Note: Descriptions are shown in the official language in which they were submitted.
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DIAGNOSTIC INDICATOR OF THYMIC FUNCTION
FIELD OF THE INVENTION
[0001] The present disclosure is in the field of immunology. In particular, it
relates to
diagnosing the ability of a thymus to be reactivated by inhibition of the
effects of sex steroids
on the thymus.
BACKGROUND OF THE INVENTION
(0002] The thymus is arguably the major organ in the immune system because it
is the
primary site of production of T lymphocytes. Its role is to attract
appropriate bone marrow-
derived precursor cells from the blood, and induce their commitment to the T
cell lineage,
including the gene rearrangements necessary for the production of the T cell
receptor for
antigen (TCR). Associated with this is a remarkable degree of cell division to
expand the
number of T cells and hence increase the likelihood that every foreign antigen
will be
recognized and eliminated. This enormous potential diversity means that for
any single
antigen the body might encounter, multiple lymphocytes will be able to
recognize it with
varying degrees of binding strength (affinity) and respond to varying degrees.
[0003] A strange feature of T cell recognition of antigen, however, is that,
unlike B
cells, the TCR only recognizes peptide fragments physically associated with
MHC molecules.
Normally this is self MHC (i.e., non-foreign MHC) and this ability is selected
for in the
thymus. This process is called positive selection and is an exclusive feature
of cortical
epithelial cells. If the TCR fails to bind to the self MHC/peptide complexes,
the T cell dies by
"neglect" - it needs some degree of signaling through the TCR for its
continued maturation.
[0004] Following selection in the cortex, the developing thymocytes acquire
functional maturation and migratory capacity and exit into the blood stream as
naive (not yet
having contacted antigen) T cells. They circulate between the lymph and blood
in search of
antigen. If after 3-4 weeks they haven't been stimulated, they become
susceptible to deletion
from the peripheral T cell pool by other recent thymic emigrants. This system
of thymic
export and peripheral T cell replacement provides a continual replenishment of
the quality of
T cells, with homeostasis maintaining the appropriate levels.
[0005] While the thymus is fundamental for a functional immune system,
releasing
~l% of its T cell content into the bloodstream per day, one of the apparent
anomalies of
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mammals is that this organ undergoes severe atrophy as a result of sex steroid
production.
This can begin even in young children but is profound from the time of
puberty. For normal
healthy individuals this loss of production and release of new T cells does
not always have
immediate clinical consequences. In fact although the aged thymus is atrophic
and consists of
less than 1 % of its young counterpart, it still continues to release a very
low level of new T
cells into the blood stream.
[0006] These are insufficient, however, to maintain the optimal levels of
peripheral T
cell subsets. But this does mean that the thymus is not completely dormant,
raising the
possibility that it could be the target of therapy. With progressive aging,
the decline in thymic
export means that the status of peripheral T cells undergoes progressive
change both
quantitatively and qualitatively. On the one hand there is a gradual decrease
in absolute T cell
numbers in the blood with age as they die off through lack of stimulation. On
the other hand,
with each antigen contact, the relevant antigen-specific naive T cells (those
which have not
yet encountered antigen) are stimulated and proliferate. A subset will
progress to be effector
cells and rid the body of the pathogen, but these eventually die through
antigen-induced cell
death. Another subset will convert to memory cells and provide long term
protection against
future contacts with that pathogen. Hence, there is a decrease in the levels
of naive T cells
and thus a reduced ability to respond to antigen.
[0007) Aging also results in a selective decline in Th cells (characterized by
expression of CD4) relative to Tc cells (expressing CD8) and imbalances in the
ratios of Thl
to Th2 cells. This does not occur in the normal young because, as mentioned
above, there is a
continual supply of new T cells being exported from the thymus, which in turn
provides a
continual replenishment of the naive T cell pool in the periphery.
[0008] Aging is not the only condition which results in T cell loss - this
also occurs
very severely for example in H1V/AIDS and following chemotherapy or
radiotherapy. Again,
in the young with an active thymus, the recovery of the immune system (through
recovery of
T cell mediated immunity) occurs relatively quickly (2-3 months), compared to
post-puberty,
when it can take 1-2 years because of the atrophic thymus.
[0009] There are thus several parameters which can influence the nature and
extent of
immune responses: the level and type of antigen, the site of vaccination, the
availability of
appropriate APC (antigen presenting cells), the general health of the
individual and the status
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of the T and B cell pools. Of these, T cells are the most vulnerable because
of the marked sex
steroid induced shut-down in thymic export which becomes profound from the
onset of
puberty.
[0010] Any vaccination program should therefore only be logically undertaken
when
S the level of potential responder T cells is optimal in terms of both the
level of naive T cells
representing a broad repertoire of specificity and the correct ratios of Thl
to Th2 cells and Th
to Tc cells. The level and type of cytokines should also be manipulated to be
appropriate for
the desired response.
[0011 ] The ability to reactivate the atrophic thymus through inhibition of
LHRH
signaling to the pituitary provides a potent means of generating a new cohort
of naive T cells
with a diverse repertoire of TCR types. This process effectively reverts the
thymus to its
prepubertal state, and does so by using the normal regulatory molecules and
pathways which
lead to optimal thymopoiesis.
[0012] The thymus is influenced to a great extent by its bi-directional
communication
1 S 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.
[0013] 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 tumor load in later life (Hirokawa, 1998).
[0014] 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
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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).
[0015] 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 (bone marrow
transplant), they
appear to show a bias towards memory cells due to the aged peripheral
microenvironment,
coupled to poor thymic production of naive T cells.
[0016] 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) 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, utilizing radiation
chimeras, have
shown that bone marrow (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 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 TCRy chain gene
rearrangement.
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Skewing of developing TCR repertoire towards, or away from, specific antigens.
[0017] The ability to enhance the uptake into the thymus of haematopoietic
precursor
cells means that the nature and type of dendritic cells can be manipulated.
For example the
precursors can be transfected with specific genes) which eventually become
expressed in the
5 dendritic cells in the thymus (and elsewhere in the body). Such genes can
include those which
encode specific antigens for which an immune response would be detrimental,
e.g.,
autoimmune diseases, allergies and graft antigens
[0018] The genes can also encode antigens (also as peptides) for which an
immune
response is desired, e.g., tumor cells and invading microorganisms. In the
latter case the level
and affinity of the peptide would be manipulated to be low enough so as not to
induce
negative selection, but high enough to promote positive selection. We have
shown that
positive selection can involve multiple cell types: the cortical epithelium
provides the specific
differentiation molecules, and third party cells the MHC/peptide ligands.
[0019] The precursors can also be genetically modified by adding or deleting
genes,
such as those coding for soluble regulatory molecules, such as chemokines,
cytokines and
other molecules affecting any aspect of thymopoiesis and T cell development,
activation,
positive or negative selection, migration, and general status. This approach
can be used to
promote or retard thymic development or T cell responsiveness. It can be used
to skew the T
cell repertoire to specific antigens to create, for example, anti-viral and
anti-tumor defenses.
This approach can also be used to modulate the nature, organization and
function of the
thymic microenvironment.
Induction of tolerance
[0020] The most effective means of generating tolerance to self is through
intra
thymic deletion (or anergy or induction of negative regulatory cells) of the
potentially self
reactive cells through negative selection, mediated most efficiently by
intrathymic dendritic
cells. As a corollary, the establishment of tolerance to exogenous or nominal
antigens could
be best achieved if dendritic cells expressing this antigen could be
incorporated into the
thymus. This form of tolerance may also be made more effective through the
advent of
inhibitory immunoregulatory cells. The mechanisms underlying the development
of the latter,
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however, are poorly understood, but again occur in the thymus and could
involve dendritic
cells.
[0021 ] In the case of hyperreactive T cells for which the target antigen is
known, the
haematopoietic stem cells can be transfected with the gene encoding the
specific antigen.
When these cells develop into dendritic cells in the thymus they will delete
any new T cells
arising which are potentially reactive to the nominal antigen.
[0022] The enormous clinical benefits to be gained through restoration of
thymic
function, represent an important strategy for the treatment of
immunodeficiencies, particularly
in the elderly, HIV patients and patients following chemotherapy. Furthermore
patients who
have functionally abnormal T cells can now be treated to remove all T cells,
thereby stopping
the disease, and then have their normal immunity restored by reactivation of
thymic function
by inhibition of sex steroid production. In the case of vaccination programs,
the reactivation
of the thymus will have profound improvements on the status of T cells and
hence the nature,
extent and quality of immune responses. Additionally, through presentation of
donor cells
during reactivation of the thymus, T cell populations can be modified to allow
for tolerance of
allogeneic and xenogeneic grafts. Moreover, regenerating populations of T
cells can be
genetically modified through gene therapy during thymic reactivation.
SUMMARY OF THE INVENTION
[0023] The present disclosure provides a diagnostic method for determining the
susceptibility of a thymus to regeneration by inhibition of sex steroid
production. In a
preferred embodiment, the method provides an early determination of this
susceptibility,
preferably within a week, more preferably within 4 to 5 days, even more
preferably within 2-3
days, and most preferably with 24 hours of initiation of inhibition.
[0024] In a particular embodiment sex steroid mediated signaling to the thymus
is
blocked by the administration of agonists or antagonists of LHRH, anti-
estrogen antibodies,
anti-androgen antibodies, passive (antibody) or active (antigen) anti-LHRH
vaccinations, or
combinations thereof ("blockers").
[0025] In a preferred embodiment, inhibition is caused by administering an
LHRH
agonist. Preferably a quick-acting antagonist such as Abarelix or Cetrorelix
is administered.
In an alternative embodiment, inhibition is caused by administering an LHRH
agonist such as
Zoladex or Leupron.
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[0026] In a preferred embodiment, the blocker(s) is administered by a
sustained
peptide-release formulation. Examples of sustained peptide-release
formulations are provided
in WO 98/08533, the entire contents of which are incorporated herein by
reference.
[0027] In one embodiment, the diagnosis is accomplished by measuring the
amount of
S thymic induced factors in a blood sample of the patient before and after
initiation of
inhibition.
[0028] In yet another embodiment, the invention is used to identify previously
unidentified thymic factors.
