Note: Descriptions are shown in the official language in which they were submitted.
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IMPROVEMENT OF T CELL MEDIATED IMMUNITY
FIELD OF THE INVENTION
The present invention concerns methods of modifying the T-cell
population make up or increasing the number of T-cells in a subject having
depressed or abnormal T-cell population or function. These methods involve
disrupting sex steroid signalling to the thymus in the subject.
BACKGROUND OF THE INVENTION
The thymus is influenced to a great extent by its bidirectional
communication with the neuroendocrine system (Kendall, 1988). Of
particular importance is the interplay between the pituitary, adrenals and
gonads on thymic function including both trophic (TSH and GH) and
atrophic effects (LH, FSH and ACTH) (Kendall, 1988; Homo-Delarche, 1991).
Indeed one of the characteristic features of thymic physiology is the
progressive decline in structure and function which is commensurate with
the increase in circulating sex steroid production around puberty (Hirokawa
and Makinodan, 1975; Tosi et al., 1982 and Hirokawa, et al., 1994). The
precise target of the hormones and the mechanism by which they induce
thymus atrophy is yet to be determined. Since the thymus is the primary site
for the production and maintenance of the peripheral T cell pool, this
atrophy has been widely postulated as the primary cause of an increased
incidence of immune-based disorders in the elderly. In particular;
deficiencies of the immune system illustrated by a decrease in T-cell
dependent immune functions such as cytolytic T-cell activity and mitogenic
responses, are reflected by an increased incidence of immunodeficiency,
autoimmunity and tumour load in later life (Hirokawa, 1998).
The impact of thymus atrophy is reflected in the periphery, with
reduced thymic input to the T cell pool resulting in a less diverse T cell
receptor (TCR) repertoire. Altered cytokine profile (Hobbs et al., 1993;
Kurashima et al., 1995); changes in CD4+ and CD8+ subsets and a bias
towards memory as opposed to naive T cells (Mackall et al., 1995) are also
observed. Furthermore, the efficiency of thymopoiesis is impaired with age
such that the ability of the immune system to regenerate normal T-cell
numbers after T-cell depletion, is eventually lost (Mackall et al., 1995).
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However, recent work by Douek et al. (1998), has shown presumably thymic
output to occur even in old age in humans. Excisional DNA products of TCR
gene-rearrangement were used to demonstrate circulating, de novo produced
naive T cells after HIV infection in older patients. The rate of this output
and
subsequent peripheral T cell pool regeneration needs to be further addressed
since patients who have undergone chemotherapy show a greatly reduced
rate of regeneration of the T cell pool, particularly CD4+ T cells, in post-
pubertal patients compared to those who were pre-pubertal (Mackall et al,
1995). This is further exemplified in recent work by Timm and Thoman
(1999), who have shown that although CD4+ T cells are regenerated in old
mice post BMT, they appear to show a bias towards memory cells due to the
aged peripheral microenvironment, coupled to poor thymic production of
naive T cells.
The thymus essentially consists of developing thymocytes interspersed
within the diverse stromal cells (predominantly epithelial cell subsets) which
constitute the microenvironment and provide the growth factors and cellular
interactions necessary for the optimal development of the T cells. The
symbiotic developmental relationship between thymocytes and the epithelial
subsets that controls their differentiation and maturation (Boyd et al.,
1993),
2o means sex-steroid inhibition could occur at the level of either cell type
which
would then influence the status of the other. It is less likely that there is
an
inherent defect within the thymocytes themselves since previous studies,
utilising radiation chimeras, have shown that BM stem cells are not affected
by age (Hirokawa, 1998; Mackall and Gress, 1997) and have a similar degree
of thymus repopulation potential as young BM cells. Furthermore,
thymocytes in older aged animals retain their ability to differentiate to at
least some degree (Mackall and Gress, 1997; George and Ritter, 1996;
Hirokawa et al., 1994). However, recent work by Aspinall (1997), has shown
a defect within the precursor CD3 CD4 CD8- triple negative (TN) population
occurring at the stage of TCR (3 chain gene-rearrangement.
The enormous clinical benefits to be gained through restoration of
thymic function, would represent an important strategy for the treatment of
immunodeficiencies, particularly in the elderly, HIV patients and following
chemotherapy.
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SUMMARY OF THE INVENTION
The present inventors have demonstrated that thymic atrophy can be
completely reversed by inhibition of sex steroid production, with full
restoration of thymic structure and function. The present inventors have also
found clinical applications for rejuvenating thymic function by disrupting
sex steroid signalling to the thymus.
Accordingly, in a first aspect, the present invention provides a method
of modifying the T-cell population makeup or increasing the number of T-
cells in a subject having depressed or abnormal T-cell population or function,
the method comprising disrupting sex steroid signalling to the thymus in the
subject.
Preferably, the modification of T-cell population makeup is
characterized by an alteration in the nature and/or ratio of T cell subsets
defined functionally and/or by expression of characteristic molecules,
wherein the characteristic molecules are selected from the group consisting
of: the T cell receptor, CD4, CDB, CD3, CD25, CD28, CD44, CD62L and CD69.
It is further preferred that increasing the number of T-cells in a subject
results in a relative increase in T cell numbers when compared to other
lymphoid cells. Preferably, the other lymphoid cells are B cells.
It is also preferred that the subject having a depressed or abnormal T-
cell population or function is suffering from a condition selected from the
group consisting of: cancer, human immunodeficiency virus infection, an
autoimmune disease, a hypersensitivity disease or endometriosis.
Preferably, the cancer sufferer has undergone chemotherapy and/or
radiation therapy and/or bone marrow transplantation.
Preferably, the subject with the human immunodeficiency virus
infection has AIDS.
In a further preferred embodiment, the subject is post-pubertal.
Autoimmune diseases are thought to arise as a polygenic trait, an
essential component of which is the participation of pathological self
reactive
T cells. By treating such subjects with chemotherapy or irradiation, with or
without bone marrow transplantation, these self reactive T cells can be
ablated. It is envisaged that disruption of sex steroid signalling to the
thymus will allow reactivation of the thymus resulting in a cohort of new
non-autoreactive T cells.
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Accordingly, in a second aspect the present invention provides a
method for treating an autoimmune disease in a subject, the method
comprising the steps; ablating the resident T cell population, and disrupting
sex steroid signalling to the thymus in the subject.
The steps of the second aspect of the present invention can be
performed in any order.
In a preferred embodiment, this method further comprises subjecting
the individual to a bone marrow transplant.
In a further preferred embodiment, the T cell population is ablated by
exposing the individual to chemotherapy or irradiation.
The present invention may also be utilized to enhance an immune
response to an antigen in a subject.
Accordingly, in a third aspect the present invention provides a method
for enhancing an immune response to an antigen in a subject, the method
comprising disrupting sex steroid signalling to the thymus in the subject, and
administering an antigen.
The antigen may be, for example, derived from an infectious agents)
or from a tumour cell.
In a preferred embodiment of the third aspect, the subject is suffering
from cancer or an infection.
In a further preferred embodiment of the third aspect, the antigen is
mixed with an adjuvant before administration.
In a fourth aspect, the present invention provides a method of
decreasing host-vs-graft reaction in a subject following transplantation of an
organ, the method comprising the following steps:
ablating T-cells in the subject;
disrupting sex steroid signalling to the thymus in the subject; and
transplanting an organ from a donor into the subject.
Preferably, the method of the fourth aspect also comprises
3o transplanting bone marrow to the subject from the donor.
With respect to each of the methods of the present invention, it is
preferred that sex steroid signalling to the thymus is disrupted by inhibiting
sex steroid production or by blocking a sex steroid receptors) within the
thymus.
Preferably, inhibition of sex steroid production is achieved by either
castration or administration of a sex steroid analogue(s).
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Preferred sex steroid analogues include, eulexin, goserelin, leuprolide,
dioxalan derivatives such as triptorelin, meterelin, buserelin, histrelin,
nafarelin, lutrelin, leuprorelin, and luteinizing hormone-releasing hormone
analogues.
5 Currently, it is preferred that sex steroid analogue is an analogue of
luteinizing hormone-releasing hormone. More preferably, the luteinizing
hormone-releasing hormone analogue is deslorelin.
In yet another preferred embodiment, the sex steroid analogues) is
administered by a sustained peptide-release formulation. Preferred sustained
peptide-release formulations are provided in WO 98/08533, the entire
contents of which are incorporated herein by reference.
In a fifth aspect, the present invention provides a composition for
enhancing an immune response to an antigen in a subject, the composition
comprising an adjuvant, the antigen, and an analogue of luteinizing
hormone-releasing hormone.
It will also be understood by the skilled addressee that the present
invention can be applied to any organism which possesses a thymus at some
stage during its development. Preferably, the organism is a mammal. More
preferably, the organism is a human.
Throughout this specification the word "comprise", or variations such
as "comprises" or "comprising", will be understood to imply the inclusion of a
stated element, integer or step, or group of elements, integers or steps, but
not the exclusion of any other element, integer or step, or group of elements,
integers or steps.
BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
Fig. 1: Changes in thymocyte number pre- and post-castration.
