growth of M. tuberculosis in mice. One experiment was carried out in CFLP mice with dose sizes of 1000 and 20000 units of IFN-X. The other experiment was performed in BALB/c mice with a single dose size of 1000 units. 4.4.1 The effect of the interferon-gamma when administered in liposomes on M. tuberculosis infection in CFLP mice In the first experiment, 62 female CFLP mice, 18-20 g in weight, were randomly allocated into 5 groups. All the mice were infected intra venously with 1.4 x 10^ cfu of M. tuberculosis per mouse. The control group was treated with PBS while the other groups were given 1000 units of IFN-V, 1000 units of IFN-* in liposomes, 20000 units of IFN-V or 20000 units of IFN-tf in liposomes. The treatments were given at 3 day intervals with the first dose 2 days prior to infection and subsequent doses on Days +1, +4 and +7. The IFN-# dilutions and liposomes were made as described in section 2.5.2.1. Due to the unknown stability of 147 the recombinant IFN-& no attempts were made to separate the free IFN-& from the encapsulated IFN-Jf. The growth of the bacilli was assessed by sacrificing 6 mice per group on Days +5 and +9 post-infection. The uptake of the inoculum was deter mined by performing viable counts on organs of 6 mice an hour after 4 5 infection. The uptake was 7.1 x 10 cfu in the lungs and 3.6 x 10 cfu in the spleen. The results of the viable counts of M. tuberculosis in the lungs are shown in Figure 4.6 and the viable counts in the spleens are represented by Figure 4.7. The mean viable counts + s.e. of the lungs and spleens are shown in Tables 4A.4 and 4A.5 respectively (Appendix 1). The Figures clearly show that both 1000 and 20000 units of IFN-V had a significant effect on the growth of M. tuberculosis in both lungs and spleens of the mice. The viable counts of the untreated controls, mice given 1000 units IFN-^ and mice given 1000 units IFN-tf in liposomes were analysed separately by a 2-way AN0VA. Orthogonal linear contrast showed that 1000 units IFN- £ had a highly significant effect in the lungs (F ratio=28.5, p < 0.001) and in the spleen (F ratio=72.2, p<0.001) when compared with the untreated controls. There was no difference between the results of treatment with 1000 units IFN-* and 1000 units IFN-V in liposomes in both organs. The administration of IFN-if in liposomes does not seem to have increased the effect in the spleens. However, in the lungs, the shape of the growth curve of the group that had been treated with 1000 units IFN-tf in liposomes suggests that encapsulation in liposomes results in a prolongation of the effect of IFN-*' while the Mean log^viable mycobacteria per lung f nefrngma n iooe. ie ee netd ih . x 0 ibe ail on bacilli viable 10 x 1.4 with infected Mice were liposomes. in interferon-gamma of en o^ f pr ug f mice. 6 of lung per cfu log^ mean Day 0 and treated with 0 u ( • ) , 1000 u (A ), 20000 u (■ ) IFN-Y , or 1000 u (A ), ), (A 1000 u or IFN-Y, ) (■ 20000 u ), (A 1000 u , ) • ( u 0 with administration treated and 0 miceafter Day CFLP of lungs in tuberculosis M. of counts Viable 4.6 Fig 00 u □ INYi lpsmso Dy -, 1 + n +. ah on rpeet the represents point Each +7. +4and +1, -2, Days liposomes.on IFN-Yin (□) 20000 u asatr infection after Days Mean loginviable mycobacteria per spleen f nefrngma n iooe. ie ee netd ih . x 0 val bcli on bacilli 106 viable x 1.4 with infected Mice were liposomes. in interferon-gamma of en og f pr pen f mice. 6 of spleen per efu g^ lo mean a 0 n tetd ih u 1000 u , ) • ( u 0 with administration treated after mice and 0 CFLP Day of spleens in tuberculosis M. of counts Viable 4.7 Fig 00 u □ INyi lpsms n as 2 +, 4ad 7 Ec pit ersns the represents point Each +7. +4and +1, -2, Days on liposomes in IFN-y (□) 20000 u as fe infection after Days (A), (A), 00 u ■) F-f o 10 u (A), 1000 u or IFN-lf, ) (■ 20000 u 150 curve of mice treated with 20000 units in liposomes suggests a decrease in the effect. Statistical evaluation of the lung and spleen counts of mice receiving IFN-Y or IFN-Y encapsulated in liposomes was performed by a three-way analysis of variance. The results of the analysis are shown in Table 4.3. TABLE 4.3 3-WAY ANALYSIS OF VARIANCE ON THE EFFECT OF INTERFERON-Y DOSE SIZE, LIPOSOMES AND TIME ON M. TUBERCULOSIS INFECTION IN CFLP MICE Source of LUNG SPLEEN variation DF MS F MS F ** IFN-Y 1 1.728 9.6 0.108 2.8 Liposomes 1 0.530 3.0 0.000 0.0 *** * Days 1 2.980 16.6 0.242 6.2 IFN-Y x Days 1 0.150 0.8 0.053 1.4 * Liposomes x Days 1 0.319 1.8 0.256 6.6 IFN-Y x Liposomes 1 0.154 0.9 0.029 0.7 Days x IFN-Y x Liposomes 1 0.146 0.8 0.000 0.0 Replicate error 40 0.179 0.039 *** p<0.001 * p < 0.05 In the 3-way analysis of variance, the sources of variation are broken down into main effects, first-order and second-order interactions. In this experiment, the main effects are IFN-Y , liposomes and time (days). The analyses showed that among the main effects; the dose size of IFN-Y 151 was highly significant (p<0.01) in the lung but not significant in the spleen; time had a significant effect in the lungs and spleens; and liposomes did not have a significant effect on either organ. There was a significant first-order interaction of time and liposomes in the spleens. A.4.2 The effect of the administration of interferon-gamma in liposomes on M. tuberculosis infection in BALB/c mice The previous experiment had shown that the administration of IFN-Y in liposomes did not have a statistically significant additional effect to that of IFN-Y even though the shape of the growth curve in the lungs of the mice given 1000 units IFN-Y in liposomes seems to indicate a prolon gation effect. It was decided to examine the effect of empty liposomes on the growth of M. tuberculosis in mice. In this experiment, an additional control group receiving PBS in lipo somes was included and a single dose size of IFN-* was tested. 65 female BALB/c mice, 18-20 g in weight, were randomly allocated into 4 groups. One control group was given PBS used for diluting IFN-^ ; the other control group received PBS encapsulated in liposomes and the other two groups were given 1000 units of IFN- V either in PBS or encapsulated in liposomes. 4 mice were sacrificed at each time point and the lungs and spleens cultured to determine the number of viable M. tuberculosis. Viable counts performed at infection showed that the number of organisms in the inoculum was 7.53 x 10^ cfu per mouse. At one hour after infection, 152 k 4 there were 7.16 x 10 cfu in the lungs and 9.23 x 10 cfu in the spleen. The results of the viable counts of the bacilli in the lungs of the various groups are shown in Figure 4.8. Similarly, the results of the viable counts in spleens are shown in Figure 4.9. For clarity, the standard errors of the means are not depicted in the graphs, but the full details of the mean counts in the lungs and spleens are shown in Tables 4A.6 and 4A.7 respectively (Appendix 1). In the lungs, there was hardly any difference between the bacillary growth curves of the groups that had been treated with PBS or PBS encapsulated in liposomes. However, there was a lowering of the lung viable counts in the groups of mice that had received either 1000 units of IFN-tf or 1000 units encapsulated within liposomes. The viable count results of the spleen (Figure 4.9) show a definite trend. There was a slight lowering of viable counts in the group that had received PBS in liposomes. Both groups that had been given 1000 units IFN-fc' showed lower spleen counts than the PBS control, with the liposomal IFN-^T group showing slightly lower counts than the IFN-fc group. Analysis of variance of the lung and spleen counts was performed by a three-way analysis of variance. The results of the AN0VA are shown in Table 4.4. ieatramnsrto fINYi iooe. iewr infected Micewere IFN-Yinliposomes.of afteradministration mice ihPS •, B nlpsms O, 00uINY() r 1000 or (♦) 1000IFN-Y u liposomes (O), in PBS (•), PBS with organisms per treated1050viableand7.5xmouseon Day with u IFN-Jr in liposomes ( O ) on Days -2, +1, +4 and +7. Each point Each on-2,IFN-JrDays and+1,u +4 +7. ) O inliposomes ( ersns thelung logiocfuperofmean represents4 mice. i. 8 ibecut oftuberculosislungscountsM. the BALB/cinof Viable .8Fig. A Mean loginviable mycobacteria per lung as fe infection after Days 153 PBS PBS,L 1000u/L lOOOu 154 Days after infection Fig. 4.9 Viable counts of M. tuberculosis in the spleens of BALB/c mice after administration of IFN-V in liposomes. Mice were infected with 7.5 x 105 viable organisms per mouse on Day 0 and treated with PBS (•), PBS in liposomes (O), 1000 u IFN-y (♦) or 1000 u IFN-Y" in liposomes (O) on Days -2, +1, +4 and +7. Each point represents the mean loglO cfu per spleen of 4 mice. 155 TABLE 4.4 THREE-WAY ANALYSIS OF VARIANCE OF THE EFFECT OF INTERFERON-GAMMA, LIPOSOMES AND TIME ON M. TUBERCULOSIS INFECTION IN BALB/C MICE Source of LUNG SPLEEN variation DF MS F MS F ** *** IFN-Y 1 0.004 11.3 0.125 35.3 *** Liposomes 1 0.004 0.1 0.125 13.9 *** *** Days 2 4.530 108 7.272 808 * IFN-V x Days 2 0.044 1.0 0.054 6.0 IFN-^ x Liposomes 1 0.007 0.2 0.001 0.1 Days x Liposomes 2 0.034 0.8 0.008 0.9 IFN-y x Days x Liposomes 2 0.026 0.6 0.001 0.1 Replicate error 48 0.042 0.009 *** p < 0.001 ** p<0.01 * p < 0.05 In the lungs, the effect of IFN-Jf and time were highly significant, and the effect of liposomes was not significant. In the spleen, all the main effects were highly significant. A synergistic effect of liposomal encapsulation and IFN-Y would have resulted in a significant interaction of liposomes x IFN-tf. However, the sum of squares for this interaction was very small and not significant, which suggests that liposomes per se had an effect irrespective of IFN-Jf. 156 4.5 The effect of pretreatment of mice with interferon-gamma on M. tuberculosis infection in BALB/c mice It was evident from the results of the earlier experiments that the effects of IFN-fr was established very early in the infection and did not seem to increase with further administration of IFN-V. This observation was confirmed by analysis of variance which showed a significant effect of IFN-X and a significant effect of time but did not reveal any signi ficant interactions of IFN-lf and days. This absence of significant interactions implies that there was no amplification of the effects of IFN-lf with further doses of IFN-JT. Examination of the growth curves of M. tuberculosis in the treated and untreated groups in lungs and spleens generally show parallel growth curves which again suggest that the effects occurred early and did not increase with time. Hence, it would appear that the preinfection dose was of paramount importance. This experiment was designed to examine the effect of preinfection trea tment of mice with several multiplicities of doses as well as schedules of administration of IFN-K. 60 female BALB/c mice, 18-20 g in weight, were randomly allocated into 6 groups. One group was used as the control; 3 groups received a single dose of IFN-Jf given on Day-3, Day-2 or Day-1; one group received two doses of IFN-f on Days -2 and -1 and the last group received three doses on Days -3, -2 and -1. IFN-& was used at 2000 units per mouse. The mice were pretreated accordingly and were then infected intravenously with the same inoculum level. Four mice were sacrificed per time point and viable counts performed 1 hour after infection, and on Days +1 and 157 +2 post-infection. The effect of pretreatment with IFN-V on the lodgement of the bacilli in the lungs and spleens after one hour was also assessed. 6 mice of the control group and another receiving 3 doses of IFN-Y were sacrificed 1 hour after infection to determine if IFN-# had affected the uptake of bacilli in lungs and spleens. Viable counts performed at infection showed that the inoculum per mouse was 4.12 x 105 cfu. The results of the viable counts in the organs of mice 1 hour after infection are shown in Table 4.5. When the values were tested by the analysis of variance they gave F ratios of 0.5 and 2.1 for the lung and the spleen respectively indicating that pretreat ment with IFN-tf did not have any significant effect on the lodgement of the organisms in the lungs and the spleen an hour after intravenous infection. TABLE 4.5 THE EFFECT OF INTERFERON-V PRETREATMENT ON THE UPTAKE OF M. TUBERCULOSIS IN ORGANS OF BALB/C MICE cfu per organ after IFN-V treatment: Log10 Mouse Lung Spleen Number 0 u 2000 u 0 u 2000 u 1 4.972 5.031 4.703 4.826 2 4.982 5.061 4.794 4.699 3 5.054 4.932 4.854 4.697 4 5.042 5.068 4.885 4.512 5 5.081 4.943 4.798 4.787 6 4.988 4.952 4.648 4.663 Mean 5.020 4.998 4.780 4.697 + s.e + 0.02 + 0.02 + 0.04 + 0.04 ** —— 158 The results of the effects of the different regimens on growth of the M. tuberculosis in the lungs are graphed in Figure 4.10 (details of the mean viable counts + s.e. are in Table 4A.8 in Appendix 1). In the first two days after infection, the lung viable counts were decreasing and there were no apparent differences between the treated groups and the untreated control. The viable counts of M. tuberculosis in the spleen of the treatment groups are shown in Figure 4.11 with full details given in Table 4A.9 (Appendix 1). The growth curves of the bacilli in the different experi mental groups show a definite trend. All the groups that had been given IFN-tf showed a slower growth of the bacilli than the control. It was also clear that there was a difference in the growth curve depending on the day of administration of IFN-V. These results showed that the admi nistration of a single dose of IFN-f on Day-1 was more effective than on Day-2, with Day-3 being the least effective. It was also obvious that a single dose at Day-1 was as effective as two doses at Days -2 and -1 or three doses at Days -3, -2 and -1. A two-way analysis of variance was performed on both the lung and spleen counts (Table 4.6). In the lungs, the effect of IFN-^ was shown to be statistically insignificant. This was probably due to the high value of the mean square of the replicate error. In the spleen, it was confirmed statistically that the pretreatment dose(s) of 2000 units of IFN-V per mouse given solely before infection had a highly significant effect on the growth of M. tuberculosis.