[0029] In another embodiment, the diagnosis is accomplished by measuring
thymic
activity. In addition to the above, this will be achieved by determining
levels of newly
produced T cells identified by the presence in these cells of small circles of
DNA termed T
cell receptor excision circles (TREC's). These TREC's are produced as a normal
part of T cell
development in the thymus, in particular as a result of gene rearrangements in
the formation
of the T cell receptor for antigen. Basic increases in total T cell number (as
measured by flow
cytometry staining for CD3, CD4 and CD8) and shifts in their in vitro
responsiveness to
stimulation with anti-CD3 cross-linking can also be used to monitor thymic
function but they
are expected to take several days to weeks before any changes may be
detectable.
DESCRIPTION OF THE FIGURES
[0030] Figure 1 A and B: Changes in thymocyte number pre- and post-castration.
Thymus atrophy results in a significant decrease in thymocyte numbers with
age. Aged (2-
year old) mice were surgically castrated and analysed for (A) thymus weight in
relation to
body weight and (B) total cells per thymus, at 2-4 weeks post-castration. A
significant
decrease in thymus weight and cellularity was seen with age compared to young
adult (2-
month) mice. This was restored by castration. At 3-weeks post-castration
thymic hypertrophy
was observed and was returned to young adult levels by 4-weeks post-
castration. Results are
expressed as mean +1 SD of 4-8 mice per group. ** = p <_O1; *** = p <_0.001
compared to
young adult and post-castration mice.
[0031 ] Figure 2 A-C: Aged (2-year old) mice were surgically castrated and
analysed
at 2 and 4 weeks post-castration for peripheral lymphocyte populations. (A)
Total lymphocyte
numbers in the spleen. Spleen numbers remain constant with age and post-
castration. (B) The
ratio of B cells to T cells did not change with age or post-castration,
however (C) a significant
decrease in the CD4+:CD8+ T cell ratio was seen with age. This was restored by
4-weeks post-
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castration. Data is expressed as mean~lSD of 4-8 mice per group. *** = p
<_0.001 compared
to young adult (2-month) and 4-week post-castrate mice.
[0032] Figure 3: Fluorescence Activated Cell Sorter (FACS) profiles of CD4 vs.
CD8
thymocyte populations with age and post-castration. Aged (2-year old) mice
were castrated
and the thymocyte subsets analysed based on the markers CD4 and CDB.
Representative
FACS profiles of CD4lCD8 dot plots are shown for CD4-CD8-DN, CD4+CD8+DP,
CD4+CD8-
and CD4-CD8+ SP thymocytes. No difference was seen in the proportions of any
CD4/CD8
defined subset with age or post-castration.
[0033] Figure 4: Aged (2-year old) mice were castrated and injected with a
pulse of
bromodeoxyuridine (BrdU) to determine levels of proliferation. Representative
histogram
profiles of the proportion of BrdU+ cells within the thymus with age and post-
castration are
shown. No difference in the proportion of proliferating cells within the total
thymus was
observed with age or post-castration.
[0034] Figure 5 A-D: 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)
However, a significant decrease in the proportion of DN (CD4-CD8-) thymocytes
proliferating was seen with age. Post-castration, this was restored and a
significant increase in
proliferation within the CD4-CD8+ SP thymocytes was observed. (C) No change in
the total
proportion of BrdU+ cells within the TN subset was seen with age or post-
castration. However
(D) the significant decrease in proliferation of the TNl (CD44+CD25-)
subpopulation with
age is not returned to normal levels by 4 weeks post.-_castration. Results are
expressed as
mean~lSD of 4-8 mice per group. * = p <0.05; *** = p <_0.001 compared to young
adult (2-
month) mice.
[0035] Figure 6 A-C: Aged (2-year old) mice were castrated and were injected
intrathymically with FITC to determine thymic export rates. The number of
FITC+ cells in the
periphery were calculated 24 hours later. (A) A significant decrease in recent
thymic emigrant
(RTE) cell numbers was observed with age. Following castration, these values
had
significantly increased by 2 weeks post-cx. (B) The rate of emigration
(export/total thymus
cellularity) remained constant with age but was significantly reduced at 2
weeks post-cx. (C)
With age, a significant increase in the ratio of CD4+ to CD8+ RTE was seen and
this was
normalised by 1-week post-cx. Results are expressed as mean~lSD of 4-8 mice
per group. **
= p <_0.01; *** = p <_0.001 compared to young adult mice. ~ = p _<0.001
compared to castrated mice.
[0036] Figure 7 A-C: Changes in thymus (A), spleen (B) and lymph node (C) cell
numbers following treatment with cyclophosphamide, a chemotherapy agent. Young
(3-
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month old) mice were depleted of lymphocytes using cyclophosphamide. Mice were
either
sham-castrated or castrated on the same day as cyclophosphamide treatment. (A)
A significant
increase in thymus cell number was observed in castrated mice compared to sham-
castrated
mice. (B) Castrated mice also showed a significant increase in spleen cell
number at 1-week
post-cyclophosphamide treatment. (C) A significant increase in lymph node
cellularity was
also observed with castrated mice at 1-week post-treatment. Results are
expressed as
meantlSD of 4-8 mice per group. *** = p <_0.001 compared to castrated mice.
[0037] Figure 8 A-C: Changes in thymus (A), spleen (B) and lymph node (C) cell
numbers following irradiation and castration on the same day. Note the rapid
expansion of the
thymus in castrated animals when compared to the non-castrate group at 2 weeks
post-
treatment. No difference in spleen (B) or lymph node (C) cell numbers was seen
with
castrated mice. Lymph node cell numbers were still chronically low at 2-weeks
post-treatment
compared to control mice. Results are expressed as mean~l SD of 4-8 mice per
group. * = p
0.05 compared to control mice; *** = p <_0.001 compared to control and
castrated mice.
[0038] Figure 9 A-C: Changes in thymus (A), spleen (B) and lymph node (C) cell
numbers following irradiation (625 Rads) one week after surgical castration. A
significant
increase in thymus regeneration was observed with castration (A). No
difference in spleen (B)
or lymph node (C) cell numbers was seen with castrated mice. Lymph node cell
numbers
were still chronically low at 2-weeks post-treatment compared to control mice.
Results are
expressed as mean + 1 SD of 4-8 mice per group. + = p __<0.05; ** = p <_0.01
compared to
control mice; *** = p <_0.001 compared to control and castrated mice.
[0039] Figure 10: Changes in thymus, spleen and lymph node cell numbers
following
treatment with cyclophosphamide, a chemotherapy agent, and surgical or
chemical castration
performed on the same day. 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. (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.
[0040] Figure 11 A and B: Lymph node cellularity following foot-pad
immunization
with Herpes Simplex Virus-1 (HSV-1). Note the increased cellularity in the
aged post-
castration as compared to the aged non-castrated group (A). Bottom graph
illustrates the
overall activated cell number as gated on CD25 vs. CD8 cells by FAGS (B).
[0041 ] Figure 12 A-C: V~310 expression on CTL (cytotoxic T lymphocytes) in
activated LN (lymph nodes) following HSV-1 inoculation. Despite the normal
V(310
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responsiveness in aged mice overall, in some mice a complete loss of V~310
expression was
observed. Representative histogram profiles are shown. Note the diminution of
a clonal
response in aged mice and the reinstatement of the expected response post-
castration.
[0042] Figure 13 A-C: Castration restores responsiveness to HSV-1
immunization.
5 (A) Aged mice showed a significant reduction in total lymph node cellularity
post-infection
when compared to both the young and post-castrate mice. (B) Representative
FAGS profiles
of activated (CD8+CD25~ cells in the LN of HSV-1 infected mice. No difference
was seen in
proportions of activated CTL with age or post-castration. (C) The decreased
cellularity within
the lymph nodes of aged mice was reflected by a significant decrease in
activated CTL
10 numbers. Castration of the aged mice restored the immune response to HSV-1
with CTL
numbers equivalent to young mice. Results are expressed as mean ~1 SD of 8-12
mice. ** = p
<_0.01 compared to both young (2-month) and non-castrated mice.
[0043] Figure 14: Popliteal lymph nodes were removed from mice immunized with
HSV-1 and cultured for 3 days. CTL assays were performed with non-immunized
mice as
control for background levels of lysis (as determined by SICr-release).
Results are expressed
as mean of 8 mice, in triplicate ~1SD. Aged mice showed a significant (p
<_0.01, *) reduction
in CTL activity at an E:T ratio of both 10:1 and 3:1 indicating a reduction in
the percentage of
specific CTL present within the lymph nodes. Castration of aged mice restored
the CTL
response to young adult levels. * = p <_0.01 compared to young adult and post-
castrate aged
mice.
[0044] Figure 15 A and B: Analysis of CD4+ T cell help and V~3 TCR response to
HSV-1 infection. Popliteal lymph nodes were removed on DS past-HSV-1 infection
and
analysed ex-vivo for the expression of (a) CD25, CD8 and specific TCRV~i
markers and (b)
CD4/CD8 T cells. (A) The percentage of activated (CD25+) CD8+ T cells
expressing either
V(310 or V/38.1 is shown as mean ~1SD for 8 mice per group. No difference was
observed
with age or post-castration. (B) A decrease in CD4/CD8 ratio in the resting LN
population
was seen with age. This was restored post-castration. Results are expressed as
meantlSD of
8 mice per group. *** = p <_0.001 compared to young and castrate mice.
[0045] Figure 16 A-D: Changes in thymus (A), spleen (B), lymph node (C) and
bone
marrow (D) 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
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per treatment group and time point). Castrated mice had significantly
increased congenic
(Ly5.2) cells compared to non-castrated animals (data not shown).
[0046] Figure 17 A and B: Changes in thymus cell number in castrated and
noncastrated mice after fetal 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
thymuses 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.
[0047] Figure 18: FACS profiles of CD4 versus CD8 donor derived thymocyte
populations after lethal irradiation and fetal liver reconstitution, followed
by surgical
castration. Percentages for each quadrant are given to the right of each plot.
The age matched
control profile is of an eight month old LyS.l 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.
[0048] Figure 19 A and B: Myeloid and lymphoid dendritic cell (DC) number
after
lethal irradiation, fetal liver reconstitution and castration. (n= 3-4 mice
for each test group.)