Thymus atrophy results in a significant decrease in thymocyte numbers with
age. By 2 weeks post- castration, cell numbers have increased to young adult
levels. By 3 weeks post-castration, numbers have significantly increased
from the young adult and they are stabilised by 4 weeks post-castration.
* * * = Significantly different from young adult (2 mth) thymus, p < 0.001
Fig. 2: (A) Spleen numbers remain constant with age and post-
castration. The B:T cell ratio in the periphery also remains constant (B),
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however, the CD4:CD8 ratio decreases significantly (p<0.001) with age and is
restored to normal young levels by 4 weeks post-ex.
Fig. 3: FRCS profiles of CD4 vs. CD8 thymocyte populations with age
and post-castration. Percentages for each quadrant are given above each plot.
Subpopulations of thymocytes remain constant with age and there is a
synchronous expansion of thymocytes following castration.
Fig. 4.1: Proliferation of thymocytes as detected by incorporation of a
pulse of BrdU. Proportion of proliferating thymocytes remains constant with
age and following castration.
Fig. 4.2: Effects of age and castration on proliferation of thymocyte
subsets. (A) Proportion of each subset that constitutes the total
proliferating
population. The proportion of CD8+ T cells within the proliferating
population is significantly increased. (B) Percentage of each subpopulation
that is proliferating. The TN and CD8 Subsets have significantly less
proliferation at 2 years than at 2 months. At 2 weeks post-castration, the TN
population has returned to normal young levels of proliferation while the
CD8 population shows a significant increase in proliferation. The level is
equivalent to the normal young by 4 weeks post-castration. (C) Overall TN
proliferation remains constant with age and post-castration, however, the
significant decrease in proliferation of the TN1 subpopulation with age, is
not returned to normal levels by 4 weeks post-castration (D). * * * =Highly
significant, p < 0.001, * * =significant, p < 0.01
Fig. 5: Migration rates from 1 year and 2 year mice as determined by
IT FITC labelling. Young adult migration rates are 1% per day. Controls
used were non-injected animals. Migration rates remain constant with age.
Fig. 6: Changes in thymus, spleen and lymph node cell numbers
following treatment with cyclophosphamide, a chemotherapy agent. Note
the rapid expansion of the thymus in castrated animals when compared to
the non-castrate (cyclo alone) group at 1 and 2 weeks post-treatment. In
addition, spleen and lymph node numbers of the castrate group were well
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increased compared to the cyclophosphamide alone group. By 4 weeks, cell
numbers are normalised. (n = 3-4 per treatment group and time point).
Fig. 7: Changes in thymus, spleen and lymph node cell numbers
following irradiation (625 Rads). Note the rapid expansion of the thymus in
castrated animals when compared to the non-castrate (irradiation alone)
group at 1 and 2 weeks post-treatment. By 4 weeks, cell numbers are
normalised. (n = 3-4 per treatment group and time point).
Fig. 8: Changes in thymus, spleen and lymph node cell numbers
following irradiation. Note the rapid expansion of the thymus in castrated
animals when compared to the non-castrate group at 1 and 2 weeks post-
treatment. However, the difference observed is not as obvious as when mice
were castrated 1 week prior to treatment (Fig. 8). By 4 weeks, cell numbers
are normalised. (n = 3-4 per treatment group and time point).
Fig. 9: Changes in thymus, spleen and lymph node cell numbers
following treatment with cyclophosphamide, a chemotherapy agent. Note
the rapid expansion of the thymus in castrated animals when compared to
the non-castrate (cyclophosphamide alone) group at 1 and 2 weeks post-
treatment. In addition, spleen and lymph node numbers of the castrate group
were well increased compared to the cyclophosphamide alone group. By 4
weeks, cell numbers are normalised. (n = 3-4 per treatment group and time
point). Chemical castration is comparable to surgical castration in
regeneration of the immune system post-cyclophosphamide treatment.
Fig. 10: Lymph node cellularity following foot-pad immunisation with
HSV-1. Note the increased cellularity in the aged post-castration as
compared to the non-castrated group. Bottom graph illustrates the overall
3o activated cell number as gated on CD25 vs. CD8 cells by FRCS.
Fig. 11: Examples of Flow cytometry dot plots illustrating activated
cell proportions in lymph nodes following HSV-1 immunisation. Activated
cells are CD25/CD8 double-positive.
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Fig. 12: V(310 expression on CTL in activated LN following HSV-1
inoculation. Note the dimunition of a clonal response in aged mice and the
reinstation of the expected response post-castration.
Fig. 13: Changes in thymus, spleen, lymph node and bone marrow cell
numbers following bone marrow transplantation of Ly5 congenic mice. Note
the rapid expansion of the thymus in castrated animals when compared to
the non-castrate group at all time points post-treatment. In addition, spleen
and lymph node numbers of the castrate group were well increased compared
to the cyclophosphamide alone group. (n = 3-4 per treatment group and time
point). Castrated mice had significantly increased congenic (Ly5.2) cells
compared to non-castrated animals (data not shown).
Fig. 14: Changes in thymus cell number in castrated and noncastrated
mice after foetal liver reconstitution. n = 3-4 for each test group. (A) At
two
weeks, thymus cell number of castrated mice was at normal levels and
significantly higher than that of noncastrated mice (*p< 0.05). Hypertrophy
was observed in thymii of castrated mice after four weeks. Noncastrated cell
numbers remain below control levels. (B) CD45.2+ cells-CD45.2+ is a marker
showing donor derivation. Two weeks after reconstitution donor-derived
cells were present in both castrated and noncastrated mice. Four weeks after
treatment approximately 85% of cells in the castrated thymus were donor-
derived. There were no donor-derived cells in the noncastrated thymus.
Fig. 15: FRCS profiles of CD4 versus CD8 donor derived thymocyte
populations after lethal irradiation and foetal liver reconstitution.
Percentages for each quadrant are given to the right of each plot. The age
matched control profile is of an eight month old Ly5.1 congenic mouse
thymus. Those of castrated and noncastrated mice are gated on CD45.2+
cells, showing only donor derived cells. Two weeks after reconstitution
subpopulations of thymocytes do not differ between castrated and
noncastrated mice.
Fig. 16: Myeloid and lymphoid dendritic cell (DC) number after lethal
irradiation, foetal liver reconstitution and castration. n= 3-4 mice for each
test group. Control (white) bars on the following graphs are based on the
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normal number of dendritic cells found in untreated age matched mice. (A)
Donor-derived myeloid dendritic cells-Two weeks after reconstitution DC
were present at normal levels in noncastrated mice. There were significantly
more DC in castrated mice at the same time point. (*p<0.05). At four weeks
DC number remained above control levels in castrated mice. (B) Donor-
derived lymphoid dendritic cells - Two weeks after reconstitution DC
numbers in castrated mice were double those of noncastrated mice. Four
weeks after treatment DC numbers remained above control levels.
Fig. 17: Changes in total and CD45.2+ bone marrow cell numbers in
castrated and noncastrated mice after foetal liver reconstitution. n=3-4 mice
for each test group. (A) Total cell number - Two weeks after reconstitution
bone marrow cell numbers had normalised and there was no significant
difference in cell number between castrated and noncastrated mice. Four
weeks after reconstitution there was a significant difference in cell number
between castrated and noncastreated mice (*p<0.05). (B) CD45.2+ cell
number - There was no significant difference between castrated and
noncastrated mice with respect to CD45.2+ cell number in the bone marrow,
two weeks after reconstitution. CD45.2+ cell number remained high in
2o castrated mice at four weeks. There were no donor-derived cells in the
noncastrated mice at the same time point.
Fig. 18: Changes in T cells and myeloid and lymphoid derived
dendritic cells (DC) in the bone marrow after foetal liver reconstitution.
n= 3-4 mice for each test group. Control (white bars on the following graphs
are based on the normal number of T cells and dendritic cells found in
untreated age matched mice. (A) T cell number - Numbers were reduced
two and four weeks after reconstitution in both castrated and noncastrated
mice. (B) Donor derived myeloid dendritic cells - Two weeks after
reconstitution DC cell numbers were normal in both castrated and
noncastrated mice. At this time point there was no significant difference
between numbers in castrated and noncastrated mice. (C) Donor-derived
lymphoid dendritic cells - Numbers were at normal levels two and four
weeks after reconstitution. At two weeks there was no significant difference
between numbers in castrated and noncastrated mice.
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Fig. 19: Change in total and CD45.2+ spleen cell numbers in castrated
and noncastrated mice after foetal liver reconstitution. n=3-4 mice for each
test group. (A) Total cell number - Two weeks after reconstitution cell
numbers were decreased and there was no significant difference in cell
5 number between castrated and noncastrated mice. Four weeks after
reconstitution cell numbers were approaching normal levels in castrated
mice. (B) CD45.2+ cell number- there was no significant difference between
castrated and noncastrated mice with respect to CD45.2+ cell number in the
spleen, two weeks after reconstitution. CD45.2+ cell number remained high
10 in castrated mice at four weeks. There were no donor-derived cells in the
noncastrated mice at the same time point.