- As the results of this experiment had shown that administration of the preinfection dose on Day-1 was more mice after pretreatment with IFN-)f.pretreatmentwith after mice tMice pretreatedwithwere iewr infected105onorganisms4.1x Day 0. Each viable with wereMice n oeo 00uINJ nDy3 □, a- () Dy (O); Dayl (O), 2000IFN-JTof Day-2udose one (□), on Day-3 on rpeet thelogio lungcfuper4 representsof mice.point mean i. .0 ibecut fM tuberculosislungsofBALB/cofcountsM.in Viable Fig. 4.10 w oe o 00u() tredsso 00u(A o nrae (#). oruntreated A) ( threedosesof2000u (A); 2000 ofutwodoses Mean log 1Qviable mycobacteria per lung as fe infection after Days 159 en o1 f pr pen f mice. 4 of spleen per log1Qmean cfu Day-3(D), IFN-Yon 2000 u of dose one with pretreated Mice were interferon-gamma. with nrae ()ad netd ih . x 0 f o Dy . ah on rpeet the represents point Each 0. Day on cfu 10 x 4.1 with infected (#).and untreated a- ( Dy1 O) to oe o 20 u F- A; he dss f 00 INYA; or IFN~Y(A); 2000 u of doses three IFN-Y(A); 2000 u of doses two ); (O Day-1 , ) (0 Day-2 Fig 4.11 Viable counts of M. tuberculosis in lungs of BALB/c mice after pretreatment pretreatment mice after BALB/c of lungs in tuberculosis M. of counts Viable 4.11 Fig Mean Iogin viable bacilli per spleen as fe infection after Days 5 161 effective than on Day-3 or Day-2 (Figure 4.11), in some of the later experiments combining IFN-Xand chemotherapy, the preinfection dose was given one day prior to infection. TABLE 4.6 ANALYSIS OF VARIANCE OF THE EFFECTS OF INTERFERON-GAMMA PRETREATMENT ON M. TUBERCULOSIS INFECTION IN BALB/C MICE Source of LUNG SPLEEN variation DF MS F MS F *** IFN-* 5 0.041 1.92 0.054 5.4 *** Days 1 0.999 46.7 0.025 2.5 IFN-)f x Days 5 0.012 0.6 0.009 0.89 Replicate error 36 0.360 0.010 *** p < 0.001 4.6 Discussion In all the in vivo experiments involving both M. microti (Chapter 3) and M. tuberculosis, the infective inocula were prepared in large quantities and stored at -70°C. It had been shown by Grover et al. (1967) that an inoculum of M. tuberculosis preserved at -70°C retained its viability and virulence for guinea-pigs over a period of one year. Subsequently it was shown that there was no decrease in viability over a three year period of storage at 70°C of several strains of mycobacteria (Kim and Kubica, 1972, 1973). The use of low temperature preservation of the infective inocula was adopted in view of the following advantages: it avoided the risk of attenuation following serial transfer on laboratory media ; it eliminated the need to periodically passage a strain through 162 an animal to regain virulence and it enabled a reproducible and precise level of infection from one experiment to another. Throughout these experiments, an infective dose size of approximately 5 x 10^ to 1 x 10^ viable M. tuberculosis H37Rv per mouse was used. This infective dose size was chosen after preliminary experiments comparing various doses. At this dose, the H37Rv proliferated in the spleen from the first day while in the lung, there was an initial lag period of about 3 days where the counts decreased before exponential growth begins around the fifth day. The initial investigations with recombinant interferon-gamma were con cerned with trying to determine an optimal dose size, schedule and frequency of administration. The IFN-Ywas obtained at a concentration of 2.5 mg ml ^ with a specific activity of 7.2 x 10^ antiviral units per mg. Due to the lack of long-term stability data, the concentrated IFN-'JJ was stored at 4°C and diluted just prior to use. Dilutions were made in PBS containing 1 mg ml ^ of homologous mouse serum albumin. A range of dose sizes from 200 units to 20,000 units per mouse were tested. With the exception of 200 units which had virtually no effect, the other doses tested had a significant inhibitory effect on the growth of M. tuberculosis in both lungs and spleens of CFLP and BALB/c mice. However, no significant differences could be detected between the effects of 1000, 2000, 5000 units. The inhibitory effect of IFN-Y was evident from the growth curves of the mycobacteria in the organs and was confirmed statistically by analysis of variance. There was a suggestion from the results of one experiment (4.4.1) that 20,000 units IFN-Y in 163 liposomes had an adverse effect (Fig 4.6). However, this was only evident in the lungs and not the spleen. Analysis of variance of the results of that experiment (Table 4.3) showed a significant effect of dose size of IFN-Y in the lungs but not the spleens. The schedule of administration most commonly used for these experiments was at three day intervals with the first dose two days prior to infect ion. Results with this schedule suggested that the effects of IFN-Y occurred early and did not seem to be potentiated by further doses. This led to an investigation of the preinfection doses of IFN- Jr. The results showed that IFN-tf given a day before infection was the most effective preinfection dose and also that multiple preinfection doses did not confer any extra benefits. There has been two reports of the effects of recombinant murine IFN-V derived from E. coli on intracellular pathogens in vivo. Kiderlan et a l . (1984) showed that IFN-y was capable of protecting mice from a systemic or local infection with L. monocytogenes. The IFN- was given intravenously at a dose of 1 x 10^ or 4 x 10^ units a day before and on the day of infection in the systemic infection model. In the local infection model, protection was observed with subcutaneous foot-pad 3 injection of a minimal dose of 10 units. The other report described the effect of IFN-5f on murine toxoplasmosis (McCabe et al., 1984). They concluded that recombinant murine IFN-y had a significant activity against toxoplasma and that the activity appeared to be associated with enhanced antibody response and activated macrophages. They used doses 3 of 5 x 10 units per mouse injected intra-peritoneally and various schedules varying from two doses given just before and after infection 164 to doses every other day. There were some similarities between these these results and the results reported in this Chapter. In both these reports, IFN-fc was given before and after infection and there did not seem to be a difference between the higher doses used. Similarly, results of this Chapter showed no detectable differences between doses ranging from 1000 to 5,000 units per mouse. The pharmacokinetics of recombinant murine IFN-V in vivo are not known. A recent clinical study of cancer patients treated intravenously and intramuscularly with recombinant human IFN-V (Kurzrock, 1985) has shown the pharmacokinetics of recombinant IFN-V in man. After intravenous administration, IFN-V was cleared exponentially from the serum with a half-life of 25-35 minutes which was independent of the dose. Intra muscular injection gave a half-life varying from 227-462 minutes indep endently of dose size. This study has confirmed the short half-life of IFN-Jf in man. Hence, if liposomes could prolong the half-life of IFN- in vivo, encapsulation in liposomes should increase the efficacy of IFN-Jf. MDP has been shown to have a very short half-life. After paren teral administration, 50% of water-soluble MDP is excreted in the urine within 30 minutes (Parant et al., 1979). Fidler et al. (1980) have also demonstrated that LKs like MAF have a very short half-life due to rapid binding to serum proteins. The enhancement of activity by liposomes has been shown in studies with lymphokines (MAFs) and immunomodulators like analogues of MDP where encapsulation in MLV liposomes had conferred a greater tumoricidal effect on experimental murine tumours (Fidler, 1980; Fidler et al., 1981; Fidler et al., 1982). 165 Another reason for using liposomes Is to exploit the natural fate of liposomes to ’target’ passively to cells of the reticuloendothelial system. Consequently, experiments were performed to investigate whether encapsulation of IFN-Y in MLV liposomes would increase its effects. The two sets of experiments where IFN-Y was administered either in PBS or encapsulated in liposomes reaffirmed that IFN-Y of 1000 Units dose size per mouse had a highly significant inhibitory effect on the growth of M. tuberculosis in both the lungs and spleens. The effect of admini stration of IFN-Y in MLV liposomes was not so clearly defined after considering both sets of results. In the first set of experiments (A.4.1), the appearance of the growth curves in the lungs (Fig. 4.6) suggested that liposomes prolonged the effect of 1000 units of IFN-Y but had an adverse effect on 20,000 units IFN-Y. However, statistical analysis (Table 4.3) did not reveal any effect of encapsulation in liposomes on the efficacy of IFN-Y. In the second set of experiments, a 3-way analysis of variance (Table 4.4) showed that in spleens, liposomes per se had a significant inhibitory effect. However, there was no significant interaction of liposomes and IFN-Y which would have been expected if liposomes enhanced the effects of IFN-Y. The control group receiving liposomes with PBS did show an effect on the mycobacterial growth in the spleen as shown by comparison of the growth curves (Fig. 4.9). This would suggest that encapsulation of IFN-Y in liposomes did not really have any additional effect over IFN-Y administered in PBS. The reason for the lack of any effect of liposomes and PBS seen in the lungs (Fig. 4.8) is probably related to the greater uptake of liposomes by spleens than lungs. 166 Despite the specific formulation of the liposomes used in these experi ments, it is known that the major proportion of liposomes is still taken up by the liver and the spleen (Poste et al., 1982). A study on experi mental chemotherapy of murine tuberculosis (Vladimirsdy and Ladigina, 1982) showed that liposomal encapsulation of streptomycin decreased the bacillary counts in the spleen but not the lungs. This finding was a bit surprising in view of reports of enhanced in vivo tumoricidal, antiviral and microbicidal activity after encapsulation in liposomes. There have been reports of increased activity after lipo somal encapsulation of anti leishmanial drugs like antimonials (Alving et a l . , 1978) in experimental visceral leishmaniasis; and antifungal drugs like amphotericin B in experimental Candida albicans infections (Fraser-Smi th et a l ., 1983; Lopez-Berestein et al., 1983). This seems to be due to the lowered toxicity of the drugs when encapsulated within liposomes thus enabling higher doses to be given. There have also been reports of the enhancement of the antiviral activity of liposome- encapsulated ribavirin against Rift Valley Fever virus infection in mice (Kende et al. , 1985) and of protection of mice against systemic herpes simplex type 2 infection by liposome encapsulated muramyl tripeptide (Koff et al., 1985). One possible explanation for the lack of enhancement of IFN-X activity reported in this Chapter could be related to the mode of activity of IFN-V. It has been shown that IFN-V reacts with cell surface receptors on murine macrophages in a specific way which initiates macrophage activation for tumoricidal activity (Celada et al., 1984). Eppstein et a l . (1985) has also reported that the antiviral activity of liposome 167 encapsulated murine IFN-Y in an in vitro system is mediated by leakage of IFN-Jf from the liposomes and subsequent interaction with a cell membrane receptor. The assumption that interaction of IFN-Y with cell- surface receptors is essential for macrophage activation could explain the lack of enhancement by liposomes cited in this thesis. IFN-V encapsulated in liposomes internalized by macrophages would not have been allowed to bind with the cell-surface receptors, thereby prevent ing the initiation of macrophage activation. It has also been reported that the in vivo effect of IFN-Y in inhibiting herpes simplex virus-2 murine infections is unchanged by encapsulation in liposomes (unpublished results cited in Eppstein et al. 1985). There is a further possible reason for the failure to detect any enhancement of effects on the in vivo growth of M. tuberculosis in mice. It could be that the lowest dose size of IFN-Y tested (1000 units) had already maximally activated macrophages thus masking any additional effects of liposomes. On retrospective consideration, a much lower dose of IFN-Y should have been used for encapsulation in liposomes. Recently it has also been documented that an inhibition instead of an enhancement of effect can been obtained in vitro with liposomes. Gilbreath et al. (1985) reported a differential inhibition of macrophage microbicidal activity by liposomes in an in vitro infection of murine resident peritoneal macrophages with L. tropica. They found that LKs induced an enhanced microbicidal activity against the protozoan amastigotes but that liposomes abrogated that enhancement. The effect was differential and depended on the type of liposome involved. MLV liposomes composed of PC and PS (molar ratio, 7:3) with and without LK, 168 completely abrogated the LK-induced enhancement of killing but not tumour cytotoxicity. In a subsequent publication (Gilbreath et al., 1985a) concluded that the inhibition of LK-enhanced killing was probably due to the interference of liposomes with one of the early stages of macrophage activation. 169 CHAPTER 5 THE IMMUNOMODULATING EFFECT OF INTERFERON-GAMMA IN COMBINATION WITH ISONIAZID AND WITH RIFAMPICIN ON MURINE M. TUBERCULOSIS INFECTION The next stage of the experiments involved the investigation of the effects of IFN-X in combination with antituberculous drugs. The effects of IFN-X in combination with isoniazid and with rifampicin were examined in an acute infection with M. tuberculosis in BALB/c mice. 5.1 Assessment of interferon-gamma and isoniazid alone and in combination on M. tuberculosis infection in BALB/c mice. 5.1.1 The effect of interferon-gamma and isoniazid when interferon- gamma is administered before and after infection of BALB/c mice with M. tuberculosis In this experiment, 110 male BALB/c mice, 18-20 g in weight, were randomly allocated into 4 groups. One group was given 2000 units of IFN-Y every three days with the first dose 2 days prior to infection; the second group was given a daily dose of isoniazid (25 mg kg starting a day after infection; the third group received both IFN-X" and isoniazid in the same schedule and dosages, and the last group comprised untreated controls. All mice were infected intravenously with a 1:1 dilution of the frozen inoculum. Viable counts set up at infection showed that the infective dose was 6.6 X 10^ cfu per mouse. The number of viable organisms in both organs were determined on Days +1, +2, +3, +4, +6 and +9 post 170 infection. Four mice were sacrificed per time point. The results of the mean viable counts in the lungs are shown in Figure 5.1. For clarity, the s.e. of the means are not depicted on the Figure but are shown in Table 5A.1 in Appendix 1. The mean viable count of 4 M. tuberculosis in the lungs was 7.2 x 10 one hour after infection. Over the next three days there was a fall in viable counts in the lungs. This was a consistent finding in all the experiments performed using this protocol of animal infection. In this experiment, the mean count 3 of the controls fell to reach 2.6 x 10 cfu on Day +3. After Day +3 the mean viable lung counts started increasing exponentially until they 4 reached 4.5 x 10 on Day +9. In the group that had been given IFN-fc there was an initial sharper decrease in mean viable counts over the 3 first two days post infection to 1.87 x 10 cfu. This levelled off over 4 Days +2 and +3 before increasing to 2.03 x 10 on Day +9. This was reflected in the growth curve of the IFN-tf treated group which was para llel to that of the untreated controls after Day +4. Thus, it seemed that IFN-tf treatment resulted in an early decrease in mean viable counts in the lungs, and this effect did not increase further with time. This was consistent with the results obtained in the earlier experiments where different dose sizes of IFN-V were investigated (Chapter 4, Fig. 4.4 and Fig. 4.6). Isoniazid was first given one day post infection and the effects were evident by Day +3. From Day +4 however, the decrease in mean viable counts became progressively larger reflecting an increasing effect with o time until by Day +9 the mean count was only 3.13 x 10 cfu. The group of mice that had received both isoniazid and IFN-if also showed a similar Mean log ^viable mycobacteria per lung 3.0“ 2.0J 2.5“ 3. 