Control (white) bars on the following graphs are based on the 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.
[0049] Figure 20 A and B: Changes in total and CD45.2+ bone marrow cell
numbers
in castrated and noncastrated mice after fetal liver reconstitution. n=3-4
mice for each test
group. (A) Total cell number-Two weeks after reconstitution bone marrow cell
numbers had
normalized 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 noncastrated 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
AM~'~JC~Ep ElIEET
IPE,q~,,~~
CA 02462758 2004-04-O1 PCT/1801/02351
Received 13 May 2002
12
number remained high in castrated mice at four weeks. There were no donor-
derived cells in
the noncastrated mice at the same time point.
[0050] Figure 21 A-C: Changes in T cells and myeloid and lymphoid derived
dendritic cells (DC) in bone marrow of castrated and noncastrated mice after
fetal 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.
[0051 ] Figure 22 A and B: Change in total and donor (CD45.2+) lymph node cell
numbers in castrated and noncastrated mice after fetal 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 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 in castrated
mice at four
weeks. There were no donor-derived cells in the noncastrated mice at.the same
time point.
[0052] Figure 23 A-C: Splenic T cells and myeloid and lymphoid derived
dendritic
cells (DC) after fetal 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 (CD45.2+)
myeloid dendritic cells-two and four weeks after reconstitution DC numbers
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 (CD45.2+)
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.
[0053] Figure 24 A and B: Changes in total and donor (CD45.2~ lymph node cell
numbers in castrated and noncastrated mice after fetal liver reconstitution.
(n=3-4 for each
AME~~ED SHEET
~~EA/AU
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13
test group.) (A) Total cell numbers-Two weeks after reconstitution cell
numbers were at
normal levels and there was no significant difference 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 donor 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.
[0054] Figure 25 A-C: Changes in T cells and myeloid and lymphoid derived
dendritic cells (DC) in the mesenteric lymph nodes of castrated and non-
castrated mice after
fetal liver reconstitution. (n=3-4 mice for each test group.) Control
(striped) bars are the
number of T cells and dendritic cells found in untreated age matched mice. (A)
T cell
numbers were reduced two and four weeks after reconstitution in both castrated
and
noncastrated mice. (B) Donor derived myeloid dendritic cells were normal in
both castrated
and noncastrated mice. At four weeks they were decreased. At two weeks there
was no
1 S 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.
[0055] Figure 26: The phenotypic composition of peripheral blood lymphocytes
was
analyzed in human patients (all >60 years) undergoing LHRH agonist treatment
for prostate
cancer. Patient samples were analyzed before treatment and 4 months after
beginning LHRH
agonist treatment. Total lymphocyte cell numbers per ml of blood were at the
lower end of
control values before treatment in all patients. Following treatment, 6/9
patients showed
substantial increases in total lymphocyte counts (in some cases a doubling of
total cells was
observed). Correlating with this was an increase in total T cell numbers in
6/9 patients.
Within the CD4+ subset, this increase was even more pronounced with 8/9
patients
demonstrating increased levels of CD4 T cells. A less distinctive trend was
seen within the
CD8+ subset with 4/9 patients showing increased levels, albeit generally to a
smaller extent
than CD4+ T cells.
[0056] Figure 27: Analysis of human patient blood before and after LHRH-
agonist
treatment demonstrated no substantial changes in the overall proportion of T
cells, CD4 or
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14
CD8 T cells, and a variable change in the CD4:CD8 ratio following treatment.
This indicates
the minimal effect of treatment on the homeostatic maintenance of T cell
subsets despite the
substantial increase in overall T cell numbers following treatment. All values
were
comparative to control values.
[0057] Figure 28: Analysis of the proportions of B cells and myeloid cells
(NK, NKT
and macrophages) within the peripheral blood of human patients undergoing LHRH
agonist
treatment demonstrated a varying degree of change within subsets. While NK,
NKT and
macrophage proportions remained relatively constant following treatment, the
proportion of B
cells was decreased in 4/9 patients.
[0058] Figure 29: Analysis of the total cell numbers of B and myeloid cells
within the
peripheral blood of human patients post-treatment showed clearly increased
levels of NK (5/9
patients), NKT (4/9 patients) and macrophage (3/9 patients) cell numbers post-
treatment.
B cell numbers showed no distinct trend with 2/9 patients showing increased
levels; 4/9
patients showing no change and 3/9 patients showing decreased levels.
[0059] Figure 30 A and B: The major change seen post-LHRH agonist treatment
was
within the T cell population of the peripheral blood. In particular there was
a selective
increase in the proportion of naive (CD45RA+) CD4+ cells, with the ratio of
naive
(CD45RA+) to memory (CD45R0+) in the CD4+ T cell subset increasing in 6/9 of
the human
patients.
[0060] Figure 31: Decrease in the impedance of skin using various laser pulse
energies. There is a decrease in skin impedance in skin irradiated at energies
as low as 10 mJ,
using the fitted curve to interpolate data.
(0061 ] Figure 32: Permeation of a pharmaceutical through skin. Permeability
of the
skin, using insulin as a sample pharmaceutical, was greatly increased through
laser irradiation.
[0062] Figure 33: Change in fluorescence of skin over time after the addition
of 5-
aminolevulenic acid (ALA) and a single impulse transient to the skin. The peak
of intensity
occurs at about 640 run and is highest after 210 minutes (dashed line) post-
treatment.
[0063] Figure 34: Change in fluorescence of skin over time after the addition
of S-
aminolevulenic acid (ALA) without an impulse transient. There is little change
in the
intensity at different time points.
CA 02462758 2004-04-O1
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[0064] Figure 35: Comparison of change in fluorescence of skin after the
addition of
5-aminolevulenic acid (ALA) and a single impulse transient under various peak
stresses. The
degree of permeabilization of the stratum corneum depends on the peak stress.
DETAILED DESCRIPTION OF THE INVENTION
5 [0065] A characteristic feature of thymic function is that while it is of
fundamental
importance to the establishment and maintenance of the immune system and hence
to the
defense against infection and disease, it characteristically undergoes a
profound age-
dependent decrease in function to less than 5% of it maximal capacity. This
becomes most
pronounced following puberty, implicating a role for sex steroids.
10 [0066] Inhibition of sex steroids results, either directly or indirectly,
in a major
reactivation of thymic function, effectively reversing the atrophy. Given the
broad range of
patient age, diseases and treatments, however, it is anticipated that many
patients will respond
differently to this treatment, including some very poorly. Hence a new
diagnostic early
indicator of this responsiveness of the thymus to activation in the absence of
sex steroids is
15 provided to formulate rational clinical management of T cell based
disorders.
(0067] Since the thymus is an endocrine organ, reactivation of thymic function
involves release of not only new T cells into the blood stream after 2-4
weeks, but prior to this
the thymus will also release increased levels of cytokines, even within hours
of reactivation.
These will be detectable in the blood or plasma. The present disclosure
utilizes these released
cells and molecules to detect the degree of response of a patient's thymus to
inhibition of sex
steroids. Provided here is a set of diagnostic techniques for making this
determination.
Disruption Of Sex Steroid Signaling To The Thymus
[0068] As will be readily understood, sex steroid signaling to the thymus can
be
disrupted in a range of ways well known to those of skill in the art, some of
which are
described herein. For example, inhibition of sex steroid production or
blocking of one or
more sex steroid receptors within the thymus will accomplish the desired
disruption, as will
administration of sex steroid agonists or antagonists, or active (antigen) or
passive (antibody)
anti-sex steroid vaccinations. Inhibition of sex steroid production can also
be achieved by
administration of one or more sex steroid analogs. In some clinical cases,
permanent removal
of the gonads via physical castration may be appropriate.
CA 02462758 2004-04-O1 PCT/IBO1/02351
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16
[0069] In a preferred embodiment the sex steroid signaling to the thymus is
disrupted
by administration of a sex steroid analog, or preferably an analog of
luteinizing hormone-
releasing hormone (LHRH). Sex steroid analogs and their use in therapies and
chemical
castration are well known. Such analogs include, but are not limited to,
Abarelix (LJS Pat. No.
6,197,337), Cetrorelix, Deslorelin (described in U.S. Patent No. 4,218,439),
Eulexin
(described in FR7923545, W086/01105 and PT100899), Goserelin (described in US
Pat. No.
4,100,274, US Pat. No. 4,128,638, GB9112859 and GB9112825), Leuprolide
(described in
US Pat. No. 4,490,291, US Pat. No. 3,972,859, US Pat. No. 4,008,209, US Pat.
No.
4,005,063, DE2509783 and US Pat. No. 4,992,421), Dioxalan derivatives such as
are
described in EP 413209, Triptorelin (described in US Pat. No. 4,010,125, US
Pat. No.
4,018,726, US Pat. No. 4,024,121, EP 364819 and US Pat. No. 5,258,492),
Meterelin
(described in EP 23904), Buserelin (described in US Pat. No. 4,003,884, US
Pat. No.
4,118,483 and US Pat. No. 4,275,001), Histrelin (described in EP217659),
Nafarelin
(described in US Pat. No. 4,234,571, W093/15722 and EP52510), Lutrelin
(described in US
Pat. No. 4,089,946), Leuprorelin (described in Plosker et al.) and LHRH
analogs such as are
described in EP181236, US Pat. No. 4,608,251, US Pat. No. 4,656,247, US Pat.
No:
4,642,332, US Pat. No. 4,010,149, US Pat. No. 3,992,365 and US Pat. No.
4,010,149. The
disclosures of each the references referred to above are incorporated herein
by reference.
[0070] While the stimulus for thymic regeneration is fundamentally based on
the
inhibition of the effects of sex steroids and/or the direct effects of the
LHRH analogues, it
may be necessary to include additional substances which can act in concert to
enhance the
thymic effect. Such compounds could include but not be limited to Interleukin
7, members of
the epithelium and fibroblast growth factor familes and keratinocyte growth
factor. It is
envisaged that these additional compounds would only be given once at the
initial LHRH
analogue application. In addition steroid receptor based modulators, which may
be targeted to
be thymic specific could be developed and used.