Fig. 20: Splenic changes in T cells and myeloid and lymphoid derived
dendritic cells (DC) after foetal liver reconstitution. n=3-4 mice for each
test
group. Control (white) bars on the following graphs are based on the normal
number of T cells and dendritic cells found in untreated age matched mice.
(A) T cell number - Numbers were reduced two and four weeks after
reconstitution in both castrated and noncastrated mice. (B) Donor derived
myeloid dendritic cells - two and four weeks after reconstitution DC numbers
2o were normal in both castrated and noncastrated mice. At two weeks there
was no significant difference between numbers in castrated and noncastrated
mice. (C) Donor-derived lymphoid dendritic cells - numbers were at normal
levels two and four weeks after reconstitution. At two weeks there was no
significant difference between numbers in castrated and noncastrated mice.
Fig. 21: Changes in total and CD45.2+ lymph node cell numbers in
castrated and noncastrated mice after foetal liver reconstitution. n=3-4 for
each test group. (A) Total cell numbers - two weeks after reconstitution cell
numbers were at normal levels and there was no significant difference in cell
number between castrated and noncastrated mice. Four weeks after
reconstitution cell numbers in castrated mice were at normal levels. (B)
CD45.2+ cell number - There was no significant difference between castrated
and noncastrated mice with respect to CD45.2+ cell number in the lymph
node, two weeks after reconstitution. CD45.2 cell number remained high in
castrated mice at four weeks. There were no donor-derived cells in the
noncastrated mice at the same point.
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Fig. 22: Changes in T cells and myeloid and lymphoid derived
dendritic cells (DC) in the mesenteric lymph nodes after foetal liver
reconstitution. n=3-4 mice for each test group. Control (white) bars on the
following graphs are based on the normal number of T cells and dendritic
cells found in untreated age matched mice. (A) T cell number - Numbers
were reduced two and four weeks after reconstitution in both castrated and
noncastrated mice. (B) Donor derived myeloid dendritic cells - Two weeks
after reconstitution DC numbers were normal in both castrated and
noncastrated mice. At four weeks they were decreased. At two weeks there
was no significant difference between numbers in castrated and noncastrated
mice. (C) Donor-derived lymphoid dendritic cells - numbers were at normal
levels two and four weeks after reconstitution. At two weeks there was no
significant difference between numbers in castrated and noncastrated mice.
DETAILED DESCRIPTION OF THE INVENTION
The phrase "modifying the T-cell population makeup" refers to altering
the nature and/or ratio of T cell subsets defined functionally and by
expression of characteristic molecules. Examples of these characteristic
molecules include, but are not limited to, the T cell receptor, CD4, CDB, CD3,
CD25, CD28, CD44, CD62L and CD69.
The phrase "increasing the number of T-cells" refers to an absolute
increase in the number of T cells in a subject in the thymus and/or in
circulation and/or in the spleen and/or in the bone marrow and/or in
peripheral tissues such as lymph nodes, gastrointestinal, urogenital and
respiratory tracts. This phrase also refers to a relative increase in T cells,
for
instance when compared to B cells.
A "subject having a depressed or abnormal T-cell population or
function" includes an individual suffering from cancer, especially one who
has undergone chemotherapy or radiation therapy, or has been subjected to a
bone marrow transplant, or breast and prostate cancer patients, or any cancer
or proliferative disorder resulting in T cell abnormalities or reduced
functional capacity of cell-mediated immunity. This phrase also includes an
individual infected with the human immunodeficiency virus, especially one
who has AIDS. Furthermore, this phrase includes any post-pubertal
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individual, especially an aged person who has decreased immune
responsiveness and increased incidence of disease as a consequence of post-
pubertal thymic atrophy. This phrase also includes a subject suffering from
endometriosis, an autoimmune disease, allergies, hypersensitivities, or any
immune dysfunction. The subject may have undergone an allogeneic bone
marrow transplantation, or be a post-chemotherapy leukaemia patient such
as CLL and low grade Non-Hogkins lymphoma patients treated with drugs
such as Fludarabine, cladrabine, dexamethasone and 2-cytodeoxyadenosine
which are severely toxic for T cells.
"Adjuvant" means one or more substances that enhances the
immunogenicity and efficacy of an antigen composition. Non-limiting
examples of suitable adjuvants include squalane and squalene (or other oils
of animal origin); block copolymers; detergents such as Tween~-80; Quil~
A, mineral oils such as Drakeol or Marcol, vegetable oils such as peanut oil;
Corynebacterium-derived adjuvants such as corynebacterium parvum;
Propionibacterium-derived adjuvants such as Propionibacterium acne;
Mycobacterium bovis (Bacillus Calmetic and Guerinn or BCG); interleukins
such as interleukin 2 and interleukin-12; monokines such as interleukin 1;
tumor necrosis factor; interferons such as gamma interferon; combinations
such as saponin-aluminium hydroxide or Quil-A aluminium hydroxide;
liposomes; iscom adjuvant; mycobacterial cell wall extract; synthetic
glycopeptides such as murarnyl dipeptides or other derivatives; Avridine;
Lipid A; dextran sulfate; DEAE-Dextran or DHAE-Dextran with aluminium
phosphate; carboxypolymethylene such as Carbopol' EMA; acrylic copolymer
emulsions such as Neocryl A640 (e.g. U.S. Pat. No. 5,047,238); vaccinia or
animal virus proteins; sub-viral particle adjuvants such as cholera toxin, or
mixtures thereof.
With regard to the method of decreasing host-vs-graft reaction in a
subject following transplantation of an organ, the host lymphocytes would be
first depleted (eg through irradiation or chemotherapy). This could be
followed by donor bone marrow/stem cell transplantation linked with
disruption of sex steroid signalling to the thymus, to establish chimeras
which would include establishment of donor cells including dendritic cells
in the host thymus to cause tolerance of newly developed host T cells to the
donor. After establishment of the central tolerance, the host would receive a
graft from the donor of the stem cells.
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As will be readily understood, sex steroid signalling to the thymus can
be disrupted in a range of ways, for example, inhibition of sex steroid
production or blocking a sex steroid receptors) within the thymus.
Inhibition of sex steroid production can be achieved, for example, by
castration, administration of a sex steroid analogue(s), and other well known
techniques. In some clinical cases permanent removal of the gonads via
physical castration may be appropriate. In a preferred embodiment, the sex
steroid signalling to the thymus is disrupted by administration of a sex
steroid analogue, preferably an analogue of luteinizing hormone-releasing
hormone. It is currently preferred that the analogue is deslorelin (described
in U.S. Patent No. 4218439).
Sex steroid analogues and their use in therapies and "chemical
castration" are well known. Examples of such analogues include Eulexin
(described in FR7923545, WO 86/01105 and PT100899), Goserelin (described
in US4100274, US4128638, GB9112859 and GB9112825), Leuprolide
(described in US4490291, US3972859, US4008209, US4005063, DE2509783
and US4992421), dioxalan derivatives such as are described in EP 413209,
Triptorelin (described in US4010125, US4018726, US4024121, EP 364819 and
US5258492), Meterelin (described in EP 23904), Buserelin (described in
US4003884, US4118483 and US4275001), Histrelin (described in EP217659),
Nafarelin (described in US4234571, W093/15722 and EP52510), Lutrelin
(described in US4089946), Leuprorelin (described in Ploskeret al.) and LHRH
analogues such as are described in EP181236, US4608251, US4656247,
US4642332, US4010149, US3992365 and US4010149. The disclosures of
each the references referred to above are incorporated herein by cross
reference.
As will be understood by persons skilled in the art at least some of the
means for disrupting sex steroid signalling to the thymus will only be
effective as long as the appropriate compound is administered. As a result,
an advantage of certain embodiments of the present invention is that once
the desired immunological affects of the present invention have been
achieved, (2-3 months) the treatment can be stopped and the subjects
reproductive system will return to normal.
As will be understood, the term "organ" is used in its broadest sense
and includes skin, kidney, liver, heart, lung etc.
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RX AMP1.RS
EXAMPLE 1 - REVERSAL OF AGED-INDUCED THYMIC ATROPHY
Materials and Methods
Animals
CBA/CAH and C57B16/J male mice were obtained from Central Animal
Services, Monash University and were housed under conventional
conditions. Ages ranged from 4-6 weeks to 26 mcnths of age and are
indicated where relevant.
Castration
Animals were anaesthetised by intraperitoneal injection of 0.3m1 of
0.3mg xylazine (Rompun; Bayer Australia Ltd., Botany NSW, Australia) and
l.5mg ketamine hydrochloride (Ketalar; Parke-Davis, Caringbah, NSW,
Australia) in saline. Surgical castration was performed by a scrotal incision,
revealing the testes, which were tied with suture and then removed along
with surrounding fatty tissue.
Bromodeoxyuridine (BrdU) incorporation
Mice received two intraperitoneal injections of BrdU (Sigma Chemical
Co., St. Louis, MO) (100mg/kg body weight in 1001 of PBS) at a 4 hour
interval. Control mice received vehicle alone injections. One hour after the
second injection, thymuses were dissected and either a cell suspension made
for FRCS analysis, or immediately embedded in Tissue Tek (O.C.T.
compound, Miles INC, Indiana), snap frozen in liquid nitrogen, and stored at
-70°C until use.