5“ 4.0“ 4.5“ 5.0“ 7pu al snai fo a+ () o eeutetd (#). untreated or were (■); fromDay+1 isoniaziddaily plus +7 IFN-Jf from Day +1; ♦) ( Days -2,onisoniazid daily +1,+4, +7; Each point represents the mean loglO cfu per lung ofloglOcfu4 thelung per mean mice.represents point Each BALB/c mice when IFN-lfBALB/cwhen mice after andadministeredbefore was tuberculosis inlungsoftheofgrowth M.on combination in isoniazidaloneandandinterferon-gamma of Effect 5.1Fig. IFN-y was given atIFN-y given was e os adamnsee F-T() on-DaysIFN-JTand+1,-2,administered+4 and per mouse (A) infection. Mice were infected with 6.6 x 105 viable organisms6.6 infected105x with viable were Mice infection. 2000 u per mouse and isoniazidat25andpermouse u mg/kg. as fe infection after Days 171 172 trend except that there was a greater decrease in mean viable counts during the first day after adminstration of IFN-X. Thereafter the curves were virtually parallel to each other. Overall, there was a more rapid decrease in mean viable counts in this group as compared with the isoniazid group. Figure 5.2 depicts the results of the mean viable counts in the spleens of the various experimental groups. The means + s.e. of this experiment are given in Table 5A.2 in Appendix 1. The Figure shows that the mean 4 viable count in the spleens was 7.7 x 10 cfu at 1 hour after infection and rose rapidly to 3.65 x 10^ by Day +9. In the group of mice treated with 2000 units of IFN-K there was an initial period of 2 days where there was hardly any increase in the mean viable count but after that period, the mean viable count started increasing in an exponential manner forming a growth curve parallel to that of the control. Again this was consistent with the results obtained in the lungs in this experiment as well as in the previous experiments described in Chapter 4. As the isoniazid was only given a day after infection, its bacteri cidal effect was only expected to become evident on or after Day +2. By Day +2, the mean viable count in the spleen had already been reduced. This effect increased with time (and doses) until by Day +9 the mean 3 viable counts in the spleen were reduced to 3.78 x 10 cfu. The same trend was seen in the mean viable spleen counts of the group of mice that had received both isoniazid and IFN-V, except that there was no initial rise in the spleen count over the first day post infection. Hence, there was an initial period of stasis followed by a fall in the counts. Thereafter, the growth curve was parallel to that of the group on isoniazid alone. Overall, there was a more rapid decrease in mean 7.0 “ 1 3 .0 J |------1------1 I I I I 01 2 31(56789 Days after infection Fig. 5.2 Effect of interferon-gamma and isoniazid alone and in combination on the growth of M. tuberculosis in spleens of BALB/c mice when IFN-Y was administered before and after infection. Mice were infected with 6.6 x 105 viable organisms per mouse and administered IFN-Y (A) on Days -2 , + 1 , +4 and + 7; daily isoniazid (♦) from Day+l; IFN-Y on Days -2, +1 , +4, +7 plus daily isoniazid from Day+l (■); or were untreated (•). IFN-Y was given at 2000 u per mouse and isoniazid at 25 mg/kg. Each point represents the mean logio cfu per spleen of A mice. 174 viable spleen counts in this group when compared with the group receiv ing isoniazid alone. Analysis of variance of both sets of results was performed by a 3-way ANOVA (Table 5.1). The main effects are isoniazid (H), interferon (IFN) and time (Days). In both organs, the main effects were all highly significant. The first-order interactions showed a highly significant interaction of isoniazid and days which could be explained by referring to Fig. 5.1 and Fig. 5.2 which showed as might be expected, that the counts for mice treated with isoniazid decreased from the untreated mice increasingly as the experiment proceededi There was no interaction of isoniazid and IFN which showed that statistically, the effects of isoniazid and IFN were independent and not additive. TABLE 5.1 THREE-WAY ANALYSIS OF VARIANCE OF THE EFFECTS OF INTERFERON-V, TIME AND ISONIAZID ON THE GROWTH OF M. TUBERCULOSIS IN BALB/C MICE Source of LUNG SPLEEN variation DF MS F MS F *** *** Isoniazid(H) 1 9.600 241 31.380 3826 *** *** IFN-S 1 0.820 20.6 0.340 41.5 *** *** Days 1 0.260 6.6 0.070 8.8 H x IFN-V 1 0.000 0.0 0.000 0.0 *** *** H x Days 1 2.960 74.6 5.168 630 IFN-Jr x Days 1 0.020 0.6 0.007 0.8 H x IFN-* x Days 1 0.020 0.5 0.013 1.5 Replicate error 60 0.040 0.008 *** p < 0.001 175 5»1.2 The effect of interferon-gamma and isoniazld when interferon- gamma was administered both before and after infection, and only after Infection of BALB/c mice with M. tuberculosis In this experiment, the effect of the timing of administration of IFN-Jf alone and in combination with isoniazid on M. tuberculosis infection was examined. In the first part of the experiment, IFN-V was given on Day-1 and Day+1 and daily isoniazid was started on Day 0. In the second part, daily isoniazid was started on Day 0 and IFN-tf was given on Day+5 and Day+7. 96 male BALB/c mice, were randomly allocated into 6 groups. All mice were infected intravenously with 3 x 10^ cfu of M. tuberculosis per mouse on Day 0. The first group was given IFN-V on Day-1 and Day+1; the second, IFN-Jf on Day+5 and Day+7; the third, daily isoniazid starting from Day 0; the fourth, IFN-V on Day-1 and Day+1 and daily isoniazid on Day 0 and Day+1; the fifth, daily isoniazid from Day 0 and IFN-V on Day+5 and Day+7. The sixth group was the untreated controls. IFN-V was used at a dose of 2000 units per mouse and isoniazid at 25 mg kg ^ . The number of viable organisms in lungs and spleens were determined on Days 0, +1, +2, +5, +7, +8 and +9 after infection. Four mice were sacrificed per group for each time point. The results of the mean viable counts in the lungs and spleens on Days 0, +1 and +2 are shown in Figures 5.3 and 5.4 respectively. The mean viable count + s.e. are detailed in Tables 5A.3 and 5A.4 in Appendix 1. Mean log^viable mycobacteria per lung Each point represents the mean logio cfu per lung of4mice.lung thecfu logio per represents mean point Each 1ad+, al snai () rmDy0 IFN-JT' from Day 0, (♦) isoniazid daily and-1 +1, -1onDays mice in the first two days after infection, when IFN-)finfection, firsttwoafter the when daysin mice given was n 1 ls snai fo a () o eeutetd (•). oruntreated were (A), from isoniazidDay 0 plusand +1 ibeognssprmueo a n ie F-f( ) onDays andIFN-lfongiven 0per Day organismsmouse viable (O IFN-/ was given at 2000 u per mouse and isoniazid at 25atisoniazidmg/kgmg.and2000 per givenatumouse IFN-/ was before and after infection. Mice were infected with 3 x 1053 x infected with were Mice infection.after beforeand in combination on the growth of M. tuberculosis in lungs of BALB/cof tuberculosis inlungs theM. of ongrowth incombination Fig. 5.3 Effect of interferon-gamma and isoniazid alone andaloneisoniazidand ofinterferon-gamma Effect Fig. 5.3 as fe infection after Days 176 , IFN-ygivenat isoniazid2000 at25andperwas umouse mg/kg. Mean log10viable mycobacteria per spleen a gvnbfr n fe ifcin Mc ee infected Micewere with givenbefore infection.and afterwas Each point representspoint theEach spleenloglOper ofcfu4 mean mice. as-,+ pu al snai rmDy0 A, orwere untreated. daily plusfrom (A), Daysisoniazid-1,Day 0 +1 3organisms105xviable pergivenIFN-ifmouse,on and0 Day (O) nDy 1 n 1 diyioizd ♦ fo a , IFN-if from0,Day on and(♦) Days isoniazid daily -1 +1, on ABcmc i the inBALB/c firstinfection, twomice after daysIFN-lf when incombinationon. the spleens in tuberculosisgrowthofM. of ofinterferon-gammaisoniazid Effectalone andand Fig. 5.4 177 + IFN +H Control H IFN 178 The mean count in the lungs one hour after infection was 2.6 x 10 cfu. As seen in all the previous experiments, there was a fall in viable counts over the first two days after infection. There was no obvious effect of isoniazid on the growth of M. tuberculosis in the lungs in the first two days. The mice that had been given IFN-Y and IFN-Y together with isoniazid showed an initial sharper decrease in viable counts than the untreated and isoniazid alone groups. 4 In the spleens, the viable count one hour after infection was 1.5 x 10 cfu. Over the next two days there was an increase in viable count in the untreated controls to reach 1.2 x 10^ cfu. The mean viable counts in the group treated with IFN-Y was lower than that of the untreated controls. On Dayf 1, there was no obvious difference between the counts of the groups that had been given IFN-Y, isoniazid, or isoniazid plus IFN-&. However, all three treatment groups had a lower mean spleen count than the untreated controls. By Day+2, the mean counts in the isoniazid groups were evidently lower than the group given IFN-Jf alone, but there was no difference between the isoniazid and the isoniazid plus IFN-Y group. Statistical analysis of the results was performed by a 3-way analysis of variance. The results are shown in Table 5.2. 179 TABLE 5.2 THREE-WAY ANALYSIS OF VARIANCE OF THE EFFECTS OF TIME, ISONIAZID AND INTERFERON-8 GIVEN BOTH BEFORE AND AFTER INFECTION OF BALB/C MICE WITH M. TUBERCULOSIS Source of LUNG SPLEEN variation DF MS F MS F * ** IFN-Y 1 0.671 7.2 0.076 9.5 *** Isoniazid (H) 1 0.075 0.8 0.316 39.5 Days 1 0.116 1.2 0.0004 0.05 Days x IFN-Y 1 0.014 0.2 O'. 003 0.4 * Days x H 1 0.248 2.7 0.042 5.3 ** IFN-Y x H 1 0.022 0.2 0.070 8.8 Days x H x IFN-Y 1 0.001 0.01 0.004 0.5 Replicate error 24 0.093 0.008 *** p < 0.001 ** p < 0.01 * p < 0.05 Among the main effects, IFN-Y and isoniazid were highly significant in the spleen and IFN-Y was significant at the 5% level in the lung. These results confirmed that IFN-Y given a day before and a day after infec tion significantly lowered the mean viable count in the spleen and lungs in the first two days of infection. The effect of isoniazid was highly significant in the spleen but not in the lung in the first two days after infection. There was a significant first-order interaction between isoniazid and IFN-JT in the spleen. However, this was not due to an additive effect between isoniazid and IFN-Y. Figure 5.4 showed that even though IFN-Y decreased the counts it did not increase the rate at which isoniazid killed the bacilli. 180 The results of the second part of the experiment investigating the effect of IFN-)| administered on Day+5 and Day+7 alone and in combination with isoniazid are shown in Figures 5.5 and 5.6. Figure 5.5 shows that mean counts of M. tuberculosis in the lungs from Day 0 to Day+9 with the full details in Table 5A.5 in Appendix 1. In 4 the untreated controls, the mean count was 2.6 x 10 cfu per lung one 3 3 hour after infection and remained in the range of 6.0 x 10 to 8.8 x 10 cfu until Day+5. The mean viable count then rose steadily to reach 2.5 4 x 10 cfu on Day+9. In the group given IFN-Y on Days +5 and +7, there was a decrease in the mean count on Day+7 followed by a steady increase parallel to the controls. Isoniazid rapidly caused a decrease in the 2 mean viable counts to reach 1.6 x 10 cfu per lung on Day+9. The addition of IFN-& on Day+5 and +7 to the isoniazid treatment did not appear to have had much effect on the counts. The corresponding mean viable counts in the spleens are shown in Figure 5.6 and the full details in Table 5A.6 in Appendix 1. A similar trend was seen in the spleen. The mean viable count in the untreated controls rose steadily to reach 3.0 x 10^ cfu per spleen on Day+9. Administra tion of IFN-Y on Day+5 and Day+7 caused a slight decrease in the mean count when compared with the controls. Isoniazid given daily caused the 4 mean viable count to decrease rapidly to reach 4.1 x 10 cfu on Day+9. The addition of IFN-Y on Day+5 and +7 to the isoniazid treatment did not cause any further decrease in viable counts. Days after infection Fig 5. 5 Effect of interferon-gamma and isoniazid alone and in combination on growth of M. tuberculosis in lungs of BALB/c mice when IFN-Y was .administered on Days +5 and +7 and isoniazid from Day 0. Mice were infected with 3 x 103 viable organisms per mouse on Day 0 and given IFN-Y (O) on Days +5 and +7, daily isoniazid (♦ ) from Day 0, daily isoniazid from Day 0 plus IFN-Y on Days +5 and +7 (A ), or were untreated ( • ) . IFN-Y was given at 2000 units per mouse and isoniazid at 25 mg/kg. Each point represents the mean log.^ cfu per lung of 4 mice. 6.5 -| Control ^-OlFN 0c) _0> a 6.0- V. u ra n 5. 0 _ uo >N E XJ ro U.5- O) o a.o- ac3 Days after infection Fig 5.6 Effect of interferon-gamma and isoniazid alone and in combination on growth of M. tuberculosis in spleens of BALB/c mice when IFN-lf was administered on Days +5 and +7 and isoniazid from Day 0. Mice were infected with 3 x 105 viable organisms per mouse on Day 0 and given IFN-y (O) on Days +5 and +7, daily isoniazid (4 ) from Day 0, daily isoniazid from Day 0 plus IFN-T on Days +5 and +7 (A), or were untreated ( •). IFN-X was given at 2000 units per mouse and isoniazid at 25 mg/kg. Each point represents the mean log-jQ cfu per spleen of 4 mice. 183 The lung and spleen counts on Days +5, +7, +8 and +9 were examined statistically by a three-way analysis of variance (Table 5.3). TABLE 5.3 THREE-WAY ANALYSIS OF VARIANCE OF THE EFFECTS OF TIME, ISONIAZID AND INTERFERON-GIVEN AFTER INFECTION OF BALB/C MICE WITH M. TUBERCULOSIS Source of LUNG SPLEEN variation DF MS F MS F IFN-* 1 0.490 2.9 0.020 2.5 *** *** Isoniazid (H) 1 30.37 178 73.4 9175 * Days 2 0.085 0.5 0.035 4.3 Days x IFN-V 2 0.073 0.4 0.005 0.6 *** *** Days x H 2 1.750 10.2 0.391 49 H x IFN-V 1 0.140 0.8 0.016 2 H x Days x IFN-^ 2 0.010 0.06 0.009 1 Replicate error 36 0.171 0.008 *** p < 0.001 * p < 0.05 These results showed that among the main effects, isoniazid was highly significant in both lungs and spleens, but IFN-V did not have any signi ficant effect in either organ. There was a significant first-order interaction between isoniazid and time in both the lungs and the spleen which was obviously due to the fact that the counts for mice treated with isoniazid decreased from the untreated mice increasingly as the experiment progressed (Figures 5.5 and 5.6). Thus, it appears that IFN-V administered on Day+5 and Day+7 did not have any significant effect on the growth of M. tuberculosis in both organs. This was indep endent of either time or isoniazid. 