Deliver~~ents For Chemical Castration
[0071 ] Delivery of the compounds of this invention can be accomplished via a
number
of methods known to persons skilled in the art. One standard procedure for
administering
chemical inhibitors to inhibit sex steroid signaling to the thymus utilizes a
single dose of an
~aMEivt~E,~ gNEE7
IP~q/.A~U
CA 02462758 2004-04-O1
WO 02/030256 PCT/IBO1/02351
17
LHRH agonist that is effective for three months. For this a simple one-time
i.v. or i.m.
injection would not be sufficient as the agonist would be cleared from the
patient's body well
before the three months are over. Instead, a depot injection or an implant may
be used, or any
other means of delivery of the inhibitor that will allow slow release of the
inhibitor.
Likewise, a method for increasing the half life of the inhibitor within the
body, such as by
modification of the chemical, while retaining the function required herein,
may be used.
[0072] Examples of more useful delivery mechanisms include, but are not
limited to,
laser irradiation of the skin, and creation of high pressure impulse
transients (also called stress
waves or impulse transients) on the skin, each method accompanied or followed
by placement
of the compounds) with or without Garner at the same locus. A preferred method
of this
placement is in a patch placed and maintained on the skin for the duration of
the treatment.
[0073] One means of delivery utilizes a laser beam, specifically focused, and
lasing at
an appropriate wavelength, to create small perforations or alterations in the
skin of a patient.
See U.S. Pat. No. 4,775,361, U.S. Pat. No. 5,643,252, U.S. Pat. No. 5,839,446,
and U.S. Pat.
No. 6,056,738, all of which are incorporated herein by reference. In a
preferred embodiment,
the laser beam has a wavelength between 0.2 and 10 microns. More preferably,
the
wavelength is between about 1.5 and 3.0 microns. Most preferably the
wavelength is about
2.94 microns. In one embodiment, the laser beam is focused with a lens to
produce an
irradiation spot on the skin through the epidermis of the skin. In an
additional embodiment,
the laser beam is focused to create an irradiation spot only through the
stratum corneum of the
skin.
[0074] Several factors may be considered in defining the laser beam, including
wavelength, energy fluence, pulse temporal width and irradiation spot-size. In
a preferred
embodiment, the energy fluence is in the range of 0.03-100,000 J/cm2. More
preferably, the
energy fluence is in the range of 0.03 - 9.6 J/cmz. The beam wavelength is
dependent in part
on the laser material, such as Er:YAG. The pulse temporal width is a
consequence of the
pulse width produced by, for example, a bank of capacitors, the flashlamp, and
the laser rod
material. The pulse width is optimally between 1 fs (femtosecond) and 1,000
~s.
[0075] According to this method the perforation or alteration produced by the
laser
need not be produced with a single pulse from the laser. In a preferred
embodiment a
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18
perforation or alteration through the stratum corneum is produced by using
multiple laser
pulses, each of which perforates or alters only a fraction of the target
tissue thickness.
[0076] To this end, one can roughly estimate the energy required to perforate
or alter
the stratum corneum with multiple pulses by taking the energy in a single
pulse and dividing
S by the number of pulses desirable. For example, if a spot of a particular
size requires 1 J of
energy to produce a perforation or alteration through the entire stratum
corneum, then one can
produce qualitatively similar perforation or alteration using ten pulses, each
having 1/1 Oth the
energy. Because it is desirable that the patient not move the target tissue
during the
irradiation (human reaction times are on the order of 100 ms or so), and that
the heat produced
during each pulse not significantly diffuse, in a preferred embodiment the
pulse repetition rate
from the laser should be such that complete perforation is produced in a time
of less than 100
ms. Alternatively, the orientation of the target tissue and the laser can be
mechanically fixed
so that changes in the target location do not occur during the longer
irradiation time.
[0077] To penetrate the skin in a manner that induces little or no blood flow,
skin is
perforated or altered through the outer surface, such as the stratum corneum
layer, but not as
deep as the capillary layer. The laser beam is focussed precisely on the skin,
creating a beam
diameter at the skin in the range of approximately 0.5 microns - 5.0 cm.
Optionally, the spot
can be slit-shaped, with a width of about 0.05-0.5 mm and a length of up to
2.5 mm. The
width can be of any size, being controlled by the anatomy of the area
irradiated and the
desired permeation rate of the fluid to be removed or the pharmaceutical to be
applied. The
focal length of the focusing lens can be of any length, but in one embodiment
it is 30 mm.
[0078] By modifying wavelength, pulse length, energy fluence (which is a
function of
the laser energy output (in Joules) and size of the beam at the focal point
(cmz)), and
irradiation spot size, it is possible to vary the effect on the stratum
corneum between ablation
(perforation) and non-ablative modification (alteration). Both ablation and
non-ablative
alteration of the stratum corneum result in enhanced permeation of
subsequently applied
pharmaceuticals.
[0079] For example, by reducing the pulse energy while holding other variables
constant, it is possible to change between ablative and non-ablative tissue-
effect. Using an
Er:YAG laser having a pulse length of about 300 ps, with a single pulse or
radiant energy and
irradiating a 2 mm spot on the skin, a pulse energy above approximately 100 mJ
causes partial
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19
or complete ablation, while any pulse energy below approximately 100 mJ causes
partial
ablation or non-ablative alteration to the stratum corneum. Optionally, by
using multiple
pulses, the threshold pulse energy required to enhance permeation of body
fluids or for
pharmaceutical delivery is reduced by a factor approximately equal to the
number of pulses.
[0080] Alternatively, by reducing the spot size while holding other variables
constant,
it is also possible to change between ablative and non-ablative tissue-effect.
For example,
halving the spot area will result in halving the energy required to produce
the same effect.
Irradiation down to 0.5 microns can be obtained, for example, by coupling the
radiant output
of the laser into the objective lens of a microscope objective. (e.g., as
available from Nikon,
Inc., Melville, NY). In such a case, it is possible to focus the beam down to
spots on the order
of the limit of resolution of the microscope, which is perhaps on the order of
about 0.5
microns. In fact, if the beam profile is Gaussian, the size of the affected
irradiated area can be
less than the measured beam size and can exceed the imaging resolution of the
microscope.
To non-ablatively alter tissue in this case, it would be suitable to use a 3.2
J/cm2 energy
1 S fluence, which for a half micron spot size would require a pulse energy of
about 5 nJ. This
low a pulse energy is readily available from diode lasers, and can also be
obtained from, for
example, the Er:YAG laser by attenuating the beam by an absorbing filter, such
as glass.
(0081 ] Optionally, by changing the wavelength of radiant energy while holding
the
other variables constant, it is possible to change between an ablative and non-
ablative tissue-
effect. For example, using Ho:YAG (holmium: YAG; 2.127 microns) in place of
the Er:YAG
(erbium: YAG; 2.94 microns) laser, would result in less absorption of energy
by the tissue,
creating less of a perforation or alteration.
[0082] Picosecond and femtosecond pulses produced by lasers can also be used
to
produce alteration or ablation in skin. This can be accomplished with
modulated diode or
related microchip lasers, which deliver single pulses with temporal widths in
the 1
femtosecond to 1 ms range. (See D. Stern et al., "Corneal Ablation by
Nanosecond,
Picosecond, and Femtosecond Lasers at 532 and 625 nm," Corneal Laser Ablation,
Vol. 107,
pp. 587-592 (1989), incorporated herein by reference, which discloses the use
of pulse lengths
down to 1 femtosecond).
(0083] Another delivery method uses high pressure impulse transients on skin
to
create permeability. See U.S. Pat. No. 5,614,502, and U.S. Pat. No. 5,658,892,
both of which
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are incorporated herein by reference. High pressure impulse transients, e.g.,
stress waves
(e.g., laser stress waves (LSW) when generated by a laser), with specific rise
times and peak
stresses (or pressures), can safely and efficiently effect the transport of
compounds, such as
those of the present disclosure, through layers of epithelial tissues, such as
the stratum
5 corneum and mucosal membranes. These methods can be used to deliver
compounds of a
wide range of sizes regardless of their net charge. In addition, impulse
transients used in the
present methods avoid tissue injury.
[0084] Prior to exposure to an impulse transient, an epithelial tissue layer,
e.g., the
stratum corneum, is likely impermeable to a foreign compound; this prevents
diffusion of the
10 compound into cells underlying the epithelial layer. Exposure of the
epithelial layer to the
impulse transients enables the compound to diffuse through the epithelial
layer. The rate of
diffusion, in general, is dictated by the nature of the impulse transients and
the size of the
compound to be delivered.
[0085] The rate of penetration through specific epithelial tissue layers, such
as the
15 stratum corneum of the skin, also depends on several other factors
including pH, the
metabolism of the cutaneous substrate tissue, pressure differences between the
region external
to the stratum corneum, and the region internal to the stratum corneum, as
well as the
anatomical site and physical condition of the skin. In turn, the physical
condition of the skin
depends on health, age, sex, race, skin care, and history. For example, prior
contacts with
20 organic solvents or surfactants affect the physical condition of the skin.
[0086] The amount of compound delivered through the epithelial tissue layer
will also
depend on the length of time the epithelial layer remains permeable, and the
size of the
surface area of the epithelial layer which is made permeable. The properties
and
characteristics of impulse transients are controlled by the energy source used
to create them.
See WO 98/23325, which is incorporated herein by reference. However, their
characteristics
are modified by the linear and non-linear properties of the coupling medium
through which
they propagate. The linear attenuation caused by the coupling medium
attenuates
predominantly the high frequency components of the impulse transients. This
causes the
bandwidth to decrease with a corresponding increase in the rise time of the
impulse transient.
The non-linear properties of the coupling medium, on the other hand, cause the
rise time to
decrease. The decrease of the rise time is the result of the dependence of the
sound and
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21
particle velocity on stress (pressure). As the stress increases, the sound and
the particle
velocity increase as well. This causes the leading edge of the impulse
transient to become
steeper. The relative strengths of the linear attenuation, non-linear
coefficient, and the peak
stress determine how long the wave has to travel for the increase in steepness
of rise time to
become substantial.
[0087] The rise time, magnitude, and duration of the impulse transient are
chosen to
create a non-destructive (i.e., non-shock wave) impulse transient that
temporarily increases
the permeability of the epithelial tissue layer. Generally the rise time is at
least 1 ns, and is
more preferably about 10 ns.