Flow Cytometric analysis
Mice were killed by COz asphyxiation and thymus, spleen and
mesenteric lymph nodes were removed. Organs were pushed gently through
a 200~.m sieve in cold PBS/1% FCS/0.02% Azide, centrifuged (650g, 5 min,
4°C), and resuspended in either PBS/FCS/Az. Spleen cells were incubated
in
red cell lysis buffer (8.9g/litre ammonium chloride) for 10 min at 4°C,
washed
and resuspended in PBS/FCS/Az. Cell concentration and viability were
determined in duplicate using a haemocytometer and ethidium
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bromide/acridine orange and viewed under a fluorescence microscope
(Axioskop; Carl Zeiss, Oberkochen, Germany).
For 3-colour immunofluorescence thymocytes were routinely labelled
with anti-a(3 TCR-FITC or anti-y8 TCR-FITC, anti-CD4-PE and anti-CD8-APC
5 (all obtained from Pharmingen, San Diego, CA) followed by flow cytometry
analysis. Spleen and lymph node suspensions were labelled with either
a(3TCR-FITC/CD4-PE/CD8-APC or B220-B (Sigma) with CD4-PE and CD8-
APC. B220-B was revealed with streptavidin-Tri-color conjugate purchased
from Caltag Laboratories, Inc., Burlingame, CA.
10 For BrdU detection, cells were surface labelled with CD4-PE and CD8-
APC, followed by fixation and permeabilisation as previously described
(Carayon and Bord, 1989). Briefly, stained cells were fixed O/N at 4°C
in 1%
PFA/0.01% Tween-20. Washed cells were incubated in 500.1 DNase (100
Kunitz units, Boehringer Mannheim, W. Germany) for 30 mins at 37°C
in
15 order to denature the DNA. Finally, cells were incubated with anti-BrdU-
FITC (Becton-Dickinson).
For 4-colour Immunofluorescence thymocytes were labelled for CD3,
CD4, CDB, B220 and Mac-1, collectively detected by anti-rat Ig-Cy5
(Amersham, U.K.), and the negative cells (TN) gated for analysis. They were
further stained for CD25-PE (Pharmingen) and CD44-B (Pharmingen)
followed by Streptavidin-Tri-colour (Caltag, CA) as previously described
(Godfrey and Zlotnik, 1993). BrdU detection was then performed as
described above.
Samples were analysed on a FacsCalibur (Becton-Dickinson). Viable
lymphocytes were gated according to 0° and 90° light scatter
profiles and data
was analysed using Cell quest software (Becton-Dickinson).
Immunohistology
Frozen thymus sections (4~.m) were cut using a cryostat (Leica) and
immediately fixed in 100% acetone.
For two-colour immunofluorescence, sections were double-labelled
with a panel of monoclonal antibodies: MTS6, 10, 12, 15, 16, 20, 24, 32, 33,
and 44 (Godfrey et al., 1990; Table 1) produced in this laboratory and the
co-expression of epithelial cell determinants was assessed with a polyvalent
35 rabbit anti-cytokeratin Ab (Dako, Carpinteria, CA). Bound mAb was revealed
with FITC-conjugated sheep anti-rat Ig (Silenus Laboratories) and anti
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cytokeratin was revealed with TRITC-conjugated goat anti-rabbit Ig (Silenus
Laboratories).
For bromodeoxyuridine detection sections were stained with either
anti-cytokeratin followed by anti-rabbit-TRITC or a specific mAb, which was
then revealed with anti-rat Ig-Cy3 (Amersham). BrdU detection was then
performed as previously described (Penitet al., 1996). Briefly, sections were
fixed in 70% Ethanol for 30 mins. Semi-dried sections were incubated in 4M
HCI, neutralised by washing in Borate Buffer (Sigma), followed by two
washes in PBS. BrdU was detected using anti-BrdU-FITC (Becton-
Dickinson).
For three-colour immunofluorescence, sections were labelled for a
specific MTS mAb together with anti-cytokeratin. BrdU detection was then
performed as described above.
Sections were analysed using a Leica fluorescent and Nikon confocal
microscopes.
Migration studies
Animals were anaesthetised by intraperitoneal injection of 0.3m1 of
0.3mg xylazine (Rompun; Bayer Australia Ltd., Botany NSW, Australia) and
l.5mg ketamine hydrochloride (Ketalar; Parke-Davis, Caringbah, NSW,
Australia) in saline.
Details of the FITC labelling of thymocytes technique are similar to
those described elsewhere (Scollay et al., 1980; Berzins et al., 1998).
Briefly,
thymic lobes were exposed and each lobe was injected with approximately
10~m of 350 ~,g/ml FITC (in PBS). The wound was closed with a surgical
staple, and the mouse was warmed until fully recovered from anaesthesia.
Mice were killed by COZ asphyxiation approximately 24h after injection and
lymphoid organs were removed for analysis.
After cell counts, samples were stained with anti-CD4-PE and anti-
CD8-APC, then analysed by flow cytometry. Migrant cells were identified as
live-gated FITC+ cells expressing either CD4 or CD8 (to omit autofluorescing
cells and doublets). The percentages of FITC+ CD4 and CD8 cells were
added to provide the total migrant percentage for lymph nodes and spleen,
respectively. Calculation of daily export rates was performed as described by
Berzins et al. (1998).
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Data was analysed using the unpaired student 't' test or
nonparametrical Mann-Whitney test was used to determine the statistical
significance between control and test results for experiments performed at
least in triplicate. Experimental values significantly differing from control
values are indicated as follows: *p<_0.05, **p_<0.01 and ***p<_0.001.
Results
The effect of age on thymocyte populations.
(i) Thymic weight and thymocyte number
With increasing age there is a highly significant (p<_0.0001) decrease in
both thymic weight (Figure 1A) and total thymocyte number (Figure 1B).
Relative thymic weight (mg thymus/g body) in the young adult has a mean
value of 3.34 which decreases to 0.66 at 18-24 months of age (adipose
deposition limits accurate calculation). The decrease in thymic weight can
be attributed to a decrease in total thymocyte numbers: the 1-2 month
thymus contains --6.7 x 10' thymocytes, decreasing to --4.5 x 106 cells by 24
months. By removing the effects of sex steroids on the thymus by castration,
regeneration occurs and by 4 weeks post-castration, the thymus is equivalent
to that of the young adult in both weight and cellularity (Figure 1A and 1B).
Interestingly, there is a significant (p<0.001) increase in thymocyte numbers
at 2 weeks post-castration (-1.2 x 10a), which is restored to normal young
levels by 4 weeks post-castration (Figure 1B).
The decrease in T cell numbers produced by the thymus is not
reflected in the periphery, with spleen cell numbers remaining constant with
age (Figure 2A). Homeostatic mechanisms in the periphery were evident
since the B cell to T cell ratio in spleen and lymph nodes was not affected
with age and the subsequent decrease in T cell numbers reaching the
periphery (Figure 2B). However, the ratio of CD4+ to CD8+ T cell
significantly decreased (p<_0.001) with age from 2:1 at 2 months of age, to a
ratio of 1:1 at 2 years of age (Figure 2C). Following castration and the
subsequent rise in T cell numbers reaching the periphery, no change in
peripheral T cell numbers was observed: splenic T cell numbers and the ratio
of B:T cells in both spleen and lymph nodes was not altered following
castration (Figure 2A and B). The decreased CD4:CD8 ratio in the periphery
with age was still evident at 2 weeks post-castration but was completely
reversed by 4 weeks post-castration (Figure 2C).
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(ii) a,QTCR, ybTCR, CD4 and CD8 expression
To determine if the decrease in thymocyte numbers seen with age was
the result of the depletion of specific cell populations, thymocytes were
labelled with defining markers in order to analyse the separate
subpopulations. In addition, this allowed analysis of the kinetics of thymus
repopulation post-castration. The proportion of the main thymocyte
subpopulations was compared with those of the normal young thymus
(Figure 3) and found to remain uniform with age. In addition, further
subdivision of thymocytes by the expression of a(3TCR and yBTCR revealed
no change in the proportions of these populations with age (data not shown).
At 2 and 4 weeks post-castration, thymocyte subpopulations remained in the
same proportions and, since thymocyte numbers increase by up to 100-fold
post-castration, this indicates a synchronous expansion of all thymocyte
subsets rather than a developmental progression of expansion.
The decrease in cell numbers seen in the thymus of aged animals thus
appears to be the result of a balanced reduction in all cell phenotypes, with
no significant changes in T cell populations being detected. Thymus
regeneration occurs in a synchronous fashion, replenishing all T cell
subpopulations simultaneously rather than sequentially.
Proliferation of thymocytes
As shown in Figure 4.1, 15-20% of thymocytes are proliferating at 4-6
weeks of age. The majority (--80%) of these are DP with the TN subset
making up the second largest population at ---6% (Figure 4.2A). Accordingly,
most division is seen in the subcapsule and cortex by immunohistology (data
not shown). Some division is seen in the medullary regions with FRCS
analysis revealing a proportion of SP cells (9% of CD4 T cells and 25% of
CD8 T cells) dividing (Figure 4.2B).