184 5.2 The effect of Interferon-gamma and rifamplcin alone and In combination on M. tuberculosis Infection In BALB/c mice In this experiment, 70 male BALB/c mice, 18-20 g in weight, were randomly allocated into 4 groups. The first group was given 2000 units of IFN-^ per mouse on Days -2 and +1; the second group was given a daily dose of rifampicin at 25 mg kg with the first dose 20 minutes after infection; the third group received both IFN-V and rifampicin in the same dosages and schedules and the last group consisted of the untreated controls. All mice were infected intravenously with 4.8 x 10^ cfu M. tuberculosis per mouse. The mean viable counts were monitored on Days +1, +2 and +3 with 4 mice per time point. The results of the mean viable counts in the lungs are shown in Figure 5.7 with the details of the means + s.e. in Table 5A.7 (see Appendix 1). As the mean counts were only monitored over the first three days post infection, Figure 5.7 depicts decreasing counts only. The mean viable 4 counts of the control untreated group decreased from 5.1 x 10 to 3 2.8 x 10 cfu per lung on Day +3. The group that had been treated with IFN-tf showed a sharper decrease over the first day and then a decrease parallel to that of the untreated group. However, the rifampicin group showed a much greater decrease over the first day which progressively 2 became larger, to a count of 4.5 x 10 cfu. The group that had been treated with both IFN-V and rifampicin showed an almost identical decrease in mean viable counts to the rifampicin group. Figure 5.8 depicts the results of the mean viable counts of the spleens of the various experimental groups. The corresponding means and 185 Days after infection Fig. 5.7 Effect of interferon-gamma and rifampicin alone and in combination on the growth of M. tuberculosis in lungs of BALB/c mice. Mice were infected with 4.8 x 105 viable organisms per mouse and given IFN-Jf ( A ) on Days -2 and +1, daily rifampicin (♦) from Day 0, IFN-/ on Days -2 and +1 plus daily rifampicin from Day 0 ( ■) or were untreated (•). IFN-)f was given at 2000 u per mouse and rifampicin at 25 mg/kg. Each point represents the mean logio cfu per lung of 4 mice. 186 Fig. 5.8 Effect of interferon-gamma and rifampicin alone and in combination on the growth of M. tuberculosis in spleens of BALB/c mice. Mice were infected with 4.8 x 105-viable organisms per mouse and given IFN-JT (A) on Days -2 and +1, daily rifampicin from Day 0 (♦), IFN-V on Days -2 and +1 plus daily rifampicin from Day 0 (■), or were untreated (•). IFN-if was given at 2000 u per mouse and rifampicin at 25 mg/kg. Each point represents the mean logio cfu per spleen of 4 mice. 187 standard errors are shown in Table 5A.8 in Appendix 1. The mean viable 4 5 spleen count rises from 6.2 x 10 to 1.7 x 10 cfu per spleen in the untreated control group. In the group treated with IFN-Y , the mean viable count showed a much slower increase over the first day but then gradually increased to reach 1.1 x 10^ cfu. This again reflects the generally observed effects of IFN-Y in the previous experiments. As seen with the mean lung counts, rifampicin was remarkably efficacious in lowering the mean count. Again, the effect became progressively larger 4 and within three days the mean count was reduced to 1.6 x 10 cfu. There was hardly any difference between the group receiving both IFN-& and rifampicin and the rifampicin group. This experiment showed that treatment of mice with IFN-tf resulted in a considerable reduction in mean viable counts in both lungs and spleens. However, the combination of IFN-Y and rifampicin did not appear to increase the bactericidal activity of rifampicin. Analysis of variance of both sets of results was performed by a 3-way ANOVA. The results of this analysis are shown in Table 5.4. The main effects in this ANOVA are rifampicin (Rf), interferon (IFN) and time (Day). All the main effects were significant in both lungs and spleen. There was a significant first-order interaction of rifampicin and time in both organs showing that as the experiment progressed, the viable counts of mice treated with rifampicin decreased from the untreated mice increasingly. There was a significant interaction of rifampicin and IFN-t in the spleen. This can be explained by referring to Figure 5.8 which shows that even though IFN-Y reduced the viable count it did not increase the rate at which rifampicin killed the tubercle bacilli. 188 TABLE 5.4 THREE-WAY ANALYSIS OF VARIANCE OF THE EFFECTS OF INTERFERON-GAMMA, RIFAMPICIN AND TIME ON M. TUBERCULOSIS INFECTION IN BALB/C MICE Source of LUNG SPLEEN variation DF MS F MS F *** *** Rifampicin (Rf) 1 3.033 132 4.075 680 * *** IFN-Y 1 0.121 5.3 0.121 20 *** *** Days 2 2.025 88 0.220 37 ** Rf x IFN-Y 1 0.024 1.0 0.079 13.2 IFN-Y x Days 2 0.006 0.3 0.009 1.5 ** *** Rf x Days 2 0.225 9.8 0.550 92 Rf x Days x IFN-Y 2 0.007 0.3 0.007 1.2 Replicate error 36 0.023 0.006 *** p< 0.001 ** p < 0.01 * p < 0.05 5.3 Discussion When two antibacterial agents are used in combination, the resulting effect usually falls into one of four categories-synergism, addition, indifference and antagonism (Hami11 o n - M i H e r , 1985). The term ’synergism’ has been used where the combined activity exceeds the sum of the separate activities. ’Addition’ has been used to describe the phenomenon where the combined activity gives an effect greater than that of the single most active component, while the term ’indifference' has been used where individual drugs in combination showed no increased or decreased activity. Where the combination results in a total effect smaller than that produced by the more active component, the term 189 'antagonism* has been used. The effect of the addition of IFN-& to isoniazid was examined with various combinations of both agents. In the first experiment (see 5.1.1), IFN-& was administered every three days with the first dose on Day-2 and isoniazid was given daily from Day+1. The results of this experiment (Figures 5.1 and 5.2) showed that there was a suggestion of an 'addition' effect of IFN-lf on the bactercidal activity of isoniazid. However, statistical analysis revealed no significant effect of IFN-V on isoniazid activity in both organs. In the second experiment (5.1.2), isoniazid was given daily from Day 0 and IFN-tf was either given on Days -1, +1 or on Days +5, +7. When IFN-fc was given both before and after infection, there was again no statis tically significant effect of IFN-& on the bactericidal activity of isoniazid. IFN-ft administered on Day+5 and Day+7 to mice did not have a significant effect on bacillary growth in both organs. Similarly, IFN-# given on Day+5 and Day+7 to a group of mice treated with isoniazid from DayO did not enhance the bactericidal effect of isoniazid. The appearance of the growth curves (Figs. 5.5 and 5.6) suggest that the effects of IFN-^f and isoniazid are 'indifferent' when IFN-& is administered on Day+5 and +7. Statistical analysis confirmed that IFN-fc given after infection did not have any significant effect on the bactericidal activity of isoniazid. The results of the combination of rifampicin with IFN-)( also did not / show any evidence of enhancement of bactericidal activity of rifampicin by IFN-Jf. In this experiment, IFN-if was administered before infection 190 (Day-2) and rifamplcin on Day 0. The growth curves (Figures 5.7 and 5.8) suggest that the effects of IFN-& and rifampicin are ’indifferent* and this was confirmed statistically. The experiments reported in this Chapter showed that IFN-tf had a significant effect on the growth of M. tuberculosis in the organs of mice when it was administered before infection but not if it was administered five days after infection. Also, there was no significant effect of IFN-lf on the activity of isoniaziti irrespective of whether it was given before or after isoniazid treatment. Similarly, IFN-^f did not increase the bactericidal effect of rifampicin when it was given before rifampicin. It has been observed that immunomodulating agents must be administered very early in the course of infection if they are to be effective (Bicker et a l . , 1979; Kokoshis et al., 1978; Block et al., 1978). The best results have generally been obtained if a compound is administered at least 24 hours before infection. The results presented in this Chapter and the previous Chapter are consistent with these findings. There have been reports of synergistic effects of combinations of immunomodulators and chemotherapeutic agents. In some of these reports, the timing and mode of administration of both agents largely affected the outcome of the combination. In one such report, Connell et al. (1985) demonstrated the synergistic effect of combinations of acyclovir and recombinant interferon-alpha against a lethal murine infection of Herpes simplex virus Type I which depended on the mode and timing of adminstration of both agents. Simultaneous parenteral administration of 191 both agents were only synergistic at certain dose combinations whereas sequential administration of parenteral IFN followed by oral acyclovir was always synergistic. Another example of the importance of sequence of administration was shown by Scott (1979) who noted synergism in a murine tumoricidal model if C . parvum was given before cyclophosphamide but not in the reverse order. However, the results described in this Chapter of the combination of IFN-^ and isoniazid did not seem to show any dependence on sequence of administration of either agent. Antibiotics are most effective in the presence of an adequate host immune response (Weinstein and Dalton, 1968). The importance of the immune response in controlling infections is emphasized by the increase in the number of infections, especially opportunistic infections, that occur in immunocompromised patients (Bodey, 1975; Atkinson et al., 1974; Kreger et al., 1980). It has been reported that several antibiotics are immunosuppressive. The immunosuppressive properties of chloramphenicol have been documented (Weisberger et al., 1964; Weisberger et al., 1966) and have been attributed to inhibition of protein synthesis. Similarly, the tetracyclines also inhibit protein synthesis and have also been shown to be immunosuppressive (Munster et al., 1977). There have been conflicting reports about the immunosuppressive properties of rifampicin. It has been cited that rifampicin caused immunosuppression of cellular and humoral responses in animal studies (Paunescu, 1970; Grassi and Pozzi, 1972; Dajani et al., 1973). Simil arly, depressed circulating T cells, suppressed in vitro PHA prolifera tive responses and diminished PPD skin reactivity responses have been detected in tuberculosis patients receiving rifampicin (Mukerjee et al., 192 1973; Ruben et a l . , 1974; Gupta et al., 1975). However, Humber et al. (1980) have shown no effect of rifampicin on humoral or cellular responses In tuberculosis patients and contacts in a controlled double blind study. Miller (1978) also reported that long-term therapy (mean duration 12.7 months) with rifampicin in tuberculosis patients did not suppress the secondary antibody response to influenza vaccination. Goldstein et al. (1976) noted that rifampicin therapy of 4-24 months in tuberculosis patients caused a depression of PHA but not Con A or pokeweed mitogen proliferative responses, and no alteration of in vivo and in vitro responses to PPD. They concluded that since a favourable therapeutic outcome was achieved, clinically, rifampicin is not significantly immunosuppressive. It is well established that depressed cell mediated immune responses occur in some tuberculosis patients (Humber et al., 1980). There is the possibility that the use of an immunomodulating agent in combination with antituberculous drugs could increase the the efficacy of the antituberculous drugs by modulating the immune response. Bicker et al. (1979) used murine models of experimental bacterial infections to inves tigate the effects of azimexon (see section 1 .3.3), chloramphenicol and cyclophosphamide. They showed that administration of cyclophosphamide alone increased the mortality among mice infected with C. albicans and Ps. aeruginosa, but the addition of azimexon reduced the mortality. Azimexon on its own had no significant effect on the survival rate in acute E. coli infection, however, a synergistic effect of azimexon and chloramphenicol, a bacteriostatic antibiotic, was demonstrated. Gillisen (1985 ) also explored the possibility of improving the 193 efficiency of antibiotics in the immunocompromised host by concomitant administration of immunomodulators. Azimexon and immunoferon (a glucopeptide) were each given to mice infected with Staphylococcus aureus and immunosuppressed by cyclophosphamide or cortisone. The results showed that low doses of azimexon stimulated the immune response while higher doses were inhibitory; in contrast, low doses of immunoferon inhibited the response while higher doses stimulated it. These variable responses illustrate the difficulty in achieving the optimal dose with immunomodulators. 194 CHAPTER 6 IN VITRO ASSESSMENT OF THE EFFECTS OF INTERFERON-GAMMA 6.1 Preliminary experiments 6.1.1 Characterization of monolayers The monolayers were stained by a modified Wright's stain and for non specific esterase (see section 2.9.3). Both staining methods confirmed that the monolayers were composed of macrophages (Fig. 6.1A and 6.IB). 6.1.2 The effect of saponin on the viable counts of L. monocytogenes and M. microti L. monocytogenes: A suspension of L. monocytogenes containing approximately 5 x 10^ organisms ml ^ was diluted 1 in 10 in duplicate with either distilled water or 2.5 % saponin (w/v in distilled water). All the tubes were incubated at 37°C for 20 minutes and left at room temperature for 2 hours before being sampled for the number of viable organisms. The results of the viable counts are shown in Table 6.1. The results showed that saponin did not grossly affect the viability of L. monocytogenes under the conditions tested. On the contrary, the counts of the bacteria suspended in saponin were slightly higher than in distilled water. 195 Fig. 6.1A Wright’s (Diff-Quik) staining of macrophage monolayers established in tissue culture 8-chambered slide for 2 days. Magnification xAOO. Fig. 6.IB Non-specific esterase staining of macrophage monolayers established in tissue culture 8-chambered slide for 2 days. Magnification x400. 196 TABLE 6.1 THE EFFECT OF SAPONIN ON VIABLE COUNTS OF L. MONOCYTOGENES Suspended in : Viable listeria per ml Distilled water A .45 x 105 Distilled water 2.95 x 105 2.5 % saponin 7.65 x 105 2.5 % saponin 4.25 x 105 M. microti: In this experiment, a suspension of M. microti containing approximately 2 x 10 ^ organisms ml * was used. 100 )il of this bacterial suspension was dispensed into duplicate tubes containing 900 jil of either distilled water, 1 % saponin or 2.5 % saponin. All the tubes were then incubated at 37°C for 20 minutes, left at room temperature for 2 hours after which they were briefly sonicated and sampled for the number of viable myco bacteria (see section 2.8.2). The results are shown in Table 6.2. These results showed that neither 1 % nor 2.5 % saponin had any gross effect on the viable counts of M. microti after 2 hours when compared with the controls. As observed in the experiment with L. monocytogenes, the viable counts obtained after suspension in saponin solutions were slightly higher. This was probably due to the surfactant activity of saponin which could have prevented the M. microti from reclumping after sonication. 197 TABLE 6.2 THE EFFECT OF SAPONIN ON VIABLE COUNTS OF M. MICROTI Bacteria suspended in: Viable mycobacteria per ml Distilled water 1.06 X 105 Distilled water 9.6 X 104 1 % saponin 1.47 X io5 1 % saponin 1.59 X 105 Ul 2.5 % saponin 1.37 X I—* o 2.5 % saponin 1.53 X io5 6.2 Listericidal Assay 6.2.1 The effect of dose size of interferon-gamma on the listericidal activity of peritoneal macrophages after previous exposure for 24 hours Duplicate monolayers in 16 mm Linbro wells were preincubated for 24 hours with either BMM or BMM containing 10, 100 or 1000 units of IFN- ml The monolayers were then infected with L. monocytogenes at a bacteria:macrophage ratio of 1:10. The viable counts immediately after the period allowed for phagocytosis (T q ) and four hours later (T^) of the monolayers are depicted in Figure 6.2. From the Figure, the control 2 monolayers showed an increase in viable count from 9.4 x 10 to 5.8 x 3 10 cfu in 4 hours, a 617 % increase of the Tq count. Treatment with 10 units resulted in a slight increase in viable count to 134 % of the base count. 