[0088] The peak stress or pressure of the impulse transients varies for
different
epithelial tissue or cell layers. For example, to transport compounds through
the stratum
corneum, the peak stress or pressure of the impulse transient should be set to
at least 400 bar;
more preferably at least 1,000 bar, but no more than about 2,000 bar.
[0089] For epithelial mucosal layers, the peak pressure should be set to
between 300
1 S bar and 800 bar, and is preferably between 300 bar and 600 bar.
[0090] The impulse transients preferably have durations on the order of a few
tens of
ns, and thus interact with the epithelial tissue for only a short period of
time.
[0091 ] Following interaction with the impulse transient, the epithelial
tissue is not
permanently damaged, but remains permeable for up to about three minutes.
[0092] In addition, the new methods involve the application of only a few
discrete
high amplitude pulses to the patient. The number of impulse transients
administered to the
patient is typically less than 100, more preferably less than 50, and most
preferably less than
10. When multiple optical pulses are used to generate the impulse transient,
the time duration
between sequential pulses is 10 to 120 seconds, which is long enough to
prevent permanent
damage to the epithelial tissue.
[0093] Properties of impulse transients can be measured using methods standard
in the
art. For example, peak stress or pressure, and rise time can be measured using
a
polyvinylidene fluoride (PVDF) transducer method as described in Doukas et
al., Ultrasound
Med. Biol., 21:961 (1995).
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22
[0094] Impulse transients can be generated by various energy sources. The
physical
phenomenon responsible for launching the impulse transient is, in general,
chosen from three
different mechanisms: (1) thermoelastic generation; (2) optical breakdown; or
(3) ablation.
[0095] For example, the impulse transients can be initiated by applying a high
energy
laser source to ablate a target material, and the impulse transient is then
coupled to an
epithelial tissue or cell layer by a coupling medium. The coupling medium can
be, for
example, a liquid or a gel, as long as it is non-linear. Thus, water, oil such
as castor oil, an
isotonic medium such as phosphate buffered saline (PBS), or a gel such as a
collagenous gel,
can be used as the coupling medium.
[0096] In addition, the coupling medium can include a surfactant that enhances
transport, e.g., by prolonging the period of time in which the stratum corneum
remains
permeable to the compound following the generation of an impulse transient.
The surfactant
can be, e.g., ionic detergents or nonionic detergents and thus can include,
e.g., sodium lauryl
sulfate, cetyl trimethyl ammonium bromide, and lauryl dimethyl amine oxide.
[0097] The absorbing target material acts as an optically triggered
transducer.
Following absorption of light, the target material undergoes rapid thermal
expansion, or is
ablated, to launch an impulse transient. Typically, metal and polymer films
have high
absorption coefficients in the visible and ultraviolet spectral regions.
[0098] Many types of materials can be used as the target material in
conjunction with
a laser beam, provided they fully absorb light at the wavelength of the laser
used. The target
material can be composed of a metal such as aluminum or copper; a plastic,
such as
polystyrene, e.g., black polystyrene; a ceramic; or a highly concentrated dye
solution. The
target material must have dimensions larger than the cross-sectional area of
the applied laser
energy. In addition, the target material must be thicker than the optical
penetration depth so
that no light strikes the surface of the skin. The target material must also
be sufficiently thick
to provide mechanical support. When the target material is made of a metal,
the typical
thickness will be 1/32 to 1/16 inch. For plastic target materials, the
thickness will be 1/16 to
1 /8 inch.
(0099] Impulse transients can be also enhanced using confined ablation. In
confined
ablation, a laser beam transparent material, such as a quartz optical window,
is placed in close
contact with the target material. Confinement of the plasma, created by
ablating the target
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material by using the transparent material, increases the coupling coefficient
by an order of
magnitude (Fabro et al., J. Appl. Phys., 68:775, 1990). The transparent
material can be
quartz, glass, or transparent plastic.
[0100] Since voids between the target material and the confining transparent
material
allow the plasma to expand, and thus decrease the momentum imparted to the
target, the
transparent material is preferably bonded to the target material using an
initially liquid
adhesive, such as carbon-containing epoxies, to prevent such voids.
[0101 ] The laser beam can be generated by standard optical modulation
techniques
known in the art, such as by employing Q-switched or mode-locked lasers using,
for example,
electro- or acousto-optic devices. Standard commercially available lasers that
can operate in a
pulsed mode in the infrared, visible, and/or infrared spectrum include Nd:YAG,
Nd:YLF, COz,
excimer, dye, Tiaapphire, diode, holmium (and other rare-earth materials), and
metal-vapor
lasers. The pulse widths of these light sources are adjustable, and can vary
from several tens
of picoseconds (ps) to several hundred microseconds. For use in the new
methods, the optical
pulse width can vary from 100 ps to about 200 ns and is preferably between
about 500 ps and
40 ns.
[0102] Impulse transients can also be generated by extracorporeal
lithotripters (one
example is described in Coleman et al., Ultrasound Med. Biol., 15:213-227,
1989). These
impulse transients have rise times of 30 to 450 ns, which is longer than laser-
generated
impulse transients. To form an impulse transient of the appropriate rise time
for the new
methods using an extracorporeal lithotripter, the impulse transient is
propagated in a non-
linear coupling medium (e.g., water) for a distance determined by equation
(1), above. For
example, when using a lithotripter creating an impulse transient having a rise
time of 100 ns
and a peak pressure of 500 barr, the distance that the impulse transient
should travel through
the coupling medium before contacting an epithelial cell layer is
approximately S mm.
[0103] An additional advantage of this approach for shaping impulse transients
generated by lithotripters is that the tensile component of the wave will be
broadened and
attenuated as a result of propagating through the non-linear coupling medium.
This
propagation distance should be adjusted to produce an impulse transient having
a tensile
component that has a pressure of only about S to 10% of the peak pressure of
the compressive
component of the wave. Thus, the shaped impulse transient will not damage
tissue.
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[0104] The type of lithotripter used is not critical. Either an
electrohydraulic,
electromagnetic, or piezoelectric lithotripter can be used.
[0105) The impulse transients can also be generated using transducers, such as
piezoelectric transducers. Preferably, the transducer is in direct contact
with the coupling
medium, and undergoes rapid displacement following application of an optical,
thermal, or
electric field to generate the impulse transient. For example, dielectric
breakdown can be
used, and is typically induced by a high-voltage spark or piezoelectric
transducer (similar to
those used in certain extracorporeal lithotripters, Coleman et al., Ultrasound
Med. Biol.,
15:213-227, 1989). In the case of a piezoelectric transducer, the transducer
undergoes rapid
expansion following application of an electrical field to cause a rapid
displacement in the
coupling medium.
[0106] In addition, impulse transients can be generated with the aid of fiber
optics.
Fiber optic delivery systems are particularly maneuverable and can be used to
irradiate target
materials located adjacent to epithelial tissue layers to generate impulse
transients in hard-to
reach places. These types of delivery systems, when optically coupled to
lasers, are preferred
as they can be integrated into catheters and related flexible devices, and
used to irradiate most
organs in the human body. In addition, to launch an impulse transient having
the desired rise
times and peak stress, the wavelength of the optical source can be easily
tailored to generate
the appropriate absorption in a particular target material.
[0107] Alternatively, an energetic material can produce an impulse transient
in
response to a detonating impulse. The detonator can detonate the energetic
material by
causing an electrical discharge or spark.
[0108] Hydrostatic pressure can be used in conjunction with impulse transients
to
enhance the transport of a compound through the epithelial tissue layer. Since
the effects
induced by the impulse transients last for several minutes, the transport rate
of a drug
diffusing passively through the epithelial cell layer along its concentration
gradient can be
increased by applying hydrostatic pressure on the surface of the epithelial
tissue layer, e.g.,
the stratum corneum of the skin, following application of the impulse
transient.
Diagnostic Indicators Of Thymic Function
A. Known Markers
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[0109] Certain markers are associated with the activation of the thymus. By
following
the concentration of any one, or any combination, of these markers, one can
monitor the level
of activation of the thymus. Changes in the levels of these marker molecules
pre-and post-
activation of thymic function can be examined using bioinformatics. For
example, two-
s dimensional gel electrophoresis of plasma (i.e., blood depleted of all cells
by centrifugation)
is performed on patients' samples pre- and post-inhibition of sex steroids.
The differentially
expressed "dots" on the gels are recorded and analyzed by computer.
1. Interleukin-7 (Il-7)
(0110] The major lymphopoietic and thymopoietic cytokine produced by thymic
10 cortical epithelial cells, IL-7 is essential for the proliferation and
differentiation of immature
thymocytes (von Freeden-Jeffry et al., 1995; Komschlies et al., 1995; Peschon
et al., 1994).
Triple negative cell development requires interaction with IL-7 (Oosterwegel
et al., 1997;
Moore et al., 1993), which acts primarily by inducing bcl-2 expression and
inhibiting
programmed cell death of immature thymocytes (Akashi et al., 1997; Maraskovsky
et al.,
1 S 1997). Treatment with IL-7 alone has been demonstrated to reverse both the
increase in
apoptosis and decline in thymopoiesis within the CD44+CD25+ (TN2) and CD44-
CD25+
(TN3) subsets, corresponding to the location of TCR (3-chain rearrangement, in
aged mice
(Andrew & Aspinall, 2001).
[0111 ] Immune recovery in mice after T cell-depleted bone marrow
transplantation
20 has been documented to be enhanced following administration of IL-7,
suggesting the
production of IL-7 may be one of the mechanisms regulating de novo production
of T cells
after bone marrow transplantation (Bolotin et al., 1996). Analysis of IL-7
serum levels in
patients before and after bone marrow transplantation by ELISA revealed an
inverse
relationship to absolute lymphocyte count (Bolotin et al., 1999). Studies
measuring IL-7
25 levels in HIV-infected pediatric and adult patients also indicate a strong
inverse correlation
between IL-7 and absolute CD4 counts and lesser but significant correlations
with CD3 and
CD8 counts (Fry et al., 2001).