3o Although cell numbers are significantly decreased in the aged thymus,
proliferation of thymocytes remains constant, decreasing to 12-15% at 2 years
(Figure 4.1), with the phenotype of the proliferating population resembling
the 2 month thymus (Figure 4.2A). Immunohistology revealed the division at
1 year of age to reflect that seen in the young adult, however, at 2 years,
proliferation is mainly seen in the outer cortex and surrounding the
vasculature (data not shown). At 2 weeks post-castration, although
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thymocyte numbers significantly increase, there is no change in the
proportion of thymocytes that are proliferating, again indicating a
synchronous expansion of cells (Figure 4.1). Immunohistology revealed the
localisation of thymocyte proliferation and the extent of dividing cells to
resemble the situation in the 2-month-old thymus by 2 weeks post-castration
(data not shown). When analysing the proportion of each subpopulation
which represent the proliferating population, there was a significant
(p < 0.001) increase in the percentage of CD8 T cells which are within the
proliferating population (1% at 2 months and 2 years of age, increasing to
--6% at 2 weeks post-castration) (Figure 4.2A).
Figure 4.2B illustrates the extent of proliferation within each subset in
young, old and castrated mice. There is a significant (p-<0.001) decay in
proliferation within the DN subset (35% at 2 months to 4% by 2 years).
Proliferation of CD8+ T cells was also significantly (p<0.001) decreased,
reflecting the findings by immunohistology (data not shown) where no
division is evident in the medulla of the aged thymus. The decrease in DN
proliferation is not returned to normal young levels by 4 weeks post-
castration. However, proliferation within the CD8+ T cell subset is
significantly (p<_0.001) increased at 2 weeks post-castration and is returning
to normal young levels at 4 weeks post-castration.
The decrease in proliferation within the DN subset was analysed
further using the markers CD44 and CD25. The DN subpopulation, in
addition to the thymocyte precursors, contains a(3TCR+CD4 CD8-
thymocytes, which are thought to have downregulated both co-receptors at
the transition to SP cells (Godfrey & Zlotnik, 1993). By gating on these
mature cells, it was possible to analyse the true TN compartment (CD3 CD4
CD8-) and these showed no difference in their proliferation rates with age or
following castration (Figure 4.2C). However, analysis of the subpopulations
expressing CD44 and CD25, showed a significant (p<0.001) decrease in
proliferation of the TN1 subset (CD44+CD25-), from 20% in the normal young
to around 6% at 18 months of age (Figure 4.2D) which was restored by 4
weeks post-castration. The decrease in the proliferation of the TN1 subset,
was compensated for by a significant (p<0.001) increase in proliferation of
the TN2 subpopulation (CD44+CD25+) which returned to normal young
levels by 2 weeks post-castration (Figure 4.2D).
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The effect of age on the thymic microenvironment
The changes in the thymic microenvironment with age were examined
by immunofluorescence using an extensive panel of mAbs from the MTS
series, double-labelled with a polyclonal anti-cytokeratin Ab.
5 The antigens recognised by these mAbs can be subdivided into three
groups: thymic epithelial subsets, vascular-associated antigens and those
present on both stromal cells and thymocytes.
(i) Epithelial cell antigens.
Anti-keratin staining (pan-epithelium) of 2 year old mouse thymus,
10 revealed a loss of general thymus architecture with a severe epithelial
cell
disorganisation and absence of a distinct cortico-medullary junction. Further
analysis using the mAbs, MTS 10 (medulla) and MTS44 (cortex), showed a
distinct reduction in cortex size with age, with a less substantial decrease
in
medullary epithelium (data not shown). Epithelial cell free regions, or
15 keratin negative areas (KNA's, van Ewijk et al., 1980; Godfrey et al.,
1990;
Bruijntjes et al., 1993).) were more apparent and increased in size in the
aged
thymus, as evident with anti-cytokeratin labelling. There is also the
appearance of thymic epithelial "cyst-like" structures in the aged thymus
particularly noticeable in medullary regions (data not shown). Adipose
20 deposition, severe decrease in thymic size and the decline in integrity of
the
cortico-medullary junction are shown conclusively with the anti-cytokeratin
staining (data not shown). As shown in Figure 2.1, the thymus is beginning
to regenerate by 2 weeks post-castration. This is evident in the size of the
thymic lobes (a), the increase in cortical epithelium as revealed by MTS 44
(b) and the localisation of medullary epithelium (c). The medullary
epithelium is detected by MTS 10 and at 2 weeks, there are still subpockets
of epithelium stained by MTS 10 scattered throughout the cortex. By 4
weeks post-castration, there is a distinct medulla and cortex and discernible
cortico-medullary junction (data not shown).
The markers MTS 20 and 24 are presumed to detect primordial
epithelial cells (Godfrey, et al., 1990) and further illustrate the
degeneration
of the aged thymus. These are present in abundance at E14, detect isolated
medullary epithelial cell clusters at 4- 6 weeks but are again increased in
intensity in the aged thymus (data not shown). Following castration, all
these antigens are expressed at a level equivalent to that of the young adult
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thymus (data not shown) with MTS 20 and MTS 24 reverting to discrete
subpockets of epithelium located at the cortico-medullary junction.
(ii) Vascular-associated antigens.
The blood-thymus barrier is thought to be responsible for the
immigration of T cell precursors to the thymus and the emigration of mature
T cells from the thymus to the periphery.
The mAb MTS 15 is specific for the endothelium of thymic blood
vessels, demonstrating a granular, diffuse staining pattern (Godfrey, et al,
1990). In the aged thymus, MTS 15 expression is greatly increased, and
reflects the increased frequency and size of blood vessels and perivascular
spaces (data not shown).
The thymic extracellular matrix, containing important structural and
cellular adhesion molecules such as collagen, laminin and fibrinogen, is
detected by the mAb MTS 16. Scattered throughout the normal young
thymus, the nature of MTS 16 expression becomes more widespread and
interconnected in the aged thymus. Expression of MTS 16 is increased
further at 2 weeks post-castration while 4 weeks post-castration, this
expression is representative of the situation in the 2 month thymus (data not
shown).
(iii) Shared antigens
MHC II expression in the normal young thymus, detected by the mAb
MTS 6, is strongly positive (granular) on the cortical epithelium (Godfrey et
al., 1990) with weaker staining of the medullary epithelium. The aged
thymus shows a decrease in MHCII expression with expression substantially
increased at 2 weeks post-castration. By 4 weeks post-castration, expression
is again reduced and appears similar to the 2 month old thymus (data not
shown).
Thymocyte emigration
Approximately 1% of T cells migrate from the thymus daily in the
young mouse (Scollay et al., 1980). We found migration was occurring at a
proportional rate equivalent to the normal young mouse at 14 months and
even 2 years of age (Figure 5) although significantly (p<_0.0001) reduced in
number. There was an increase in the CD4:CD8 ratio of the recent thymic
emigrants from -~-3:1 at 2 months to --7:1 at 26 months. By 1 week post-
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castration, cell number migrating to the periphery has substantially increased
with the overall rate of migration remaining constant at 1-1.5%.
EXAMPLE 2 - REVERSAL OF CHEMOTHERAPY- OR RADIATION-INDUCED
THYMIC ATROPHY.
Castrated mice (either one-week prior to treatment, or on the same day
as treatment), showed substantial increases in thymus regeneration rate
following irradiation or cyclophosphamide treatment.
In the thymus, irradiated mice show severe disruption of thymic
architecture, concurrent with depletion of rapidly dividing cells. Cortical
collapse, reminiscent of the aged/hydrocortisone treated thymus, reveals loss
of DN and DP thymocytes. There is a downregulation of a(3-TCR expression
on CD4+ and CD8+ SP thymocytes - evidence of apoptosing cells. In
comparison, cyclophosphamide-treated animals show a less severe disruption
of thymic architecture, and show a faster regeneration rate of DN and DP
thymocyte s .
By 1 week post-treatment castrated mice showed significant thymic
regeneration even at this early stage (Figures 6, 7 and 8). In comparison, non-
2o castrated animals, showed severe loss of DN and DP thymocytes (rapidly-
dividing cells) and subsequent increase in proportion of CD4 and CD8 cells
(radio-resistant). This is best illustrated by the differences in thymocyte
numbers with castrated animals showing at least a 4-fold increase in thymus
size even at 1 week post-treatment. By 2 weeks, the non-castrated animals
showed relative thymocyte normality with regeneration of both DN and DP
thymocytes. However, proportions of thymocytes are not yet equivalent to
the young adult control thymus. Indeed, at 2 weeks, the vast difference in
regulation rates between castrated and non-castrated mice was maximal (by 4
weeks thymocyte numbers were equivalent between treatment groups).
Interestingly, thymus size appears to 'overshoot' the baseline of the
control thymus. Indicative of rapid expansion within the thymus, with the
migration of these newly derived thymocytes not yet occurring (it takes --3-4
weeks for thymocytes to migrate through and out into the periphery).