100 units caused a decrease in the T^ counts to 96 % of the Tq count while 1000 units resulted in a decrease to 70 %. Monolayers that or. ahmnlyrws infected monolayer Each waswith hours. activity of peritoneal macrophages after prior exposure forafterexposureofperitonealpriormacrophages activity 2 Fig. for Viable listeria per monolayer monolayers after infectionafter monolayers 40 6.2 minutes and each bar represents theeachs.eandbarcfuof mean minutes + feto oe sizeofinterferon-gammaofdoselistericidalonEffect nt Itreo / ml / Interferon Units To 198 ( Q ) andafter 2.4 x 104 4 hours cfu listeriacfu T 4 ( 24 0>- 199 had been treated with IFN-Y appeared to show a dose-related listericidal activity. Analysis of variance was performed on the viable counts of the monolayers that had been treated with 10, 100 or 100 units of IFN-Y. The results (Table 6.3) showed that linear regression of dose (IFN-)f concentration) on time was highly significant confirming the existence of dose-related listericidal activity. TABLE 6.3 ANALYSIS OF VARIANCE OF THE EFFECT OF INTERFERON-GAMMA DOSE SIZE AND TIME ON LISTERICIDAL ACTIVITY Source of variation DF MS F Time (Hours) 1 0.0005 0.3 * Dose 2 0.0152 7.6 * Time x Dose 2 0.0207 10.4 ** Linear dose regression 1 0.0414 20.7 Replicate error 6 0.0020 ** p < 0.01 * p < 0.05 200 6.2.2 The effect of varying the dose size and pretreatment period of interferon-gamma on listericidal activity of peritoneal macrophages In this experiment, the monolayers were pretreated with 0, 100, 1000 units IFN-Y ml ^ for 24 hours or 0, 10, 100, 1000 units IFN-Jf ml ^ for 48 hours. The results are shown in Figure 6.3. As was seen in the previous experiment, both 100 and 1000 units of IFN-# activated the macrophages to be listericidal after previous exposure for 24 or 48 hours. Pretreatment with 10 units IFN-* for 48 hours did not activate for listericidal activity. The % decrease of viable count over 4 hours for both treated groups was slightly greater in the 24 hour than 48 hour group. This difference in the magnitude of the response seemed to be related to the ability of the untreated monolayers in the 48 hour pretreatment group to allow extensive proliferation of the bacilli. In the 48 hour pretreatment monolayers the % increase in viable count over 4 hours was 817 % compared to an increase of only 332 % in the 24 hour monolayers. Thus, pretreatment of the monolayers for 48 hours did not result in an increased magnitude of listericidal activity which was probably directly related to the decreased microbicidal activity of macrophages the longer it has been cultured in vitro. 201 li 3x10 4 p 2x10 A A 4 A i. 1x10 □ T o • H r , , J2 A o A c 5x1 03 o A E ^ A u 0) a JT. to T A / £ A A l/J / 1x1 03 A / ?- _a> * 1 A / y A ✓ A y .2 / > A y ✓ / c A / y (0 / y Fig* 6.3 Effect of varying the dose size and pretreatment period of interferon-gamma on listericidal activity of macrophages. Monolayers were preincubated with IFN- for either 24 or 48 hours and infected with 3.3 x 104 cfu listeria per monolayer. Each bar represents the mean + s.e cfu of 2 monolayers after infection TO ( □ ) and after 4 hours T4 ([23) • 202 6*3 In vitro assessment of effects of interferon-gamma on the growth of M. microti The effects of IFN-V on the growth of mycobacteria in macrophages was investigated. M. microti was a natural choice as it is virulent for mice and has the advantage of ease of handling, being a non-human pathogen. In the first experiments, various doses of IFN-V were added to macrophage monolayers for various periods before and at daily intervals after infection with M. microti. The next set of experiments assessed the effects of various doses of IFN-V when added after infection. Finally, the effects of IFN-V in combination with isoniazid and with rifampicin were investigated in the latter model. 6.3.1 Treatment of macrophage monolayers with interferon-gamma before and after infection 6.3.1.1 The effect of dose size of interferon-gamma added both before and after infection on the growth of M. microti in macrophages Duplicate monolayers in 16 mm Linbro wells each containing 5 x 10 macrophages were preincubated for 72 hours with either BMM or BMM containing 10, 100 or 1000 units of IFN-V ml The monolayers were infected with 4.4 x 10^ viable M. microti for 60 minutes. The number of viable mycobacteria per monolayer was determined by disruption of monolayers by scraping (section 2.9.4) and performing viable counts. There was no significant loss of cells from the control or IFN-V treated monolayers as assessed by their DNA content. This was confirmed by 203 daily microscopic examination of the monolayers. Figure 6.4A shows a monolayer that had been been incubated with BMM for 3 days and Figure 6.4B shows a monolayer that had been incubated with BMM plus 100 units IFN-X per ml for 3 days with daily medium changes. The mean viable counts were determined 1 hour, 1 day and 3 days after infection. The results of the mean viable mycobacterial counts are shown in Figure 6.5. The treated monolayers appeared to show a dose related response to previous exposure to IFN-Y, 1000 units having the greatest effect. The bactericidal effects of IFN-Y were evident 1 hour after infection and did not appear to increase with time. The number of viable bacteria detected in the supernatants after the phagocytosis period and the last rinses was approximately 5% of the monolayer-associated counts. Throughout the three days the number of bacteria in the supernatants did not rise above 2% of the monolayer-associated counts. Figure 6.6 shows the results of an experiment which differed only in the length of time allowed for exposure to IFN-Y and for phagocytosis. The wells which had been previously exposed to 0, 100 or 1000 units IFN-Y for 48 hours were infected with 3.6 x 10^ cfu each and incubated at 37°C for 2 hours. At the end of 2 hours there were considerably more bacilli in these monolayers than in the previous experiment. As seen earlier, the bactericidal effect of IFN-X was evident after the phagocytosis period and did not increase further with time. However, there were differences between Figures 6.5 and 6.6. In Figure 6.6, there were hardly any differences between the monolayers treated with 100 or 1000 units of IFN-Y except immediately after the phagocytosis period. 204 Fig. 6.4A Macrophage monolayer after 3 days incubation in BMM (with daily medium changes). Magnification x250. Fig. 6.4B Macrophage monolayer after 3 days incubation in BMM plus 100 u IFN-tf per ml (with daily medium changes). Magnification x250. M.microti in macrophagesafter prior exposure for 72 hours. Monolayerswere infected with 4.4 x105 viable organisms each i . . EffectFig.of 6.5dose size of interferon-gamma on growth of 0 □, or IFfHf1000 100 Eachper(□), u upoint(O) ml.represents themean s.e. + cfu of for 60 minutesafter prior exposure to ) 0 ! u ( , Mean viable mycobacteria per monolayer 2 monolayers. 205 10 u(A), Control lOOOu IFN 1OOu IFN O IFNlOu M. microti in macrophages after prior exposure for 48 hours. Eachpoint represents the mean 4-s.e. cfu of 3 monolayers. Monolayers wereexposed to or 100 1000 0u u (□), (•), u(^)IFN-X' Fig. before infection with3.6 x105 viable organisms each for Mean viable mycobacteria per monolayer 6.6 Effectof dose size of interferon-gamma on growth of 206 2 hours. Control 0 u100 1 000 u 207 6.3»1.2 The effect of previous exposure to interferon-gamma on the phagocytosis of M. microti The previous set of experiments had shown that immediately after the period allowed for phagocytosis, the mean viable counts in treated monolayers were considerably lower than counts in untreated monolayers. In order to exclude the possibility that this was due to a decrease in uptake of organisms during phagocytosis, an experiment was performed in which both viable counts and counts of stained bacilli were done on macrophages previously exposed to IFN-Y. This experiment was performed in tissue culture 8-chambered slides with approximately 2 x 10~* macrophages per well. The monolayers were preincubated with BMM or BMM containing 100 or 500 units IFN-Y ml ^ for 48 hours. Infection was achieved with 2 x 10 cfu per well and phagocy tosis allowed to proceed for 2 hours. The higher infectivity ratio was chosen to yield countable numbers of bacilli in the stained monolayers. Mean viable counts mmediately after phagocytosis (T^) and 24 hours later (T24) are shown in Table 6.4. The viable count results showed the same trend as in previous experiments with the difference in mean viable count between treated and untreated groups apparent immediately after the period allowed for phagocytosis. 208 TABLE 6.4 THE EFFECT OF PREVIOUS EXPOSURE TO INTERFERON-GAMMA ON VIABLE COUNTS OF M. MICROTI IN MONOLAYERS IN 8-CHAMBERED SLIDES 4 IFN-* Mean + s.e.(xlO ) cfu per monolayer units per ml T T 0 *24 0 9.5 + 0.9 19.4 + 0.9 100 6.7 + 0.9 12.1 + 0.9 500 6.6 + 0.7 12.5 + 0.5 The monolayers were stained for acid-fast bacilli (AFB) immediately after phagocytosis. In each monolayer, 300 macrophages were scored as the number of macrophages containing no AFB, 1-2 AFB, 3-5 AFB and > 5 AFB. There were hardly any macrophages containing >5 AFB and conse quently this catergory was excluded from the results. The distribution of uptake of AFB by the monolayers as assessed by staining is depicted in Figure 6.7. The figure showed clearly that there was no detectable difference in uptake of M. microti between the untreated and treated monolayers. From the bacillary counts, the percentage of macrophages infected, the mean number of AFB per macrophage and the mean number of AFB per infected macrophage were also calculated (Table 6.5). These indices of infection also confirmed that there were no detectable differences between the untreated and treated groups. The results showed that the reduced viable counts immediately after the period allowed for phagocytosis was not due to reduced uptake of bacilli. bar represents the mean s.e.+ number of macrophages containing macrophagesafter pretreatment with interferon-gamma. Distribution Fig. Each 6.7of uptake of acid-fast bacilli by 0 Number of macrophages (□), , ) □ ( 1-2 ), □ or 3-5 (JHJJ) ( bacilli per macrophage. nt Itreo pr ml per Interferon Units 209 210 TABLE 6.5 THE EFFECT OF PREVIOUS EXPOSURE TO INTERFERON-GAMMA ON COUNTS OF ACID-FAST BACILLI IN MACROPHAGE MONOLAYERS Index of Treatment with IFN-Y ( units/ml) infection 0 100 500 % macrophages infected 28.7 33.3 27.0 Mean AFB per macrophage 0.48 0.62 0.48 Mean AFB per infected macrophage 1.68 1.87 1.50 6.3.1.3 The effect of previous exposure of macrophages to interferon- gamma on viable intracellular M. microti at intervals during the phagocytosis period. The previous experiments had shown that previous exposure of macrophages to IFN-Y for 48 or 7 2 hours caused a decrease in the number of viable intracellular M. microti immediately after a phagocytosis period of 1 or 2 hours and that this was not due to a decrease in bacillary uptake. In order to investigate the events occurring during the period allowed for phagocytosis, the number of viable intracellular M. microti was determined at intervals during this period. Monolayers in 16 mm Linbro wells were pretreated for 48 hours with BMM or BMM containing 100 units IFN-Y ml ^ and were then infected with 3.1 x 10^ cfu M. microti per well. The monolayers were lysed with 1 % saponin after periods of 15, 30, 60 and 120 minutes. One set of wells that had been infected for 120 minutes were overlaid with fresh media and reincubated for a further 24 hours before being lysed. 211 The results are shown in Figure 6.8 . At 15 minutes after infection, there was not much difference between the number of viable intracellular M. microti in the untreated and treated monolayers. There were 1.54 3 3 x 10 cfu per untreated monolayer and 1.25 x 10 cfu per treated mono- layer. However, by 30 minutes after infection, there was a substantial difference between the treated and untreated monolayers. At 30 minutes after infection, the mean count of M. microti in the control monolayers was 6.1 x 10 cfu compared with a mean of 3.6 x 10 in the IFN-tf treated monolayers. The increase in the number of viable bacilli in both sets of monolayers were approximately parallel from 30-120 minutes after infection. The results of the mean viable counts of both sets of monolayers at the end of the 120 minute phagocytosis period and 24 hours later were consistent with the previous results (Figures 6.5 and 6.6). In the next experiment, monolayers in Linbro wells were prepared and overlaid with either BMM or BMM containing 100 units IFN-X ml The monolayers were all infected with 3.6 x 10^ cfu M. microti per well for 15, 30 or 60 minutes. At each of these time points, after thorough washing to remove unattached bacilli, half the monolayers were lysed w‘ith 1 % saponin to determine the number of cfu while the other half were overlaid with fresh BMM or BMM containing 100 units IFN-V ml * and reincubated. After 24 hours, the mean number of viable M. microti per monolayer was determined. The results are shown in Figure 6.9. In this experiment, even at 15 minutes after infection there was a difference between the number of viable bacilli in the treated and untreated monolayers. The mean viable count in the untreated monolayers 15 minutes after infection was extracellularbacilli from the well. viable intracellular M.microti at intervals during the phagocytosis per ml for 48 hours before infection with3.1 x 105 viable bacilli eidad ■ ■)period representsand (■ the period after the removal of e el Eachper point well.represents the mean s.e. + cfu of 3 monolayers. (- — -) represents the events occurring during the phagocytosis Fig. e id Monolayersperiod. were exposed to 0 (u or IFN-lf 100 •) u (O) Mean viable mycobacteria per monolayer x0 I “ 3x10 x0 - 1x10 5x103- 5x102_ IxlO3- 6.8 U H Effectof previous exposure to interferon-gamma on I I I I 1 3 60 30 15 0 ----- minutes ie fe infection after Time 1 - 212 ~T~ 120 n --- hours 24 1 and ( were exposed ) or IFN-K to100 0 peru u (O (#) ml for 48hours bacilli from the well. beforeinfection with3.6 x 105 viable bacilli Each per well. viable intracellular M.microti at intervals during the phagocytosis represents the events occurring during the phagocytosis period on rpeet h en+se cuo ooaes ) point represents the mean s.e.+ cfu of 3 monolayers. periodand their subsequent fate 24 Monolayershours later. EffectFig.of 6.9previous exposure to interferon-gamma on Mean viable mycobacteria per monolayer x0 - - 6x10H x0 “ 5x10 x0 — 4x10 3x1 3x10 2x10 _ _ 2x10 x0-- 1x10 5X102- ---- °J —i i i— i— °3J u 4 4 4_ 4 4 ) represents the period after the removal of extracellular 1 3 60 30 15 0 minutes ----- ie fe infection after Time r 213 hours "1 24 0 u (15m) u 100 0 u (30m) u 100 u (15m) u 0 30m) ( u 0 0 u (60m) u 100 214 3 3 8.5 x 10 cfu and increased slightly to 9.1 x 10 after 24 hours. However, in the treated monolayers, the count after 15 minutes was 4.7 3 3 x 10 cfu and decreased slightly to 4.0 x 10 . At 30 minutes after 4 infection the mean count of untreated monolayers was 1.4 x 10 cfu and 4 increased to 1.7 x 10 cfu. In the treated monolayers, the mean counts 3 were 6.1 x 10 at 30 minutes after infection and increased slightly to 3 6.9 x 10 cfu after 24 hours. However, at 60 minutes after infection, 4 the mean viable count in the untreated monolayers was 3.2 x 10 and 4 increased substantially to 4.7 x 10 cfu after 24 hours. The correspon- 4 4 ding counts in the treated monolayers were 1.4 x 10 and 1.7 x 10 cfu. Both experiments showed that as early as 15 to 30 minutes after infection, differences in the number of viable intracellular bacilli were evident between untreated and treated monolayers. The previous experiment had shown that IFN-V did not reduce bacillary uptake and no significant loss of macrophages from monolayers was seen in any experi ment. All the earlier results indicate that macrophages were activated to be bactericidal and the results from both these experiments showed that the bactericidal activity occurs as early as 15-30 minutes after infection. 