[0112] The mechanism underlying the increase in circulating IL-7 are unclear
but it
has been suggested that decreased T cell numbers result in diminished IL-7
receptor
availability leading to increased levels of free IL-7 with no change in IL-7
production. That
is, binding to lymphocytes that express IL-7 receptors (Bolotin et al., 1999)
homeostatically
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regulates circulating IL-7 levels. An alternative mechanism is the direct
upregulation of IL-7
in response to lymphopoenia through the interaction of T cells and IL-7-
producing cells via a
soluble mediator or through direct contact within the lymphoid
microenvironment (Fry et al.,
2001 ).
S Normal IL-7levels
[0113] In children aged 6-months to 5.5 years, the normal mean concentration
of IL-7
is 10.7 ~ 3.9 pg/ml. In adults aged 22.2 to 53.5 years the mean is appreciably
lower, at 3.1 ~
2.5 pg/ml. It has thus been suggested that IL-7 levels may be determined by
age since IL-7
levels are highest in infants less than one year of age and lower in children
and adults (Bolotin
et al., 1999). This would support previous studies which demonstrated an age-
dependent
decline in thymopoietic capacity in chemotherapy and bone marrow transplant
patients
beginning in adolescence (Mackall et al., 1995; Weinberg et al., 1995).
Moreover studies of
bone marrow stroma from aged mice have shown decreased secretion of IL-7 with
age
(Stephan et al., 1998).
[0114] According to an embodiment of the present disclosure, concentration of
IL-7 in
a patient's blood or serum is monitored before and after administration of the
agents) that
block sex steroid mediated signaling to the thymus. Rise in the concentration
of IL-7 within
2-3 days, preferably within 24 hours, more preferably within 2-3 hours, of
administration of
the agents) signifies that the thymus is responding to blockage of the sex
steroid activity.
Concentration of IL-7 is periodically monitored to determine the level of
activation of the
thymus over time.
2. Facteur Thymique Serique (FTS)
[0115] It is now largely established that the immune and neuroendocrine
systems
cross-talk by using similar ligands and receptors. The thymic-
hypothalamus/pituitary axis
constitutes a bi-directional circuit where the ascending feedback loop is
effected by thymic
factors of epithelial origin. Aside from modulating the release of peptidic
hormones and
neuropeptides, thymic hormones act mainly to promote the phenotypic maturation
of
progenitor cells from the bone marrow and to modulate mature T cell function
(Bitter and
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Crispe, 1992). Hence thymic hormones may be important in a large spectrum of
pathological
conditions ranging from immunodeficiencies to neuroendocrine diseases.
[0116] FTS or thymulin is a nonapeptide hormone secreted exclusively by the
thymic
subcapsular and medullary cells (Bitter and Crispe, 1992). Essential for both
early and late
stages of T cell differentiation as well as T cell function, FTS also induces
expression of
several T cell markers, and promotes T cell functions such as allogeneic
cytotoxicity,
suppressor functions and IL-2 production (Bitter and Crispe, 1992).
[0117] FTS titers in children gradually increase with increasing age from 2.69
~ 1.10
at a few days of age to 4.77 ~ 0.44 at a few years of age, then gradually
decrease to 0.66 ~
0.26 at 36 years of age to old age (Consolini et al., 2000). As the thymus is
physiologically
under neuroendocrine control, peptide hormones and neuropeptides influence age-
related
fluctuations in FTS levels. As noted above, impaired hormonal activity has
been shown to be
associated with age-related thymic atrophy (Consoloni et al., 2000). In
particular, thymic
atrophy is most evident following the rise in serum sex steroid levels
following puberty
(Fabris et al., 1997). Moreover FTS secretion by thymic epithelial cells is
enhanced by
growth hormone (Mocchegiani et al., 1990).
(0118] Zinc has been shown to be important in cellular immunity (Prasad et
al., 1988),
which is not surprising since FTS is biologically activated upon binding one
molecule of zinc
(Zn-FTS) (Bach, 1983). As zinc turnover is usually reduced with age (Panerai
and Sacerdote,
1997), it has been postulated that the low FTS levels in old age can be
related to a zinc
deficiency (Mocchegiani and Fabris, 1995). Indeed it was found zinc treatment
in elderly
patients restores thymic secretory activity (Morcchegiani et al., 1990).
However, in vitro
studies on addition of zinc ions to plasma from adolescent patients did not
restore the
biological activity of FTS, indicating that the decreased FTS levels in
adolescence is more
likely related to the decline of thymic activity than zinc deficiency
(Consolini et al., 2000).
[0119] In an embodiment of the present disclosure, the concentration of FTS in
a
patient's blood or serum is monitored before and after administration of the
agents) that block
sex steroid mediated signaling to the thymus. Rise in the concentration of FTS
within 2-3
days, preferably within 24 hours, more preferably within 2-3 hours, of
administration of the
agents) signifies that the thymus is responding to blockage of the sex steroid
activity.
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Concentration of FTS is periodically monitored to determine the level of
activation of the
thymus over time.
3. Thymosin And Thymopoietin
[0120] In contrast to FTS which begins to decline after 20 years of age in
humans,
thymosin-alpha l and thymopietin serum levels seem to decline as early as 10
years of age
(reviewed in Bodey et al., 1997). Castration appears to increase thymosin-
alpha 1 and
thymosin-beta 4 serum levels as found in male rats (Windmill and Lee, 1999).
[0121 ] In an embodiment of the invention, the concentration of thymopoietin,
thymosin-alpha 1, thymosin-beta 4, or combinations thereof are measured before
and after
administration of the agents) that block sex steroid mediated signaling to the
thymus. Rise in
the concentration of any of these compounds or combinations within 2-3 days,
preferably
within 24 hours, more preferably within 2-3 hours of administration of the
agents) signifies
that the thymus is responding to blockage of the sex steroid activity.
Concentration of any of
these compounds or combinations is periodically monitored to determine the
level of
activation of the thymus over time.
B. Newly Identified Markers
[0122] In addition to the known markers for thymic activation, several
additional
markers have been identified and used, based on the methods of the present
disclosure.
[0123] Procedures for obtaining these markers can mimic those for following
the
already identified markers. For example, 2D gel electrophoresis can be used
and the intensity
of the various spots monitored over time. The spots will usually correspond to
individual
proteins, although occasionally there may be overlap or concurrence of spots
from two or
more different proteins. The identity of the molecules is revealed by solid
phase amino acid
sequencing. A new molecules) so identified as being altered in expression
(increase or
decrease) as a result of thymic activation will form the basis of a new
diagnostic test for
thymic responsiveness to loss of sex steroids.
T Cell Analysis
[0124] Monitoring of T cell production is another method that may be used to
determine activation of the thymus. Techniques such as flow cytometric
analysis of whole
peripheral blood, detection of proliferating cells by monitoring the marker
Ki67, and TREC
analysis are among the methods known to those of skill in the field for such
monitoring. In an
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embodiment of the invention, numbers of T cells, as well as proliferating T
cells, are
determined before and after administration of the agents) that block sex
steroid mediated
signaling to the thymus. Rise in the number of any of these T cells or
combinations within 2-
3 days, preferably within 24 hours, more preferably within 2-3 hours of
administration of the
agents) signifies that the thymus is responding to blockage of the sex steroid
activity.
Concentration of any of these T cells or combinations is periodically
monitored to determine
the level of activation of the thymus over time.SMALL ANIMAL STUDIES
Materials and Methods
Animals
[0125] 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 months of age and are indicated where relevant.
Castration
[0126] Animals were anesthetized by intraperitoneal injection of 0.3 ml of 0.3
mg
xylazine (Rompun; Bayer Australia Ltd., Botany NSW, Australia) and 1.5 mg
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
[0127] Mice received two intraperitoneal injections of BrdU (Sigma Chemical
Co., St.
Louis, MO) (100 mg/kg body weight in 100p1 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 FACS 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
[0128] Mice were killed by COZ asphyxiation and thymus, spleen and mesenteric
lymph nodes were removed. Organs were pushed gently through a 200pm 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/liter
ammonium
chloride) for 10 min at 4°C, washed and resuspended in PBS/FCS/Az. Cell
concentration and
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viability were determined in duplicate using a hemocytometer and ethidium
bromide/acridine
orange and viewed under a fluorescence microscope (Axioskop; Carl Zeiss,
Oberkochen,
Germany).
[0129] For 3-color immunofluorescence thymocytes were routinely labeled with
anti-
s a(3TCR-FITC or anti-y8 TCR-FITC, anti-CD4-PE and anti-CD8-APC (all obtained
from
Pharmingen, San Diego, CA) followed by flow cytometry analysis. Spleen and
lymph node
suspensions were labeled 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 [0130] For BrdU detection, cells were surface labeled with CD4-PE and CD8-
APC,
followed by fixation and permeabilization 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 SOOpI DNase (100 Kunitz units, Boehringer Mannheim, W. Germany)
for 30
mins at 37°C in order to denature the DNA. Finally, cells were
incubated with anti-BrdU-
15 FITC (Becton-Dickinson).
[0131 ] For 4-color Immunofluorescence thymocytes were labeled for CD3, CD4,
CDB, B220 and Mac-l, 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
20 described (Godfrey and Zlotnik, 1993). BrdU detection was then performed as
described
above.
[0132) Samples were analyzed on a FacsCalibur (Becton-Dickinson). Viable
lymphocytes were gated according to 0° and 90° light scatter
profiles and data was analyzed
using Cell quest software (Becton-Dickinson).
25 Immunohistology
[0133] Frozen thymus sections (4~m) were cut using a cryostat (Leica) and
immediately fixed in 100% acetone.
[0134] For two-color immunofluorescence, sections were double-labeled with a
panel
of monoclonal antibodies: MTS6, 10, 12, 15, 16, 20, 24, 32, 33, 35 and 44
(Godfrey et al.,
30 1990; Table 1) produced in this laboratory and the co-expression of
epithelial cell
determinants was assessed with a polyvalent rabbit anti-cytokeratin Ab (Dako,
Carpinteria,
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CA). Bound mAb was revealed with FITC-conjugated sheep anti-rat Ig (Silenus
Laboratories) and anti-cytokeratin was revealed with TRITC-conjugated goat
anti-rabbit Ig
(Silenus Laboratories).