Therefore, although proportions within each subpopulation are equal,
numbers of thymocytes are building before being released into the periphery.
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Figure 9 illustrates the use of chemical castration compared to surgical
castration in enhancement of T cell regeneration. The kinetics of chemical
castration are much slower than surgical, that is, mice take about 3 weeks
longer to decrease their circulating sex steroid levels. However, chemical
castration is still effective in regenerating the thymus as illustrated in
Figure
9.
EXAMPLE 3 - THYMIC REGENERATION FOLLOWING INHIBITION OF SEX
1o STEROIDS RESULTS IN RESTORATION OF DEFICIENT PERIPHERAL T
CELL FUNCTION.
To determine whether castration can enhance the immune response,
Herpes Simplex Virus (HSV) immunisation was examined as it allows the
study of disease progression and role of CTL (cytotoxic) T cells. Castrated
mice have a qualitatively and quantitatively improved responsiveness to the
virus. Mice were immunised in the footpad and the popliteal (draining)
lymph node analysed at D5 post-immunisation. In addition, the footpad is
removed and homogenised to determine the virus titre at particular time-
points throughout the experiment.
2o At D5 post-immunisation, the castrated mice have a significantly larger
lymph node cellularity than the aged mice (Figure 10). In addition, activated
cell numbers within the lymph nodes are significantly increased when
compared to the aged controls (Figure 10 and 11). Further, activated cell
numbers correlate with that found for the young adult indicating that CTLs
are being activated to a greater extent in the castrated mice, but the young
adult may have an enlarged lymph node due to B cell activation.
There is a 40% bias post-immunisation for V(310 usage for the CTLs in
response to HSV. When aged and castrated mice were analysed for their V(3
expression, it was found that this was predominant in the young adult and
castrated mice. However, no such bias was observed with the aged mice
(Figure 12). This illustrates the vital need for increased production of T
cells
from the thymus throughout life, in order to get maximal immune
responsiveness.
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EXAMPLE 4 - INHIBITION OF SEX STEROIDS ENHANCES UPTAKE OF
NEW HAEMOPOIETIC PRECURSOR CELLS INTO THE THYMUS WHICH
ENABLES CHIMERIC MIXTURES OF HOST AND DONOR LYMPHOID
CELLS (T, B, AND DENDRITIC CELLS)
Previous experiments have shown that microchimera formation plays
an important role in organ transplant acceptance. Dendritic cells have also
been shown to play an integral role in tolerance to graft antigens. Therefore,
the effects of castration on thymic chimera formation and dendritic cell
number was studied.
For the syngeneic experiments, 4 three month old mice were used per
treatment group. All controls were age matched and untreated. For congenic
experiments, 3-4 eight month old mice were used per treatment group. All
controls were age matched and untreated.
Thymic changes following lethal irradiation, foetal liver reconstitution and
castration of syngeneic mice
The total thymus cell numbers of castrated and noncastrated
reconstituted mice were compared to untreated age matched controls and are
summarised in Figure 12. One week after treatment total leukocyte numbers
of both castrated and noncastrated mice were lower than the control but did
not differ significantly from each other. At 3 weeks cell number remained
below control levels, however, those of castrated mice was three fold higher
than the noncastrated mice (p<0.05) (Figure 13A).
Splenic changes following lethal irradiation, syngeneic foetal liver
reconstitution and castration.
Total cell numbers in the spleen were greatly decreased 1 and 3 weeks
after irradiation and reconstitution, in both castrated and noncastrated mice.
There was no statistically significant difference in total spleen cell number
between castrated and noncastrated treatment groups (Figure 13B).
Mesenteric lymph nodes following lethal irradiation, syngeneic foetal liver
reconstitution and castration
Mesenteric lymph node cell numbers were greatly decreased 1 week
after irradiation and reconstitution, in both castrated and noncastrated mice.
However, by the 3 week time point cell numbers had reached control levels.
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There was no statistically significant difference in lymph node cell number
between castrated and noncastrated treatment groups (Figure 13C).
Thymic changes following lethal irradiation, foetal liver reconstitution and
5 castration of congenic mice
In noncastrated mice, there was a profound decrease in thymocyte
number over the 4 week time period, with little or no evidence of
regeneration (Figure 14A). In the castrated group, however, by two weeks
there was already extensive thymopoiesis which by four weeks had returned
1o to control levels, being 10 fold higher than in noncastrated mice. Flow
cytometeric analysis of the thymii with respect to CD45.2 (donor-derived
antigen) demonstrated that no donor derived cells were detectable in the
noncastrated group at 4 weeks, but remarkably, virtually all the thymocytes
in the castrated mice were donor - derived at this time point (Figure 14B).
15 Given this extensive enhancement of thymopoiesis from donor-derived
haemopoietic precursors, it was important to determine whether T cell
differentiation had proceeded normally. CD4, CD8 and TCR defined subsets
were analysed by flow cytometry. There were no proportional differences in
thymocytes subset proportions 2 weeks after reconstitution (Figure 15). This
2o observation was not possible at 4 weeks, because the noncastrated mice were
not reconstituted with donor derived cells. However, at this time point the
thymocyte proportions in castrated mice appear normal.
Two weeks after foetal liver reconstitution there were significantly
more donor-derived, myeloid dendritic cells (defined as CD45.2+ Mac1+
25 CD11C+) in castrated mice than noncastrated mice, the difference was 4-fold
(p<0.05). Four weeks after treatment the number of donor-derived myeloid
dendritic cells remained above the control in castrated mice (Figure 16A).
2 weeks after foetal li ver reconstitution the number of donor derived
lymphoid dendritic cells (defined as CD45.2+Mac1-CD11C+) in the thymus
of castrated mice was double that found in noncastrated mice. Four weeks
after treatment the number of donor-derived lymphoid dendritic cells
remained above the control in castrated mice (Figure 16B).
Immunofluorescent staining for CD11C, epithelium (antikeratin) and
CD45.2 (donor-derived marker) localised dendritic cells to the
corticomedullary junction and medullary areas of thymii 4 weeks after
reconstitution and castration. Using colocalisation software donor-derivation
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of these cells was confirmed (data not shown). This was supported by flow
cytometry data suggesting that 4 weeks after reconstitution approximately
85% of cells in the thymus are donor derived.
Changes in the bone marrow following lethal irradiation, foetal liver
reconstitution and castration
Cell numbers in the bone marrow of castrated and noncastrated
reconstituted mice were compared to those of untreated age matched controls
and are summarised in Figure 17A. Bone marrow cell numbers were normal
two and four weeks after reconstitution in castrated mice. Those of
noncastrated mice were normal at two weeks but dramatically decreased at
four weeks (p<0.05). Although, at this time point the noncastrated mice did
not reconstitute with donor-derived cells.
Flow cytometeric analysis of the bone marrow with respect to CD45.2
(donor-derived antigen) established that no donor derived cells were
detectable in the bone marrow of noncastrated mice 4 weeks after
reconstitution, however, almost all the cells in the castrated mice were
donor- derived at this time point (Figure 17B).
Two weeks after reconstitution the donor-derived T cell numbers of
both castrated and noncastrated mice were markedly lower than those seen
in the control mice (p<0.05). At 4 weeks there were no donor-derived T cells
in the bone marrow of noncastrated mice and T cell number remained below
control levels in castrated mice (Figure 18A).
Donor-derived, myeloid and lymphoid dendritic cells were found at
control levels in the bone marrow of noncastrated and castrated mice 2
weeks after reconstitution. Four weeks after treatment numbers decreased
further in castrated mice and no donor-derived cells were seen in the
noncastrated group (Figure 18B).
Splenic changes following lethal irradiation, foetal liver reconstitution and
castration
Spleen cell numbers of castrated and noncastrated reconstituted mice
were compared to untreated age matched controls and the results are
summarised in Figure 19A. Two weeks after treatment, spleen cell numbers
of both castrated and noncastrated mice were approximately 50% that of the
control. By four weeks, numbers in castrated mice were approaching normal
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levels, however, those of noncastrated mice remained decreased. Analysis of
CD45.2 (donor-derived) flow cytometry data demonstrated that there was no
significant difference in the number of donor derived cells of castrated and
noncastrated mice, 2 weeks after reconstitution (Figure 19B). No donor
derived cells were detectable in the spleens of noncastrated mice at 4 weeks,
however, almost all the spleen cells in the castrated mice were donor
derived.
Two and four weeks after reconstitution there was a marked decrease
in T cell number in both castrated and noncastrated mice (p<0.05) (Figure
20A). Two weeks after foetal liver reconstitution donor-derived myeloid and
Lymphoid dendritic cells (Figures 20 A and B respectively) were found at
control levels in noncastrated and castrated mice. At 4 weeks no donor
derived dendritic cells were detectable in the spleens of noncastrated mice
and numbers remained decreased in castrated mice.
The effects of lethal irradiation, foetal liver reconstitution and castration
on
mesenteric lymph node numbers.