215 6.3.2 The effect of interferon-gamma added after M. microti infection of peritoneal macrophages In these experiments, triplicate monolayers in 16 mm Linbro wells were established for 48 hours before infection with M. microti. The monolayers were infected with 5.6 x 10^ viable organisms and phagocy tosis allowed to proceed for 2 hours. monolayers were disrupted by scraping and the remaining monolayers were overlaid with BMM or BMM containing either 100 or 1000 units IFN-Y ml The mean viable counts were performed after 1 , 2 and 3 days. The results are shown in Figure 6.10. In the control untreated monolayers, the viable count increased from 5 x 10 immediately after infection to 1.5 x 10^ on Day +3. There was hardly any difference between all monolayers at Day +1 but by Day +2, both the treated mono- layers showed an inhibition of growth of M. microti. The monolayers treated with either 100 or 1000 units of IFN-Y showed fairly similar viable counts. The results showed that IFN-Y added after infection of macrophages with M . microti had a bacteriostatic effect on the mycobacteria. The bacteriostatic effect was not seen immediately, but was reflected in the viable counts one day later, and there was no apparent difference between monolayers that had been treated with 100 or 1000 units IFN-Y. After phagocytosis and all the washes the number of M. microti detected in the supernatants of the monolayers represented only approximately 1 % of the number of monolayer-associated bacteria. Throughout the 3 days of infection, the number of bacteria present in the supernatants did not rise above 1 % of the monolayer-associated viable counts. macrophages with M. Monolayers microti. were each infected with •, 0 □, or 1001000 IFN-V Each u(□), 0 u(•), upoint per (A) ml. i . .0 EffectFig.of 6.10interferon-gamma addedafter infection of represents the mean s.e. + of 3 monolayers. x105 5.6viable bacilli for 2 hours before the addition of Mean viable mycobacteria per monolayer x0 J r “J 1x10 ----- as fe infection after Days 1 1 ------ 1 2 ----- Control 217 Analysis of variance of all the results confirmed that IFN-X treatment of the monolayers had a highly significant inhibitory effect on the growth of M. microti (F ratio = 11.4; p<0.001). 6.3.3 The effect of interferon-gamma and isoniazid alone and in combination on the growth of M. microti in vitro Monolayers in 16 mm Linbro wells were established for 48 hours and infected with 2.4 x 10^ viable M. microti per well for 90 minutes. The Tq monolayers were disrupted by scraping and the remaining monolayers were overlaid with BMM, BMM with IFN-y, BMM with isoniazid or BMM with IFN-V plus isoniazid. IFN-V' was used at 100 units ml ^ and isoniazid at 1 p g ml Viable counts were determined on Days +1, +2 and +3 by lysing with 1% saponin. The results are shown in Figure 6.11. 4 After phagocytosis, the mean count in the monolayers was 1.3 x 10 cfu per monolayer. The treated monolayers showed a distinct inhibition of bacillary proliferation after Day +1 when compared to the untreated monolayers. The isoniazid treated monolayers inhibited the growth of M. microti by Day +1 and the inhibition became greater by Day +2. The monolayers that had been treated with IFN-X and isoniazid showed a similar inhibition to the isoniazid treated ones. A three-way analysis of variance was performed on all the results. The results of the analysis are shown in Table 6.6 and showed a significant interaction of isoniazid and IFN-X. The interaction was present because the counts with IFN-V alone were less than those for the control, whereas those with IFN-Y and isoniazid were marginally higher than those were each infected with 2.4 x105 viable bacilli for 90 minutes. l snai ( ). isoniazidml (A Eachpoint s.e. represents cfu of 3 replicates the mean of in combination on growth of M. microti in Monolayersmacrophages. untreatedmonolayers or after (•), the addition of 100 IFN-ifu i* .1 EffectFig* of 6.11 interferon-gamma and isoniazid alone and (O), Mean viable mycobacteria per monolayer x0 — 2x10 x0 — 3x10 5x10 x0 — 4x10 IxlO4— 5x 103__ 4 4 4 Pr isoniazidor 100 (♦), u IFN-V plus 1 pgper Per 1 4 _ 1 3 2 1 0 as fe infection after Days 218 219 with isoniazid alone. However, although the findings suggest slight 'antagonism' between IFN-V and isoniazid, the difference between the counts at 1 , 2 and 3 days for IFN-Y and isoniazid alone do not attain statistical significance. TABLE 6.6 THREE-WAY ANALYSIS OF VARIANCE OF THE IN VITRO EFFECTS OF TIME, ISONIAZID AND INTERFERON-t ON M. MICROTI INFECTION OF MACROPHAGES Source of variation DF MS F IFN-y 1 0.007 2.6 *** Isoniazid (H) 1 0.332 123 *** Days 2 0.059 22 Days x IFN-df 2 0.004 1.5 Days x H 2 0.010 3.6* ** H x IFN-JT 1 0.028 10.4 Days x H x IFN-* 2 0.008 3.0 Replicate error 24 0.0027 *** p < 0.001 ** p < 0.01 * p <0.05 As in the previous experiments, the number of M. microti present in the supernatants did not exceed 5% of the monolayer-associated count and after Day +1, was less than 1%. Phase contrast microscopy did not show any significant loss of macrophages in any monolayer throughout the duration of the experiment. 220 6.3.A The effect of rifampicin and interferon-gamma alone and in combination on the in vitro growth of M» microti 48 hour monolayers in 16 mm Linbro wells were infected with 2.2 x 10^ cfu of M. microti per well. After 90 minutes phagocytosis, the Tq monolayers were disrupted by scraping while the remaining monolayers were overlaid with BMM, BMM with IFN-Y, BMM with rifampicin, or BMM with IFN-Y and rifampicin. IFN-Y was used at 100 units ml ^ and rifampicin at 10 jjg ml Viable counts were performed on Days +1, +2 and +3 by disrupting the monolayers with 1 % saponin. The mean viable counts are shown in Figure 6.12. 4 After phagocytosis, there were 1.3 x 10 viable mycobacteria in the monolayers. In the untreated monolayers, the bacilli proliferated to 4 reach 3.9 x 10 cfu per monolayer by Day+3. As was seen in the previous results, the IFN-Y treated monolayers showed an inhibition of the pro liferation after Day+1. The ri f ampicin-treated monolayers showed a 3 dramatic decrease in the mean counts to 3 x 10 cfu by Day+1, and 1 x 3 10 cfu by Day+3. The monolayers that had been treated with both rifampicin and IFN-Yshowed a similar inhibition to the monolayers treated with rifampicin alone, but to a lesser extent. A three-way analysis of variance was performed on all the above results (Table 6.7). The results of the analysis showed a highly significant interaction of IFN-Y and rifampicin. The interaction was because the counts for mice treated with IFN-Y alone were less than those for control mice, whereas counts with IFN-Y and rifampicin were slightly higher than those with rifampicin alone. Even though these findings ml rifampicinml (A). were each infected with 2.2 x 105 viable bacilli for 90 minutes. Eachpoint represents the mean s.e. + cfuof 3 replicates of untreated ormonolayers after the (•), addition of 100 uIFN-JT i* .2 Effect ofFig*interferon-gamma 6.12 and rifampicin alone and incombination on growth of M. microti in macrophages. Monolayers O, 10 pg/mlrifampicin(O), or100 uIFN-£(♦), plus 10 pgper Mean viable mycobacteria per monolayer as fe infection after Days 221 222 suggest 'antagonism' between IFN-Y and rifampicin, the difference between the counts at 1, 2 and 3 days for IFN-V plus rifampicin and rifampicin alone was not statistically significant. As in the previous experiments, the number of viable bacteria in the supernatants did not exceed 5% of the monolayer-associated count after phagocytosis and was less than 2% after the first day. Phase contrast microscopy did not show any significant loss of macrophages in any monolayer throughout the duration of the experiment. TABLE 6.7 THREE-WAY ANALYSIS OF THE IN VITRO EFFECTS OF TIME, RIFAMPICIN AND INTERFERON-* ON THE GROWTH OF M. MICROTI IN MACROPHAGES Source of variation DF MS F ifn-3 1 0.000 0 *** Rifampicin (R) 1 5.733 940 ** Days 2 0.045 7.4 Days x IFN-Y 2 0.000 0 *** Days x R 2 0.211 35 ** R x IFN-Y 1 0.062 10 R x Days x IFN-Y 2 0.016 2.6 Replicate error 24 0.006 *** p< 0.001 ** p < 0.01 223 6.4 Discussion All the experiments in this Chapter were performed on murine resident peritoneal cells. In the L. monocytogenes and M. microti assays, a low bacteria : macrophage ratio was used. This ensured that extracellular bacteria were easily removed after phagocytosis and that the assays were not compromised by extracellular events. In all of the experiments, the extracellular component only accounted for about 1-5 % of the monolayer- associated bacteria at the end of phagocytosis and after the first day they rarely accounted for more than 1%. The low numbers of bacteria in the supernatants meant that there was no need to resort to the use of antibiotics at any time. This was important because it had been shown that antibiotics influence the results of intracellular killing assays (Hart, 197A; Cole and Brostoff, 1975; Biroum-Noerjasin, 1977). The listericidal assays established that IFN-Y activated macrophages for listericidal activity. Preincubation of macrophages for 24 hours was sufficient for activation and 48 hours did not seem to increase the magnitude of the listericidal activity. However, this was probably due to the decreased microbicidal activity of macrophages the longer it has been cultured in vitro. Nathan et al. (1983) had observed that the effects of IFN-Y were less dramatic when added to freshly isolated monocytes and that this was due to the release of copious ^2^2' ^he activation of macrophages to be listericidal by IFN-Y was shown to be significantly dose-related. 10 units IFN-Y ml ^ was insufficient and a dose between 10-100 units ml ^ was required for activation with 1000 units giving the biggest response. 224 The experiments with M. microti established that IFN-V could activate macrophages for antimycobacterial activity. The effect of IFN-tf was investigated when it was added both before and after infection and also when it was added only after infection. IFN-X added 48 or 72 hours before infection caused a decrease in the viable monolayer-associated counts of M. microti. This bactericidal effect was evident as early as an hour after infection and did not increase with further doses of IFN-V. There were several explanations for the phenomenon. It could have been due to a loss of macrophages in the treated monolayers, a decrease in uptake of bacilli by treated cells or a rapid kill of bacilli occurring during phagocytosis. The monitor ing of macrophage numbers by phase-contrast microscopy and DNA analysis confirmed that there was no significant loss of macrophages in the treated monolayers. The experiment where both viable counts and acid- fast bacilli staining were performed showed that the reduction in viable counts in the treated monolayers was not accompanied by a similar reduction in acid-fast bacillary counts, indicating that there was no reduced uptake of bacilli. All the results suggest that the most likely explanation for the phenomenon was rapid kill of bacilli occurring during phagocytosis. In an attempt to elucidate the events during the period allowed for phagocytosis, viable counts were performed at short intervals after infection. The results showed that the bactericidal effects of previous exposure to IFN-V was seen as early as 15-30 minutes after infection. These results suggest that the bactericidal activity of macrophages occurs simultaneously with phagocytosis. Lefford and Runft (1984) have 225 recently reported the rapid killing of 90% of an inoculum of BCG or M. smegmatis in vivo in 1 hour by peritoneal macrophages in BCG infected mice. Early bactericidal activity was also observed in all the listericidal assays. Phagocytosis periods of 40 minutes were employed and without exception, treated monolayers contained fewer monolayer-associated bacteria than control monolayers. It has been estimated (Davies, 1983a) that L. monocytogenes could be killed by activated rat peritoneal macro phages approximately 3 minutes after the bacterium makes contact with the cell. In a study of the listericidal activity of activated human macrophages, Biroum-Noerjasin (1977) reported that the bactericidal activity started early and was of short duration. He showed that 15 minutes after in vitro infection, activated macrophages had a faster and greater killing capacity than unactivated macrophages. Blanden (1968) also reported killing of S. typhimurium by murine peritoneal macrophages within minutes of phagocytosis. This is not totally unexpected because it has been demonstrated that completion of phagocytosis (Hirsch, 1962), stimulation of oxidative metabolism (Cohen et al., 1981; Smith et al., 1980) , and the formation of digestive vacuoles (Zucker-Franklin et al., 1964; De Heer et al., 1980) occur within minutes of phagocyte-particle contact. The addition of IFN-JT after infection demonstrated that IFN-2T could activate macrophages for antimycobacterial activity without the need for previous exposure before infection. There were differences in the response when compared to the pretreatment experiments. The major difference was that when IFN-V’ was added after infection, a bacterio 226 static instead of a bactericidal effect was seen. Another difference was the delay of the onset of the IFN-V effect. There was no effect 24 hours after addition of IFN-Y, but by 48 hours, there was an obvious difference in viable counts between the control and IFN-y treated monolayers. The minimal dose of IFN- that could activate macrophages for antilist- erial and antimycobacterial activity was between 10-100 unit ml This is in accord with the doses shown to be effective in several other murine studies. Murray et al . (1985) reported that preincubation of murine peritoneal macrophages with 100 units ml ^ recombinant IFN- )( caused an almost complete inhibition of T. gondii and that 65-75% of intracellular L. donovani were killed by preincubation with 10-100 units per ml. They also found that extending the pretreatment period to 72 hours or increasing the concentrations to above 100 units did not further enhance antiprotozoal activity. In another study, Wirth et al. (1985) found that preincubation with 125 units ml ^ recombinant IFN-Jf enhanced the phagocytosis and killing of Trypanosoma cruzi in mouse peritoneal macrophages as well as a macrophage-like cell line P388D1. They showed that preincubation of at least 24 hours is needed for the enhancement of phagocytosis and suggested that IFN-jT triggered time- dependent cellular events