[0135] For BrdU 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 (Penh et
al., 1996).
Briefly, sections were fixed in 70% Ethanol for 30 mins. Semi-dried sections
were incubated
in 4M HCI, neutralized by washing in Borate Buffer (Sigma), followed by two
washes in
PBS. BrdU was detected using anti-BrdU-FITC (Becton-Dickinson).
[0136] For three-color immunofluorescence, sections were labeled for a
specific MTS
mAb together with anti-cytokeratin. BrdU detection was then performed as
described above.
[0137] Sections were analyzed using a Leica fluorescent and Nikon confocal
microscopes.
Migration studies
[0138] Animals were anesthetized by intraperitoneal injection of 0.3m1 of
0.3mg
xylazine (Rompun; Bayer Australia Ltd., Botany NSW, Australia) and l.Smg
ketamine
hydrochloride (Ketalar; Parke-Davis, Caringbah, NSW, Australia) in saline.
[0139] Details of the FITC labeling 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
inj ection and lymphoid organs were removed for analysis.
[0140] After cell counts, samples were stained with anti-CD4-PE and anti-CD8-
APC,
then analyzed 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).
[0141 ] Data analyzed using the unpaired student 't' test or nonparametrical
Mann-
Whitney test was used to determine the statistical significance between
control and test results
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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
[0142] With increasing age there is a highly significant (p<_0.0001) decrease
in both
thymic weight (Figure lA) 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 107 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 lA and 1B). Interestingly, there is a significant (p<_
0.001) increase in
thymocyte numbers at 2 weeks post-castration (~1.2 x 108), which is restored
to normal young
levels by 4 weeks post-castration (Figure 1 B).
[0143] 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).
(ii) a(3TCR, yBTCR, CD4 and CD8 expression
[0144] To determine if the decrease in thymocyte numbers seen with age was the
result of the depletion of specific cell populations, thymocytes were labeled
with defining
markers in order to analyze the separate subpopulations. In addition, this
allowed analysis of
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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.
[0145] 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
[0146] As shown in Figure 4, 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 SA). Accordingly, most division is seen in the
subcapsule and
cortex by immunohistology (data not shown). Some division is seen in the
medullary regions
with FAGS analysis revealing a proportion of SP cells (9% of CD4 T cells and
25% of CD8 T
cells) dividing (Figure SB).
[0147] Although cell numbers are significantly decreased in the aged thymus,
proliferation of thymocytes remains constant, decreasing to 12-15% at 2 years
(Figure 4), with
the phenotype of the proliferating population resembling the 2 month thymus
(Figure SA).
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 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).
Immunohistology
revealed the localization 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 analyzing the proportion of each subpopulation which represent
the
proliferating population, there was a significant (p<0.001) increase in the
percentage of CD8
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34
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 SA).
(0148] Figure SB 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.
(0149] The decrease in proliferation within the DN subset was analyzed further
using
the markers CD44 and CD25. The DN subpopulation, in addition to the thymocyte
precursors, contains a~iTCR+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 analyze the true TN
compartment (CD3-CD4-
CD8-) and these showed no difference in their proliferation rates with age or
following
castration (Figure SC). 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 SD)
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 SD).
The effect of age on the thymic microenvironment.
(0150] The changes in the thymic microenvironment with age were examined by
immunofluorescence using an extensive panel of MAbs from the MTS series,
double-labeled
with a polyclonal anti-cytokeratin Ab.
(0151 ] The antigens recognized by these MAbs can be subdivided into three
groups:
thymic epithelial subsets, vascular-associated antigens and those present on
both stromal cells
and thymocytes.
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(i) Epithelial cell antigens.
[0152] Anti-keratin staining (pan-epithelium) of 2 year old mouse thymus,
revealed a
loss of general thymus architecture with a severe epithelial cell
disorganization and absence of
a distinct cortico-medullary junction. Further analysis using the MAbs, MTS 10
(medulla)
5 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
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 labeling. There is also the appearance of thymic epithelial "cyst-
like" structures
10 in the aged thymus particularly noticeable in medullary regions (data not
shown). Adipose
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). The
thymus is beginning to regenerate by 2 weeks post-castration. This is evident
in the size of
the thymic lobes, the increase in cortical epithelium as revealed by MTS 44,
and the
1 S localization of medullary epithelium. 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).
[0153] The markers MTS 20 and 24 are presumed to detect primordial epithelial
cells
20 (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
thymus (data not
shown) with MTS 20 and MTS 24 reverting to discrete subpockets of epithelium
located at
25 the cortico-medullary junction.
(ii) Vascular-associated antigens.
[0154] The blood-thymus barner 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.
30 [0155] 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,
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36
MTS 15 expression is greatly increased, and reflects the increased frequency
and size of blood
vessels and perivascular spaces (data not shown).
[0156] 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
[0157] 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 MHC
II
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
[0158] 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-castration, cell number migrating to the periphery has
substantially increased
with the overall rate of migration remaining constant at 1-1.5%.
EXAMPLES
EXAMPLE 1: T Cell Depletion
[0159] In order to remove the abnormal T cells, the patient underwent T cell
depletion. One standard procedure for this step is as follows: The human
patient received
anti-T cell antibodies in the form of a daily injection of l5mg/kg of Atgam
(xeno anti-T cell
globulin, Pharmacia Upjohn) for a period of 10 days in combination with an
inhibitor of T cell
activation, cyclosporin A, 3mg/kg, as a continuous infusion for 3-4 weeks
followed by daily
tablets at 9mg/kg as needed. This treatment did not affect early T cell
development in the
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Received 13 May 2002
37
patient's thymus, as the amount of antibody necessary to have such an affect
cannot be
delivered due to the size and configuration of the human thymus. The treatment
was
maintained for approximately 4-6 weeks to allow the loss of sex steroids
followed by the
reconstitution of the thymus. The prevention of T cell reactivity may also be
combined with
inhibitors of second level signals such as interleukins or cell adhesion
molecules to enhance
the T cell ablation.
[0160] Because in many cases it is not possible to reduce only the antigen-
specific T
cells which cause the disease, the whole population of T cells, including the
pathological
ones, is depleted. This depletion of peripheral T cells markedly retards the
disease.
Simultaneously, however, because of the lack of T cells, it induces a state of
generalized
immunodeficiency which means the patients are highly susceptible to infection,
particularly
viral. Even B cell responses will not function normally in the absence of
appropriate T cell
help.
EXAMPLE 2: Sex Steroid Ablation Therapy
[0161 ] The patient was given sex steroid ablation therapy in the form of
delivery of an
LHRH agonist. This was given in the form of either Leucrin (depot injection;
22.Smg) or
Zoladex (implant; 10.~ mg), either one as a single dose effective for 3
months. This was
effective in reducing sex steroid levels sufficiently to reactivate the
thymus. In some cases it
is also necessary to deliver a suppresser of adrenal gland production of sex
steroids, such as
Cosudex (Smglday) as one tablet per day for the duration'of the sex s~'eroid
ablation therapy.
Adrenal gland production of sex steroids makes up around 10-15% of a human's
steroids.
[0162] Reduction of sex steroids in the blood to minimal values took about 1-3
weeks;
concordant with this was the reactivation of the thymus. In some cases it is
necessary to
extend the treatment to a second 3 month injection/implant.
EXAMPLE 3: Alternative Delivery Method
[0163] In place of the 3 month (or 3 times one month) depot or implant
administration
of the LHRH agonist, alternative methods can be used. In one example the
patient's skin may
be irradiated by a laser such as an Er:YAG laser, to ablate or alter the skin
so as to reduce the
impeding effect of the stratum corneum.
[0164] A. Laser Ablation or Alteration: An infrared laser radiation pulse was
formed using a solid state, pulsed, Er:YAG laser consisting of two flat
resonator mirrors, an
~~~~~aED SHEET
IPF'A/,A,~
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38
Er:YAG crystal as an active medium, a power supply, and a means of focusing
the laser
beam. The wavelength of the laser beam was 2.94 microns. Single pulses were
used.
[0165] The operating parameters were as follows: The energy per pulse was 40,
80 or
120 mJ, with the size of the beam at the focal point being 2 mm, creating an
energy fluence of
1.27, 2.55 or 3.82 J/cm2. The pulse temporal width was 300 ps, creating an
energy fluence
rate of 0.42, 0.85 or 1.27 x 104 W/cm2.
[0166] Subsequently, an amount of LHRH agonist is applied to the skin and
spread
over the irradiation site. The LHRH agonist may be in the form of an ointment
so that it
remains on the site of irradiation. Optionally, an occlusive patch is placed
over the agonist in
order to keep it in place over the irradiation site.
[0167] Optionally a beam sputter is employed to split the laser beam and
create
multiple sites of ablation or alteration. This provides a faster flow of LHRH
agonist through
the skin into the blood stream. The number of sites can be predetermined to
allow for
maintenance of the agonist within the patient's system for the requisite
period of time.
[0168] B. Pressure Wave: A dose of LHRH agonist is placed on the skin in a
suitable container, such as a plastic flexible washer (about 1 inch in
diameter and about 1/16
inch thick), at the site where the pressure wave is to be created. The site is
then covered with
target material such as a black polystyrene sheet about 1 mm thick. A Q-
switched solid state
ruby laser (20 ns pulse duration, capable of generating up to 2 joules per
pulse) is used to
generate the laser beam, which hits the target material and generates a single
impulse
transient. The black polystyrene target completely absorbs the laser radiation
so that the skin
is exposed only to the impulse transient, and not to laser radiation. No pain
is produced from
this procedure. The procedure can be repeated daily, or as often as required,
to maintain the
circulating blood levels of the agonist.
EXAMPLE 4: Sample Collection
[0169] Selected patients were bled immediately prior to receiving the LHRH
analogue
to inhibit sex steroid production, and at short time intervals (typically
during the first 24-72
hours) after the application of the LHRH analogue. Blood was centrifuged
(750gav) to
sediment cells and the plasma collected. The plasma samples were compared by
subjecting
them to analysis of concentration of particular thymic marker molecules.