Lymph node cell numbers of castrated and noncastrated, reconstituted
mice were compared to those of untreated age matched controls and are
summarised in Figure 21A. Two weeks after reconstitution cell numbers
were at control levels in both castrated and noncastrated mice. Four weeks
after reconstitution, cell numbers in castrated mice remained at control
levels but those of noncastrated mice decreased significantly (Figure 21B).
Flow cytometry analysis with respect to CD45.2 suggested that there was no
significant difference in the number of donor-derived cells, in castrated and
noncastrated mice, 2 weeks after reconstitution (Figure 21B). No donor
derived cells were detectable in noncastrated mice 4 weeks after
reconstitution. However, virtually all lymph node cells in the castrated mice
were donor-derived at the same time point.
Two and four weeks after reconstitution donor-derived T cell numbers
in both castrated and noncastrated mice were lower than control levels. At 4
weeks the numbers remained low in castrated mice and there were no donor-
derived T cells in the lymph nodes of noncastrated mice (Figure 22).
Two weeks after foetal liver reconstitution donor-derived, myeloid and
lymphoid dendritic cells were found at control levels in noncastrated and
castrated mice (Figures 22 A & B respectively). Four weeks after treatment
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the number of donor-derived myeloid dendritic cells fell below the control,
however, lymphoid dendritic cell number remained unchanged.
General Discussion of the Examples
We have shown that aged thymus, although severely atrophic,
maintains its functional capacity with age, with T cell proliferation,
differentiation and migration occurring at levels equivalent to the young
adult mouse. Although thymic function is regulated by several complex
interactions between the neuro-endocrine-immune axes, the atrophy induced
by sex steroid production exerts the most significant and prolonged effects
illustrated by the extent of thymus regeneration post-castration both of
lymphoid and epithelial cell subsets.
Thymus weight is significantly reduced with age as shown previously
(Hirokawa and Makinodan, 1975, Aspinall, 1997) and correlates with a
significant decrease in thymocyte numbers. The stress induced by the
castration technique, which may result in further thymus atrophy due to the
actions of corticosteroids, is overridden by the removal of sex steroid
influences with the 2-week castrate thymus increasing in cellularity by 20-30
fold from the pre-castrate thymus. By 3 weeks post-castration, the aged
thymus shows a significant increase in both thymic size and cell number,
surpassing that of the young adult thymus presumably due to the actions of
sex steroids already exerting themselves in the 2 month old mouse.
Our data confirms previous findings that emphasise the continued
ability of thymocytes to differentiate and maintain constant subset
proportions with age (Aspinall, 1997). In addition, we have shown
thymocyte differentiation to occur simultaneously post-castration indicative
of a synchronous expansion in thymocyte subsets. Since thymocyte numbers
are decreased significantly with age, proliferation of thymocytes was
analysed to determine if this was a contributing factor in thymus atrophy.
Proliferation of thymocytes was not affected by age-induced thymic
atrophy or by removal of sex-steroid influences post-castration with ---14% of
all thymocytes proliferating. However, the localisation of this division
differed with age: the 2 month mouse thymus shows abundant division
throughout the subcapsular and cortical areas (TN and DP T cells) with some
division also occurring in the medulla. Due to thymic epithelial
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disorganisation with age, localisation of proliferation was difficult to
distinguish but appeared to be less uniform in pattern than the young and
relegated to the outer cortex. By 2 weeks post-castration, dividing
thymocytes were detected throughout the cortex and were evident in the
medulla with similar distribution to the 2 month thymus.
The phenotype of the proliferating population as determined by CD4
and CD8 analysis, was not altered with age or following castration. However,
analysis of proliferation within thymocyte subpopulations, revealed a
significant decrease in proliferation of both the TN and CD8+ cells with age.
Further analysis within the TN subset on the basis of the markers CD44 and
CD25, revealed a significant decrease in proliferation of the TN1
(CD44+CD25-) population which was compensated for by an increase in the
TN2 (CD44 CD25+) population. These abnormalities within the TN
population, reflect the findings by Aspinall (1997). Surprisingly, the TN
subset was proliferating at normal levels by 2 weeks post-castration
indicative of the immediate response of this population to the inhibition of
sex-steroid action. Additionally, at both 2 weeks and 4 weeks post-castration,
the proportion of CD8+ T cells that were proliferating was markedly
increased from the control thymus, possibly indicating a role in the re-
establishment of the peripheral T cell pool.
Thymocyte migration was shown to occur at a constant proportion of
thymocytes with age conflicting with previous data by Scollay et al (1980)
who showed a ten-fold reduction in the rate of thymocyte migration to the
periphery. The difference in these results may be due to the difficulties in
intrathymic FITC labelling of 2 year old thymuses or the effects of adipose
deposition on FITC uptake. However, the absolute numbers of T cells
migrating was decreased significantly as found by Scollay resulting in a
significant reduction in ratio of RTES to the peripheral T cell pool. This
will
result in changes in the periphery predominantly affecting the T cell
repertoire (Mackall et al., 1995). Previous papers (Mackall et al, 1995) have
shown a skewing of the T cell repertoire to a memory rather than naive T cell
phenotype with age. The diminished T cell repertoire however, may not
cope if the individual encounters new pathogens, possibly accounting for the
rise in immunodeficiency in the aged. Obviously, there is a need to re-
establish the T cell pool in immunocompromised individuals. Castration
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allows the thymus to repopulate the periphery through significantly
increasing the production of naive T cells.
In the periphery,. T cell numbers remained at a constant level as
evidenced in the B:T cell ratios of spleen and lymph nodes, presumably due
5 to peripheral homeostasis (Mackall et al., 1995; Berzins et al., 1998).
However, disruption of cellular composition in the periphery was evident
with the aged thymus showing a significant decrease in CD4:CD8 ratios from
2:1 in the young adult to 1:1 in the 2 year mouse, possibly indicative of the
more susceptible nature of CD4+ T cells to age or an increase in production of
10 CD8+ T cells from extrathymic sources. By 2 weeks post-castration, this
ratio
has been normalised, again reflecting the immediate response of the immune
system to surgical castration.
The above findings have shown firstly that the aged thymus is capable
of functioning in a nature equivalent to the pre-pubertal thymus. In this
15 respect, T cell numbers are significantly decreased but the ability of
thymocytes to differentiate is not disturbed. Their overall ability to
proliferate and eventually migrate to the periphery is again not influenced by
the age-associated atrophy of the thymus. However, two important findings
were noted. Firstly, there appears to be an adverse affect on the TN cells in
20 their ability to proliferate, correlating with findings by Aspinall (1997).
This
defect could be attributed to an inherent defect in the thymocytes
themselves. Yet our data, and previous work has shown thymocyte
differentiation, although diminished, still occurs and stem cell entry from
the
BM is also not affected with age (Hirokawa, 1998; Mackall and Gress, 1997).
25 This implicates the thymic stroma as the target for sex steroid action and
consequently abnormal regulation of this precursor subset of T cells.
Secondly, the CD8+ T cells were significantly diminished in their
proliferative capacity with age and, following castration, a significantly
increased proportion of CD8+ T cells proliferated as compared to the 2 month
30 mouse. The proliferation of mature T cells is thought to be a final step
before
migration (Suda and Zlotnik, 1992), such that a significant decrease in CD8+
proliferation would indicate a decrease in their migrational potential. This
hypothesis is supported by our finding that the ratio of CD4:CD8 T cells in
RTES increased with age, indicative of a decrease in CD8 T cells migrating.
Alternatively, if the thymic epithelium is providing the key factor for the
CD8
T cell maintenance, whether a lymphostromal molecule or cytokine
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influence, this factor may be disturbed with increased sex-steroid production.
By removing the influence of sex-steroids, the CD8 T cell population can
again proliferate optimally. Thus, it was necessary to determine, in detail,
the status of thymic epithelial cells pre- and post-castration.
The cortex appears to 'collapse' with age due to lack of thymocytes
available to expand the network of epithelium. The most dramatic change in
thymic epithelium post-castration was the increased network of cortical
epithelium detected by MTS 44, illustrating the significant rise in thymocyte
numbers. At 2 weeks post-castration, KNAs are abundant and appear to
1o accommodate proliferating thymocytes indicating that thymocyte
development is occurring at a rate higher than the epithelium can cope with.
The increase in cortical epithelium appears to be due to stretching of the
thymic architecture rather than proliferation of this subtype since no
proliferation of the epithelium was noted with BrdU staining by
immunofluorescence.
Medullary epithelium is not as susceptible to age influences most
likely due to the lesser number of T cells accumulating in this area (>95% of
thymocytes are lost at the DP stage due to selection events). However, the
aged thymus shows severe epithelial cell disruption distinguished by a lack
of distinction of the cortico-medullary junction with the medullary
epithelium incorporating into the cortical epithelium. By 2 weeks post-
castration, the medullary epithelium, as detected by MTS 10 staining is re-
organised to some extent, however, subpockets are still present within the
cortical epithelium. By 4 weeks post-castration, the cortical and medullary
epithelium is completely reorganised with a distinct cortico-medullary
junction similar to the young adult thymus.