leading to enhanced phagocytosis. Recombinant IFN-& has also been shown to activate human monocytes in vitro. Murray et al. (1983) showed that treatment for 72 hours with 300 units ml ^ IFN-V primed monocytes to generate 7-8 fold more H2O2 and enhanced ant i lei shmanial activity. Similarly, Nathan et al. (1983) established that the incubation of monocytes with 100-300 units 227 ml * IFN-V for 2-3 days resulted in increased peroxide generation and toxoplasmacidal activity. There have been conflicting reports about the role of LPS in enhancement of tumoricidal activity by IFN-^f. Pace et al. (1985) found that IFN-^f could not activate peritoneal mouse macrophages to kill P815 cells unless LPS was present as a second signal. However Varesio et al. (1984) found significant levels of cytolytic activity induced by the same source of recombinant IFN-X in the absence of LPS. Subsequently, both sets of investigators collaborated in some experiments to unravel the controversies and concluded that the strain of mouse and assay conditions influences the role of LPS in IFN-& activation for tumoricidal activity (Pace et al., 1985). The level of LPS contamination of the IFN-2T used in the results reported in Chapter 6 was examined by testing the levels of endotoxin in the media used in some experiments. BMM with and without IFN-V was tested for endotoxin by the limulus lysate assay (assays were performed by J. McConnell, Bacteriology Department, RPMS). Most of the samples tested revealed no presence of endotoxin (<0.1 ng ml ^) and the highest level recorded was 10 ng ml The amount of endotoxin that has been shown to influence activation of macrophages for tumoricidal activity by MAF is 500 ng ml ^ (Svedersky et al., 1984). It was thus rather unlikely that LPS played a role in the antilisterial and antimycobacterial activities reported in this Chapter. Although the growth curves of M. microti suggested that IFN-X might have been slightly 'antagonistic* to isoniazid and to rifampicin, this effect 228 was not statistically significant. Hence, the effect of the combination of IFN-Jf with isoniazid and rifampicin should be considered as one of 'indifference'. The suggestion of slight 'antagonism' observed was not totally unexpected. Both isoniazid and rifampicin are bactericidal agents and it is known that bactericidal agents can be adversely affected by agents or conditions that slow down bacterial growth. Lowrie et al. (1979a) have demonstrated that the activity of penicillin, a bactericidal agent, on S. typhimurium within macrophages was directly related to the bacterial growth rate as well as the penicillin concen tration. Similarly, Dickinson and Mitchison (1981) have shown that when the growth rate of M. tuberculosis was reduced uniformly by lowering the incubation temperature or the pH of the culture medium, the bactericidal activities of rifampicin and isoniazid were decreased to a similar extent. There was a distinct difference in the activity of isoniazid and rifampicin in this in vitro system. Unlike rifampicin which immediately decreased the number of M. microti, isoniazid permitted the mycobacteria to grow within the macrophages for the first day. This was not due to resistance of M. microti to isoniazid as in vitro sensitivity tests had shown that, like most strains of M. tuberculosis, the minimal inhibitory concentration was 0.1 ^g ml This difference in early bactericidal activity between isoniazid and rifampicin had been previously observed in in vitro studies on these drugs. An increase in viable counts of a culture of M. tuberculosis occurred in the first day after exposure to 1 }ig ml * isoniazid (Dickinson and Mitchison, 1966). In contrast, the bactericidal action of 0.2 jig ml ^ rifampicin was evident after periods of exposure as short as 2 and 6 hours (Dickinson and Mitchison, 1970a). 229 CHAPTER 7 GENERAL DISCUSSION In the four decades since the discovery of streptomycin, remarkable advances have been made in the chemotherapy of tuberculosis. However, despite all of these, there is still room for improvement. Shortening the duration of treatment and the use of fully supervised intermittent regimens have helped to overcome some, but not all of the problems of chemotherapy. In technically advanced countries, the most serious problem with short-course chemotherapy is non-compliance (Addington, 1979). It is thus desirable to try to shorten regimens further. The situation is rather different in poor developing countries. Despite all the improvements made in short-course regimens, their prohibitive cost and the lack of organizational facilities in the Health Services have limited the applications of short-course chemotherapy in most developing countries. Chemotherapy in poor developing countries still largely rely on standard regimens of isoniazid with streptomycin and thiacetazone given for 12-24 months. Even though such regimens have been shown to ensure a long and lasting cure, in actual practice, it falls short of the ideal (New Delhi Tuberculosis Centre, 1977; East Af ri ca/Bri t i sh Medical Research Council Tanzania Tuberculosis Survey, 1977; Second East Africa/British Research Council Kenya Tuberculosis Survey, 1979). The main reasons are the lack of treatment centres making it difficult for patients to obtain the drugs, and non-compliance Non-compliance is a particularly large problem in developing countries because of the long duration of chemotherapy. 230 Immunomodulating agents might be of value in two ways. First, by increasing the speed with which organisms are killed by antituberculous drugs and second, by killing persisting organisms remaining dormant in the lesions after completion of chemotherapy. In either case, the result of effective immunomodulating activity would be to shorten the duration of chemotherapy necessary to achieve a stable cure. Although there have been a few reports of levamisole in conjunction with chemotherapy augmenting the clinical response to tuberculosis, the evidence is not very convincing (see section 1.7). The findings reported in this thesis showed that levamisole did not have any signifi cant effect on the growth of M. microti in mice. The preliminary results of clinical trials in Zambia and Kenya also showed no evidence of augmentation of response when levamisole was added to the chemothera peutic regimen (Kenyan/Zambian/British Medical Research Council Collabo rative Study, in preparation). It would appear unlikely that levamisole will have a role in the therapy of tuberculosis. The results obtained in the in vivo experiments with murine tuberculosis in Chapter 4 showed that 1000 units of recombinant IFN-V administered intravenously every three days with the first dose one or two days prior to infection, had an inhibitory effect on the growth of M. tuberculosis. However, even though the results were statistically significant, the effects were not very large. An increase in dose size, multiple daily dosage before infection and encapsulation in MLV liposomes did not increase the effect. The results of the experiments described in Chapter 5 did not show any 231 significant *synerglstlc1 effect of IFN-Y with either Isoniazld or rifampicin. Although there was a suggestion of an Addition* effect of IFN-'tf with isoniazid In one experiment, this was not shown to be statistically significant. The effects observed in the combination of IFN-Y and isoniazid and also with rifampicin in vivo can be described as ’indifferent'. This was apparent irrespective of the timing of adminis tration of IFN-V. The experiments in Chapter 6 showed that IFN-Y could activate murine peritoneal macrophages in vitro for microbicidal activity. Listericidal activity could be achieved by incubating macrophages with IFN-Y for 24 hours prior to infection. The assays with M. microti revealed that exposure of macrophage monolayers to IFN-Y for 2 days prior to infection resulted in a bactericidal effect that occurred mainly in the first 15-30 minutes after infection. However, the addition of IFN-Y to macrophages which had already been infected with M. microti resulted in a bacteriostatic effect on bacillary growth that was delayed by 24 hours. As was seen in the in vivo experiments, the addition of IFN-Y in vitro did not increase the bactericidal activities of isoniazid or rifampicin. Although the growth curves of M. microti in macrophages obtained in the in vitro experiments suggested that IFN-Y might have been slightly ’antagonistic’ to the bactericidal activities of isoniazid and rifampi cin, this effect was not statistically significant. Hence the in vitro effects of IFN-Y in combination with isoniazid and with rifampicin can be considered as one of ’indifference’. From the results obtained in both the in vivo and in vitro experiments, it would seem that it 232 is unlikely that IFN-8 will have a major impact on the chemotherapy of active tuberculosis, that is to say, in the early stages of drug treatment. The effect of IFN-}f on established tuberculosis was only assessed in one experiment (see section 5.1.2, Figures 5.5 and 5.6). The results showed that IFN-V did not have any significant effect on the growth of M. tuberculosis when given 5 days after infection. This was consistent with the findings of others that immunomodulating agents were most effective when administered shortly before infection. Similarly, when IFN-tf was given to mice that had been treated with isoniazid for 5 days, the effect of IFN-)( was ’indifferent' to the bactericidal activity of isoniazid. Mitchison and Selkon (1956) had shown that BCG vaccination of guinea-pigs increased the bactericidal activity of isoniazid in an ’additive' manner. The results that they obtained suggested that the immune response in the guinea-pigs is primarily bactericidal. However, Rees and Hart (1960) had shown that in chronic murine tuberculosis, the immune response was primarily bacteriostatic. It would be of consider able interest to assess the effects of IFN-tf in combination with isoniazid and with rifampicin in a model of established tuberculosis in mice where its modification of the immune response might influence the outcome of the interactions of IFN-V and antituberculous drugs. In the past, there was considerable interest in the use of corticoste roids in the treatment of tuberculosis. Steroids cause immunosuppres sion which might allow dormant bacilli to multiply, thereby becoming susceptible to the bactericidal action of antituberculosis drugs. There have been conflicting reports on the role of corticosteroids in the 233 treatment of tuberculosis. Steroids accelerate experimental disease in steroid-sensitive animals such as mice (Hart and Rees, 1950; Batten and McCune, 1957), albino-rats (Michael et al., 1950) and rabbits (Bunn and Drobeck, 1952) and have been administered in the post-chemotherapeutic period to assess the relapse rates in murine experimental chemotherapy (McCune et al., 1966; Grumbach, 1975). Batten and McCune (1957a) reported that steroids given simultaneously with antituberculous drugs did not enhance nor diminish drug activity. It was noted that steroids did not enhance the sterilizing ability of the combination of pyrazin- amide and isoniazid. Early controlled clinical trials on the influence of corticosteroids on treatment of pulmonary tuberculosis (Horne, 1960; United States Public Health Service Tuberculosis Therapy Trials, 1960; British Tuberculosis Association, 1961) suggested that steroids increased the rate of sputum conversion slightly in the early months and hastened the rate of radiographic improvement. In a review of the literature, Horne (1966) concluded that except for the moribund patient, there is no indication for the use of corticosteroids in routine treatment of pulmonary tuberculosis. The role of steroids in short-course chemotherapy for pulmonary tuberculosis was recently examined in a trial in India (Tuberculosis Research Centre, 1983). The results showed that predniso lone did not influence the speed of sputum conversion. The overall relapse rates were low but there was little difference between the prednisolone group and the controls. It appears unlikely that there will be a role for steroids in reducing even further the duration of short-course chemotherapy. 234 Immunomodulating agents could possibly have a role In the elimination of dormant organisms. Kanal and Kondo (1981) Investigated the ability of an immunomodulator, lentinan (see section 1.3.1) to influence the relapse rate in experimental murine chemotherapy. Mice that had been infected with M. tuberculosis were treated with a combination of either streptomycin, isoniazid and rifampicin or ethambutol, isoniazid and rifampicin for five months. This was followed by lentinan treatment for one month, one month without treatment and then another month of lentinan. The relapse rates were then assessed over the following four months by the number of organs with organisms detectable by culture. Among the 20 control mice, 11 lungs and 14 spleens were culture-positive compared with 4 positive lungs and 10 positive spleens among 20 mice treated with lentinan. It would seem that lentinan treatment in the post-chemotherapeutic period had reduced the relapse rate in the lungs but not in the spleens. The problem of dormant tubercle bacilli persisting in lesions is of considerable importance in the control of tuberculosis. An agent effective against the last remaining bacilli in tuberculous lesions would have a direct and immediate effect on the chain of transmission of M. tuberculosis and would have important epidemiological consequences. Chemoprophylaxis with isoniazid has been impractical on a massive scale because of the duration of treatment needed. However, an immunomodula- ting agent which acts by stimulating the immune response could be effective against dormant bacilli without the need for extended treatment. The effectiveness of IFN-V on dormant tubercle bacilli when administered after chemotherapy should be explored. This role of IFN-'K could be examined in a post-chemotherapeutic murine model similar to one 235 described by the Cornell group (McCune et al. , 1956; McCune et al., 1966) or by Kanai and Kondo (1981). Immunotherapy in tuberculosis has also been attempted with transfer factor. Whitcomb and Rocklin (1973) administered transfer factor to a patient with progressive primary tuberculosis refractory to standard chemotherapy and in whom a defect in cell mediated immune response to tuberculosis was shown. The administration of transfer factor restored immunologic reactivity accompanied by a dramatic clinical improvement. Rubinstein and colleagues (1977) also successfully used transfer factor to treat a patient with progressive tuberculosis refractory to eight drugs. Clinical recovery was accompanied by recovery of cell-mediated immune function. Transfer factor has also been used to treat a patient with disseminated non-reactive infection with M. kansasii (Daniel et al. , 1975). The patient had failed to respond to 5 weeks of appro priate chemotherapy and manifested cutaneous anergy and depressed lymphoproliferative responses. Treatment with transfer factor resulted in dramatic clinical recovery and restoration of DTH and lymphoproli- ferative response to PHA. IFN-X might conceivably have a role in the treatment of infections caused by other mycobacteria like M. avium which are refractory to standard