Subtle changes were also observed following castration, most evident
in the decreased expression of MHC class II and blood-thymus barrier
antigens when compared to the pre-castrate thymus. MHCII (detected by
MTS6) is increased in expression in the aged thymus possibly relating to a
decrease in control by the developing thymocytes due to their diminished
numbers. Alternatively, it may simply be due to lack of masking by the
thymocytes, illustrated also in the post-irradiation thymus (Randle and Boyd,
1992) which is depleted of the DP thymocytes. Once thymocyte numbers are
increased following castration, the antigen binding sites are again blocked by
the accumulation of thymocytes thus decreasing detection by
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immunofluorescence. The antigens detecting the blood-thymus barrier
(MTS12, 15 and 16) are again increased in the aged thymus and also revert to
the expression in the young adult thymus post-castration. Lack of masking
by thymocytes and the close proximity of the antigens due to thymic atrophy
may explain this increase in expression. Alternatively, the developing
thymocytes may provide the necessary control mechanisms over the
expression of these antigens thus when these are depleted, expression is not
controlled. The primordial epithelial antigens detected by MTS 20 and MTS
24 are increased in expression in the aged thymus but revert to subpockets of
1o epithelium at the cortico-medullary junction post-castration. This
indicates a
lack of signals for this epithelial precursor subtype to differentiate in the
aged mouse. Removing the block placed by the sex-steroids, these antigens
can differentiate to express cortical epithelial antigens.
The above findings indicate a defect in the thymic epithelium
rendering it incapable of providing the developing thymocytes with the
necessary stimulus for development. However, the symbiotic nature of the
thymic epithelium and thymocytes makes it difficult to ascertain the exact
pathway of destruction by the sex steroid influences. The medullary
epithelium requires cortical T cells for its proper development and
maintenance. Thus, if this population is diminished, the medullary
thymocytes may not receive adequate signals for development. This
particularly seems to affect the CD8+ population. IRFr/-- mice show a
decreased number of CD8+ T cells. It would therefore, be interesting to
determine the proliferative capacity of these cells.
The defect in proliferation of the TN1 subset which was observed
indicates that loss of cortical epithelium affects thymocyte development at
the crucial stage of TCR gene rearrangement whereby the cortical epithelium
provides factors such as IL-7 and SCF necessary for thymopoiesis (Godfrey
and Zlotnik, 1990; Aspinall, 1997). Indeed, IL-7~/- and IL-7R-/- mice show
similar thymic morphology to that seen in aged mice (Wiles et al., 1992;
Zlotnik and Moore, 1995; von Freeden-Jeffry, 1995). Further work is
necessary to determine the changes in IL-7 and IL-7R with age.
In conclusion, the aged thymus still maintains its functional capacity,
however, the thymocytes that develop in the aged mouse are not under the
stringent control by thymic epithelial cells as seen in the normal young
mouse due to the lack of structural integrity of the thymic
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microenvironment. Thus the proliferation, differentiation and migration of
these cells will not be under optimal regulation and may result in the
increased release of autoreactive/immunodysfunctional T cells in the
periphery. The defects within both the TN and particularly, CD8+
populations, may result in the changes seen within the peripheral T cell pool
with age. In addition, we have described in detail, the effects of castration
on
thymic epithelial cell development and reorganisation. The mechanisms
underlying thymic atrophy utilising steroid receptor binding assays and the
role of thymic epithelial subsets in thymus regeneration post-castration are
1o currently under study. Restoration of thymus function by castration will
provide an essential means for regenerating the peripheral T cell pool and
thus in re-establishing immunity in immunosuppressed individuals.
The impact of castration on thymic structure and T cell production
was investigated in animal models of immunodepletion. Specifically,
Example 2 examined the effect of castration on the recovery of the immune
system after sublethal irradiation and cyclophosphamide treatment. These
forms of immunodepletion act to inhibit DNA synthesis and therefore target
rapidly dividing cells. In the thymus these cells are predominantly immature
cortical thymocytes, however all subsets are effected (Fredrickson and Basch
2o 1994). In normal healthy aged mice, the qualitative and quantitative
deviations in peripheral T cells seldom lead to pathological states. However,
major problems arise following severe depletion of T cells because of the
reduced capacity of the thymus for T cell regeneration. Such insults occur in
HIV/AIDS, and particularly following chemotherapy and radiotherapy in
cancer treatment (Mackall et al. 1995).
In both sublethally irradiated and cyclophosphamide treated mice,
castration markedly enhanced thymic regeneration. Castration was carried
out on the same day as and seven days prior to immunodepletion in order to
appraise the effect of the predominantly corticosteroid induced, stress
3o response to surgical castration on thymic regeneration. Although increases
in thymus cellularity and architecture were seen as early as one week after
immunodepletion, the major differences were observed two weeks after
castration. This was the case whether castration was performed on the same
day or one week prior to immunodepletion.
Immunohistology demonstrated that in all instances, two weeks after
castration the thymic architecture appeared phenotypically normal, while
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that of noncastrated mice was disorganised. Pan epithelial markers
demonstrated that immunodepletion caused a collapse in cortical epithelium
and a general disruption of thymic architecture in the thymii of noncastrated
mice. Medullary markers supported this finding. Interestingly, one of the
first features of castration-induced thymic regeneration was a marked
upregulation in the extracellular matrix, identified by MTS 16.
Flow cytometry analysis data illustrated a significant increase in the
number of cells in all thymocyte subsets in castrated mice, corresponding
with the immunofluorescence. At each time point, there was a synchronous
increase in all CD4, CD8 and a(3-TCR - defined subsets following
immunodepletion and castration. This is an unusual but consistent result,
since T cell development is a progressive process it was expected that there
would be an initial increase in precursor cells (contained within the
CD4-CD8- gate) and this may have occurred before the first time point.
Moreover, since precursors represent a very small proportion of total
thymocytes, a shift in their number may not have been detectable. The
effects of castration on other cells, including macrophages and granulocytes
were also analysed. In general there was little alteration in macrophage and
granulocyte numbers within the thymus.
2o In both irradiation and cyclophosphamide models of immunodepletion
thymocyte numbers peaked at every two weeks and decreased four weeks
after treatment. Almost immediately after irradiation or chemotherapy,
thymus weight and cellularity decreased dramatically and approximately 5
days later the first phase of thymic regeneration begun. The first wave of
reconstitution (days 5-14) was brought about by the proliferation of
radioresistant thymocytes (predominantly double negatives) which gave rise
to all thymocyte subsets (Penit and Ezine 1989). The second decrease,
observed between days 16 and 22 was due to the limited proliferative ability
of the radioresistant cells coupled with a decreased production of thymic
precursors by the bone marrow (also effected by irradiation). The second
regenerative phase was due to the replenishment of the thymus with bone
marrow derived precursors (Huiskamp et al. 1983).
It is important to note that in adult mice the development from a HSC
to a mature T cell takes approximately 28 days (Shortman et al. 1990).
Therefore, it is not surprising that little change was seen in peripheral T
cells
up to four weeks after treatment. The periphery would be supported by some
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thymic export, but the majority of the T cells found in the periphery up to
four weeks after treatment would be expected to be proliferating
cyclophosphamide or irradiation resistant clones expanding in the absence of
depleted cells. Several long term changes in the periphery would be
5 expected post-castration including, most importantly, a diversification of
the
TCR repertoire due to an increase in thymic export. Castration did not effect
the peripheral recovery of other leukocytes, including B cells, macrophages
and granulocytes.
Example 4 shows the influence of castration on sygeneic and congenic
10 bone marrow transplantation. Starzl et al. (1992) reported that
microchimeras evident in lymphoid and nonlymphoid tissue were a good
prognostic indicator for allograft transplantation. That is it was postulated
that they were necessary for the induction of tolerance to the graft (Starzl
et
al. 1992). Donor-derived dendritic cells were present in these chimeras and
15 were thought to play an integral role in the avoidance of graft rejection
(Thomson and Lu 1999). Dendritic cells are known to be key players in the
negative selection processes of thymus and if donor-derived dendritic cells
were present in the recipient thymus, graft reactive T cells may be deleted.
In order to determine if castration would enable increased chimera
2o formation, a study was performed using syngeneic foetal liver
transplantation. The results showed an enhanced regeneration of thymii of
castrated mice. These trends were again seen when the experiments were
repeated using congenic (Ly5) mice. Due to the presence of congenic
markers, it was possible to assess the chimeric status of the mice. As early
as
25 two weeks after foetal liver reconstitution there were donor-derived
dendritic
cells detectable in the thymus, the number in castrated mice being four-fold
higher than that in noncastrated mice. Four weeks after reconstitution the
noncastrated mice did not appear to be reconstituted with donor derived
cells, suggesting that castration may in fact increase the probability of
30 chimera formation. Given that castration not only increases thymic
regeneration after lethal irradiation and foetal liver reconstitution and that
it
also increases the number of donor-derived dendritic cells in the thymus,
along-side stem cell transplantation this approach increases the probability
of
graft acceptance.
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It will be appreciated by persons skilled in the art that numerous
variations and/or modifications may be made to the invention as shown in
the specific embodiments without departing from the spirit or scope of the
invention as broadly described. The present embodiments are, therefore, to
be considered in all respects as illustrative and not restrictive.
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