antituberculous drugs. Usuda and colleagues (1981) have used lentinan with limited success in three patients excreting drug-resistant tubercle bacilli for 10 years. There was an improvement in phagocytic ability of neutrophils and lymphoproliferative responses in all 3 patients, but only 1 patient ceased bacillary excretion. There is a possibility that other immunomo- dulating agents like IFN-X might prove more effective in treatment of 236 drug-resistant tuberculosis. The encapsulation of IFN-Y in MLV liposomes did not confer any enhance ment of the effect of IFN-Y. The reasons for the lack of enhancement have been discussed in Section 4.6. There are many advantages of using liposomes as drug carriers. They are made from natural constituents and thus are biodegradable and relatively non-toxic. Liposomes are also easily made from a variety of components which lead to a wide variety of biological properties. The sizes of liposomes can also be altered by using different methods of preparation and encapsulation is achieved without the need for chemical modification as no bond-formation is needed. However, there are also several disadvantages in the use of liposomes as drug carriers. Their biodegradability results in a relati vely short half-life compared to other synthetic carriers. Another disadvantage is the natural fate of the liposomes to target mainly to macrophages of the liver and spleen, although this might not be a disadvantage in certain applications. Thus, liposomes could be very effective in delivering drugs or immunomodulating agents in diseases caused by intracellular parasites, in neoplastic disorders of histiocytes and lysosomal storage diseases. Much of the interest in using liposomes has centred on the possibility of ’targeting' these structures to specific cell types by incorporating ligands into the liposomal membrane that would selectively bind to specific cell surface determinants. Leserman et al. (1981) demonstrated a method of covalent coupling of monoclonal antibodies to liposomes that enabled a specific delivery of methotrexate to target murine cells. There is the exciting possibility to targeting immunomodulators and 237 antituberculous drugs specifically to macrophages harbouring tubercle bacilli by incorporating monoclonal antibodies to M. tuberculosis into the liposomal membrane. It would seem that the area of liposome targeting offers considerable clinical potential. There is also the possibility that combinations of immunomodulators will be more effective than single agents. An example is the combination of C. parvum and levamisole which were effective together but not alone in murine neoplastic models (Anaclerio et al. , 1977). As in the other reports of combination of agents, the timing of administration of both levamisole and C. parvum was crucial to the synergy. Brehmer et al. (1981) demonstrated the synergism of MDP and TDM (trehalose dimycolate) on resistance of mice against aerogenic infection with M. tuberculosis and influenza-A virus. MDP was ineffective on its own but when combined with TDM, it completely protected against influenza-A and inhibited the growth of M. tuberculosis in the lungs. One of the major problems with immunotherapy has been that most of the potent agents are derived from bacteria and other microorganisms. These natural agents have been largely macromolecular in nature and chemically not defined. A large number have been pyrogenic and consequently have been excluded from clinical use. Some of these problems have been resolved by unravelling the chemical nature of potent agents and chemical synthesis of analogous molecules. The synthesis of MDP and its analogues is a good example. Unfortunately, the synthetic molecule is often not biologically active. In the case of MDP, there have been hundreds of derivatives with different biological properties. Other synthetic derivatives of natural agents are bestatin and tuftsin. There 238 is the possibility that by protein engineering, derivatives of agents could be produced that are biologically more active than the original agents. New forms of interferons have been engineered by recombinant DNA technology. One example is the artificially engineered IFN-beta ser which differs from natural IFN-beta by substitution of serine for cysteine in position 17 (Khosrovi, 1983). This engineered product has biological properties similar to the native product but has superior specific activity and stability. Other examples are 'hybrid' IFNs formed by joining the N-terminal and C-terminal halves of different IFN- alpha subtypes (Streuli et al., 1980; Week et al., 1981; Rehberg et al., 1982). The 'hybrid' molecules were shown to have biological properties different from their parent molecules. One other problem is that immunomodulating agents must be administered very early in the course of infection if they are to be effective. This means that in clinical practice they might be of greater value in prophylaxis of opportunistic infections in patients at risk than in the treatment of already established infections. There is difficulty in selecting the dosage, frequency and intervals between administrations. Part of the difficulty is because the dose-response curve for immuno- therapeutic agents is different from that of other chemotherapeutic agents. It has been shown that supraoptimal doses can produce paradoxical effects. Another problem with immunomodulating agents is that in addition to the desired effect on the immune system there could be other undesirable effects like hypersensitivity or aggravation of autoimmune responses. The ideal immunomodulating agent should have a defined chemical corapo- 239 sltion to eliminate batch to batch variation and be devoid of carcino genic or antigenic activity. 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MICROTI IN LUNGS OF CFLP MICE Days Log viable mycobacteria per lung after after treatment with levamisole (mg/kg)* infection 0 2.5 25 3 3.904 + 0.08 7 3.886 + 0.21 15 4.667 + 0.10 4.668 + 0.14 4.316 + 0.09 22 5.376 + 0.13 5.482 + 0.13 5.220 + 0.09 29 5.993 + 0.25 5.842 + 0.11 6.342 + 0.26 36 6.822 + 0.08 6.620 + 0.15 6.604 + 0.23 43 7.031 + 0.09 7.141 + 0.06 7.466 + 0.12 * Mean + s.e. of 4 mice TABLE 3A.2 EFFECT OF LEVAMISOLE ON THE GROWTH OF M. MICROTI IN SPLEENS OF CFLP MICE Days Log viable mycobacteria per spleen after after treatment with levamisole (mg/kg)* infection 0 2.5 25 3 5.447 + 0.17 7 5.267 + 0.08 15 5.606 + 0.13 5.610 + 0.08 5.607 + 0.16 22 6.004 +0.10 6.107 + 0.12 5.867 + 0.06 29 6.348 + 0.06 6.132 + 0.13 6.484 + 0.11 36 6.095 + 0.20 5.936 + 0.22 6.102 + 0.14 43 5.885 + 0.16 5.906 + 0.19 6.106 + 0.22 Mean + s.e. of 4 mice 290 TABLE 4A.1 EFFECT OF SIZE OF INFECTIVE DOSE ON GROWTH OF M. TUBERCULOSIS IN MICE Infective dose Days after viable mycobacteria/organ Lo8io. (orgs/mouse) infection Lung Spleen 5 x 106 1 5.323 + 0.04 6.316 + 0.08 8 6.188 + 0.05 6.924 + 0.04 15 8.023 + 0.04 7.138 + 0.04 22 9.155 + 0.27 6.962 + 0.24 5 x 105 1 3.926 + 0.09 5.247 + 0.05 8 5.074 + 0.10 6.303 + 0.15 15 6.814 + 0.09 6.376 + 0.15 22 7.874 + 0.26 6.460 + 0.09 29 7.847 + 0.08 5.986 + 0.24 4 5 x 10 1 3.074 + 0.07 4.132 + 0.12 8 3.873 + 0.09 5.484 + 0.05 15 5.954 + 0.08 6.228 + 0.09 22 6.564 + 0.13 5.793 + 0.10 29 6.698 + 0.05 5.438 + 0.16 43 6.906 -f 0.39 5.138 + 0.21 * Mean + s.e. of 4 mice TABLE 4A.2 EFFECT OF SIZE OF DOSE OF INTERFERON-GAMMA ON THE GROWTH OF M. TUBERCULOSIS IN LUNGS OF INFECTED MICE Log viable mycobacteria per lung* after treatment Days after witn interferon-gamma (units /mouse) infection 0 200 1000 5000 0 5.679 + 0.04 3 4.470 + 0.04 4.323 + 0.12 4.216 + 0.24 4.087 + 0.17 5 4.232 + 0.05 4.456 + 0.09 4.166 + 0.21 4.104 + 0.21 9 5.116 + 0.12 4.806 + 0.14 4.696 + 0.08 4.729 + 0.13 * Mean + s.e. of 5 mice 291 TABLE 4A.3 EFFECT OF DOSE SIZE OF INTERFERON-GAMMA ON THE GROWTH OF M. TUBERCULOSIS IN SPLEENS OF INFECTED MICE Log._ viable mycobacteria per spleen after treatment Days after witn interferon-gamma (units/mouse) infection 0 200 1000 5000 0 5.154 + 0.05 3 5.552+0.04 5.514 + 0.04 5.367 + 0.05 5.377 + 0.03 5 5.887 + 0.04 5.914 + 0.04 5.776 + 0.04 5.703 + 0.02 9 6.674 + 0.05 6.648 + 0.06 6.560 + 0.04 6.549 + 0.05 * Mean + s.e. of 5 mice TABLE 4A.4 EFFECT OF ADMINISTRATION OF INTERFERON-GAMMA IN LIPOSOMES ON GROWTH OF M. TUBERCULOSIS IN LUNGS OF CFLP MICE L o g ^ viable mycobacteria per lung* Days after infection Treatment group 0 (1 hour) 5 9 Control 4.974 + 0.15 5.605 + 0.13 1000 u 4.243 + 0.15 4.903 + 0.10 1000 u, liposomes 4.614 + 0.22 4.727 + 0.14 5.551 + 0.12 20.000 u 4.508 + 0.24 5.172 + 0.12 20.000 u, liposomes 4.885 + 0.26 5.442 + 0.12 * Mean + s.e. of 6 mice 292 TABLE 4A.5 EFFECT OF ADMINISTRATION OF INTERFERON-GAMMA IN LIPOSOMES ON GROWTH OF M. TUBERCULOSIS IN SPLEENS OF CFLP MICE L°gio viable mycobacteria per spleen* Days after infection Treatment group 0 (1 hour) 5 9 Control 4.851 + 0.06 5.822 + 0.08 6.316 + 0.07 1000 u 5.286 + 0.06 5.931 + 0.06 1000 u, liposomes 5.377 + 0.08 5.730 + 0.04 20.000 u 5.397 + 0.13 5.910 + 0.07 20.000 u, liposomes 5.589 + 0.06 5.808 + 0.04 * Mean + s.e. of 6 mice TABLE 4A.6 EFFECT OF ADMINISTRATION OF INTERFERON-GAMMA IN LIPOSOMES ON GROWTH OF M. TUBERCULOSIS IN LUNGS OF BALB/C MICE viable mycobacteria per lung* ment group Days after PBS PBS 1000 u 1000 u infection liposomes liposomes 0 4.855 + 0.08 3 3.506 + 0.1 3.558 + 0.15 3.377 + 0.09 3.540 + 0.13 6 3.678 + 0.05 3.712 + 0.12 3.511 + 0.05 3.372 + 0.08 9 4.441 + 0.04 4.474 + 0.14 4.268 + 0.12 4.237 + 0.08 * Mean + s.e. of 4 mice TABLE 4A.7 EFFECT OF ADMINISTRATION OF INTERFERON-GAMMA IN LIPOSOMES ON GROWTH OF M. TUBERCULOSIS IN SPLEENS OF BALB/C MICE viable mycobacteria per lung* Tr tliment group Days after PBS PBS 1000 u 1000 u infection liposomes liposomes 0 4.965 + 0.06 3 5.239 + 0.03 5.206 + 0.03 5.172 + 0.05 5.111 + 0.05 6 5.867 + 0.07 5.779 + 0.05 5.792 + 0.04 5.672 + 0.07 9 6.586 + 0.03 6.455 + 0.03 6.312 + 0.06 6.199 + 0.05 * Mean + s.e. of 4 mice TABLE 4A.8 EFFECT OF PRETREATMENT WITH INTERFERON-GAMMA ON THE GROWTH OF M. TUBERCULOSIS IN THE LUNGS OF BALB/C MICE Treatment Mean log viable mycobacteria per lung* on: group Day 0 (I hr) Day 1 Day 2 Control 5.020+0.02 3.783 + 0.04 3.512 + 0.04 2000 u, Day -3 3.836 + 0.02 3.661 + 0.06 2000 u, Day -2 3.718 + 0.03 3.416 + 0.04 2000 u, Day -1 3.768 + 0.07 3.530 + 0.09 2000 u x 2, Day -2,-1 3.728 + 0.04 3.365 + 0.07 2000 u x 3, Day -3,-2,-1 3.841 + 0.10 3.458 + 0.06 * Mean + s.e. of 4 mice 294 TABLE 4A.9 EFFECT OF PRETREATMENT WITH INTERFERON-GAMMA ON THE GROWTH OF M. TUBERCULOSIS IN THE SPLEENS OF BALB/C MICE Treatment Mean log viable mycobacteria per spleen* on: group Day 0 hr) Day 1 Day 2 Control 4.780+0.04 4.966 + 0.04 5.108 + 0.02 2000 u, Day -3 4.923 + 0.03 4.980 + 0.03 2000 u, Day -2 4.845 + 0.05 4.933 + 0.03 2000 u, Day -1 4.876 + 0.05 4.823 + 0.04 2000 u x 2, Day -2,-1 4.831 + 0.05 4.853 + 0.04 2000 u x 3, Day -3,-2,-1 4.813 + 0.02 4.832 + 0.06 * Mean + s.e. of 4 mice TABLE 5A.1 THE EFFECTS OF INTERFERON-GAMMA AND ISONIAZID ALONE AND IN COMBINATION ON THE GROWTH OF M. TUBERCULOSIS IN LUNGS OF BALB/C MICE Mean log.^ viable mycobacteria per lung* after treatment with: Days after Untreated IFN-tf + infection controls IFN-V Isoniazid isoniazid 0 (1 hr) 4..858 + 0.09 1 3.983 + 0.12 3.865 + 0.2 2 3.584 + 0.13 3.263 + 0.10 3.656 + 0.11 3.379 + 0.16 3 3.419 + 0.15 3.273 + 0.06 3.318 + 0.09 3.226 + 0.02 4 3.562 + 0.10 3.364 + 0.13 3.065 + 0.06 2.854 + 0.05 6 3.673 + 0.09 3.634 + 0.06 2.854 + 0.09 2.624 + 0.10 9 4.651 + 0.12 4.308 + 0.10 2.496 + 0.06 2.344 + 0.05 * Mean + s.e. of 4 mice 295 TABLE 5A.2 THE EFFECT OF INTERFERON-GAMMA AND ISONIAZID ALONE AND IN COMBINATION ON THE GROWTH OF M. TUBERCULOSIS IN SPLEENS OF BALB/C MICE Mean log^ viable mycobacteria per spleen* after treatment with: Days ------:------after Untreated IFN-Y + infection control IFN-Y Isoniazid isoniazid 0 (1 hr) 4.887 + 0.04 1 5.071 + 0.04 4.930 + 0.06 2 5.043 + 0.03 4.937 + 0.02 4.938 + 0.05 4.886 + 0.04 3 5.196 + 0.05 5.062 + 0.03 4.721 + 0.05 4.593 + 0.06 4 5.352 + 0.03 5.274+0.06 4.471 + 0.07 4.264 + 0.06 6 5.999 + 0.05 5.892 + 0.02 4.128 + 0.04 4.014 + 0.04 9 6.562 + 0.07 6.282 + 0.02 3.578 + 0.04 3.478 + 0.03 * Mean + s.e. of 4 mice TABLE 5A.3 THE EFFECT OF INTERFERON-GAMMA AND ISONIAZID ALONE AND IN COMBINATION ON THE GROWTH OF M. TUBERCULOSIS IN LUNGS OF BALB/C MICE IN THE FIRST TWO DAYS AFTER INFECTION WHEN IFN-Y WAS GIVEN BEFORE AND AFTER INFECTION Mean log viable mycobacteria per lung* after treatment with: Days after Untreated i fn-Y + infection control IFN-Y Isoniazid isoniazid 0 (1 hr) 4.416 + C1.10 1 3.860 + 0.06 3.651 + 0.02 3.979 + 0.16 3.691 + 0.06 2 3.944 + 0.21 3.678 + 0.10 3.737 + 0.25 3.340 + 0.18 * Mean + s.e. of 4 mice 296 TABLE 5A.4 THE EFFECT OF INTERFERON-GAMMA AND ISONIAZID ALONE AND IN COMBINATION ON THE GROWTH OF M. TUBERCULOSIS IN SPLEENS OF BALB/C MICE IN THE FIRST TWO DAYS AFTER INFECTION WHEN IFN-V WAS GIVEN BEFORE AND AFTER INFECTION Mean log.^ viable mycobacteria per spleen* after treatment with: Days after Untreated IFN-Y + infection control IFN-X Isoniazid isoniazid 0 (1 hr) 4.171 + 0.06 1 5.013 + 0.03 4.824 + 0.03 4.813 + 0.09 4.770 + 0.05 2 5.094 + 0.02 4.900 + 0.01 4.708 + 0.03 4.744 + 0.06 * Mean + s.e. of 4 mice TABLE 5A.5 THE EFFECTS OF INTERFERON-GAMMA AND ISONIAZID ALONE AND IN COMBINATION ON THE GROWTH OF M. TUBERCULOSIS IN LUNGS OF BALB/C MICE WHEN IFN-y WAS ADMINISTERED ON DAYS +5 AND +7. Mean log^Q viable mycobacteria per lung* after treatment with: Days after Untreated ifn-Y + infection controls IFN-X Isoniazid isoniazid 0 (1 hr) 4.416 + 0.10 1 3.860 + 0.06 3.979 + 0.16 2 3.944 + 0.21 3.737 + 0.25 5 3.778 + 0.20 3.100 + 0.20 7 3.977 + 0.06 3.777 + 0.18 2.906 + 0.33 2.666 + 0.10 8 4.275 + 0.02 4.157 + 0.17 2.445 + 0.27 2.534 + 0.28 9 4.402 + 0.13 4.152 + 0.30 2.206 + 0.34 2.076 + 0.18 * Mean + s.e. of 4 mice 297 TABLE 5A.6 THE EFFECT OF INTERFERON-GAMMA AND ISONIAZID ALONE AND IN COMBINATION ON THE GROWTH OF M. TUBERCULOSIS IN SPLEENS OF BALB/C MICE WHEN IFN-tf WAS ADMINISTERED ON DAYS +5 AND +7 Mean log.^ viable mycobacteria per spleen* after treatment with: Days after Untreated IFN-Y + infection control iFN-y Isoniazid isoniazid 0 (1 hr) 4.171 + 0.06 1 5.013 + 0.03 4.813 + 0.09 2 5.094 + 0.02 4.708 + 0.03 5 5.692 + 0.03 4.123 + 0.03 7 6.016 + 0.03 6.014 + 0.05 3.864 + 0.04 3.855 + 0.02 8 6.221 + 0.07 6.129 + 0.03 3.717 + 0.03 3.670 + 0.04 9 6.480 + 0.04 6.328 + 0.05 3.610 + 0.07 3.639 + 0.05 * Mean + s.e. of 4 mice TABLE 5A.7 THE EFFECT OF INTERFERON-GAMMA AND RIFAMPICIN ALONE AND IN COMBINATION ON GROWTH OF M. TUBERCULOSIS IN LUNGS OF BALB/C MICE Mean log^ viable mycobacteria per lung* after treatment with: Days after Untreated IFN-V + i nfection controls IFN-Y Ri fampicin rifampicin 0 (1 hr) 4.707 + 0.06 1 3.898 + 0.04 3.769 + 0.05 3.548 + 0.1 3.563 + 0.09 2 3.617 + 0.03 3.487 +_0.1 3.141 + 0.13 3.085 + 0.11 3 3.447 + 0.07 3.245 + 0.04 2.654 + 0.04 2.585 + 0.09 * Mean + s.e. of 4 mice 298 TABLE 5A.8 THE EFFECT OF INTERFERON-GAMMA AND RIFAMPICIN ALONE AND IN COMBINATION ON GROWTH OF M. TUBERCULOSIS IN SPLEENS OF BALB/C MICE Mean log^Q viable mycobacteria per spleen* after treatment with: Days after Untreated IFN-JT + infection controls IFN-# Rifampicin rifampicin 0 (1 hr) 4.792 + 0.005 1 5.877 + 0.04 4.930 + 0.05 4.760 + 0.02 4.751 + 0.03 2 5.060 + 0.05 4.913 + 0.02 4.491 + 0.02 4.473 + 0.03 3 5.242 + 0.06 5.029 + 0.06 4.218 + 0.08 4.147 + 0.01 * Mean + s.e. of 4 mice