EXAMPLE 5: Flow Cytometry Analysis Of Whole Peripheral Blood
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[0170] 201 of the appropriate antibody cocktail was added to 200p1 whole blood
and
incubated in the dark, RT for 30min. For removal of RBC, 2m1 of FACS lysis
buffer
(Becton-Dickinson, USA) was then added to each tube, vortexed and incubated
lOmin, RT in
the dark. Samples were centrifuged at 600gmaX, supernatant removed and cells
washed twice
in FACS buffer. Finally, cells were resuspended in 1%PFA for FACS analysis.
EXAMPLE 6: Ki67 Analysis
[0171 ] For detection of proliferating cells, lysed samples were incubated for
20min,
RT, in the dark in 5001 of lx FACS permeabilising solution (Becton-Dickinson,
USA).
Washed samples were incubated with either anti-Ki67-PE or anti-Ki67-FITC (or
the
appropriate isotype controls) for 30min at RT, in the dark. Samples were then
washed and
resuspended in 1%PFA for analysis.
(0172]
Antibody Cocktails:
1. CD27/CD45RA/CD45R0/CD4 or CD8
2. CD62L/CD45RA/CD45R0/CD4 or CD8
3. yBTCR/a(3TCR/CD28/CD4 or CD8
4. CD69/CD25/CD152/CD3
5. CDllb/CDllc/CD56/CD3
6. CD19/CD117/CD34/CD3
7. CD3/CD4/CD8/HLA-DR
8. For Ki67:
a) CD4 or CD8/CD45R0/CD27 followed by Ki67-PE or IgGI-PE
b) a(3TCR/CD8a/CDBb followed by Ki67-FITC or IgGl-PE
EXAMPLE 7: Detection Of Intracellular Cytokines
[0173] 2001 of whole blood was stimulated with soluble purified anti-CD3
(5~g/ml)
and anti-CD28 (lOwg/ml) for 6 hours at 37°C, 5%C02. Brefeldin A (final
concentrationl0~g/ml) was added during the final 4 hours to limit cytokine
secretion from the
activated cells. Following stimulation, samples were incubated for l5min, RT
with 20p1 of
20mM EDTA in PBS. Samples were then surface stained with anti-CD4-FITC and
anti-CD8-
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CyChrome. Following lysis and permeabilisation, cells were stained with anti-
IL-4-PE and
anti-IFNy-APC or the appropriate isotype controls. Unstimulated cells were
used as a control
for activation.
EXAMPLE 8: Preparation Of PBMC
S [0174] Purified lymphocytes were used for T-cell stimulation assays and TREC
analysis. 10-SOmI of peripheral blood was diluted 1:1 with RPMI-Heparin.
Diluted blood
was carefully layered over ficoll-hypaque at a ratio of 2:1 blood:ficoll.
Tubes were
centrifuged at (SOOgmaX) for 25 min at RT. Following centrifugation, the
plasma layer was
removed and stored at -20°C for analysis of sex steroid levels. The
buffy coat layer was
10 removed and diluted with RPMI-Heparin. Tubes were centrifuged at
25°C for 1 Smin at
(600gmaX), followed by a second wash at 400gmaX for lOmin. Supernatant was
removed and
cell counts performed in duplicate using a haemocytometer. Cells not used for
stimulation
assays were resuspended in freezing media and stored at -70°C
overnight, before transfernng
to Liquid Nitrogen prior to TREC analysis. Plasma collected following ficoll
purification was
15 stored at -20°C prior to analysis of sex steroid levels.
EXAMPLE 9: T Lymphocyte Stimulation Assay
[0175] For mitogen stimulation, PBMC were plated out in 96-well round-bottom
plates at a concentration of 1 x 105/well in 100p1 of RPMI-FCS. Cells were
incubated at
37°C, 5% COZ with PHA in doses from 1-lOpg/ml. For TCR-specific
stimulation, cells were
20 incubated for 48 hours on plates previously coated with purified anti-CD3
(1-lOpg/ml) and
anti-CD28 (lOpg/ml). Following plaque formation (48-72 hours), lpCi of 3H-
Thymidine
was added to each well and plates incubated for a further 16-24 hours. Plates
were harvested
onto filter mats and incorporation of 3H-Thymidine was determined using liquid
scintillation
on a (3-counter (Packard-coulter, USA).
25 EXAMPLE 10: TREC Analysis
[0176] Detection of TRECs is perfomed by purifying new helper T cells (Th;
e.g.,
CD4+, CD45RA+ CD27+) and cytotoxic T cells (Tc; e.g., CD8+, CD45RA+ CD27+) by
flow
cytometry and then TREC analysis using specific DNA probes and RT-PCR.
A. Cell Sorting
30 [0177] Frozen samples were rapidly thawed, washed in FACS buffer
containinglmM
EDTA and 1% Human Serum and centrifuged (600gmaX, Smin., 4°C). Cells
were incubated
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41
with anti-CD4-FITC, anti-CD3-APC and anti-CD45RA-PE for 30min., RT, washed and
fixed
by the drop-wise addition of lml of 3% Formalin in PBS. Samples were incubated
for a
further 30min., washed and resuspended in 5001 FACS buffer for sorting. Four
populations
were obtained: CD3+CD4+CD45RA+; CD3+CD4+CD45RA-; CD3+CD4-CD45RA+ and
CD3+CD4-CD45RA-.
B. DNA Isolation
[0178] Cells were sorted directly into PCR grade O.SmI eppendorfs, centrifuged
(8min, 2500gmaX) and resuspended in Proteinase K (PK) digestion buffer (2x 10
5 cells/ 20w1 of
a 0.8mg/mL solution). Proteinase K (PK) was added to the PCR digestion buffer
just prior to
use. Samples were incubated for 1 hour at 56°C followed by lOmin at
95°C to inactivate the
proteinase. Lysed samples were stored at -70°C prior to RT-PCR.
C. Real Time-PCR using Molecular Beacons
[0179] This technique is described in Zhang et al., 1999. Primers for signal
joint
TRECs were 5'-AAAGAGGGCAGCCCTCTCCAAGGCAAA-3' (SEQ ID NO:1) and 5'-
AGGCTGATCTTGTCTGACATTTGCTCCG-3' (SEQ ID N0:2). Primers for coding joint
TRECs were 5'-CCTGTTTGTTAGGGCACATTAGAATCTCTCACTG-3' (SEQ ID N0:3)
and 5'-CTAATAATAAGATCCTCAAGGGTCGAGACTGTC-3' (SEQ ID N0:4). DNA
was extracted from the cells using Proteinase K digestion. PCR conditions
were: 95°C for 5
min, followed by 90°C, 60°C and 72°C, each for 30s, for
30 or 35 cycles as indicated. Each
PCR reaction contained lU platinum Taq polymerase, l.8mM MgClz, 0.2mM dNTPs,
l2.SpM each primer and 1.7 nmol (SpCi) 3zP-labelled dCTP in SOpI platinum Taq
buffer.
EXAMPLE 11: Radioimmunoassay
[0180] Detection of sex steroid levels in patient sera (frozen following
Ficoll-Paque
centrifugation) was performed using a ~zSI-Testosterone radioimmunoassay
(RIA). Prior to
the assay, all reagents, samples and controls were brought to room
temperature. Control tubes
had either buffer alone - non-specific binding (NSB) tube or Ong/ml
testosterone standard
(B°). Buffer alone, standards (0-lOng/ml testosterone) or test samples
were added to each
tube, followed by sex binding globulin inhibitor (SBGI) to limit non-specific
binding of the
radio-labelled testosterone. The lzsl-testosterone was added to each tube
followed by an anti-
testosterone antibody (except for the NSB tubes). Tubes were then incubated at
37°C for 2
hours. Following this, a secondary antibody was added to all tubes which were
incubated for
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42
a further 60 mins following vortexing. Tubes were centrifuged (1000gm~) for 15
mins,
supernatant removed and the precipitate counted on a Packard Cobra auto-y
counter.
Triplicate cpm results were averaged and a standard curve constructed using
the formula for
percent bound Testosterone (B/Bo):
Sample - NSB
%B/Bo -
Bo - NSB
Sample = average cpm of particular test sample
NSB = average cpm of non-specific binding tube
Bo = average cpm of Ong/ml standard (total binding tube)
The level of testosterone in each test sample was determined from the standard
curve. The
plasma was subjected to protein analysis based on 2D gel electrophoresis
followed by
1 S computer based bioinformatics to determine the presence of indicators of
thymic function.
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REFERENCES
Aspinall, R. 1997. Age-associated thymic atrophy in the mouse is due to a
deficiency affecting rearrangement of the TCR during intrathymic T cell
development. J.
Immunol. 158:3037.
Berzins, S.P., Boyd, R.L. and Miller, J.F.A.P. 1998. The role of the thymus
and recent
thymic migrants in the maintenance of the adult peripheral lymphocyte pool.
JExp. Med.
187:1839.
Boyd, R.L., Tucek, C.L., Godfrey, D.L, Wilson, T.J, Davidson, N.J., Bean,
A.G.D., Ladyman,
H.M., Bitter, M.A. and Hugo, P. 1993. The thymic microenvironment. Immunology
Today
14:445.
Bruijntjes, J.P., Kuper, C.J., Robinson, J.E. and Schutirman, H.J. 1993.
Epithelium-free area in the thymic cortex of rats. Dev. Immunol. 3:113.
Carayon, P., and Bord, A. 1992. Identification of DNA-replicating lymphocyte
subsets using
a new method to label the bromo-deoxyuridine incorporated into the DNA. J.
Imm. Methods
147:225.
Douek, D.C., McFarland, R.D., Keiser, P.H., Gage, E.A., Massey, J.M., Haynes,
B.F., Polis,
M.A., Haase, A.T., Feinberg, M.B., Sullivan, J.L., Jamieson, B.D., Zack, J.A.,
Picker, L.J.
and Koup, R.A. 1998. Changes in thymic function with age and during the
treatment of HIV
infection. Nature 396:690.
Fredrickson, G.G. and Basch, R.S. 1994. Early thymic regeneration after
irradiation.
Development and Comparative Immunology 18:251.
George, A. J. and Bitter, M.A. 1996. Thymic involution with ageing:
obsolescence or good housekeeping? Immunol. Today 17:267.
CA 02462758 2004-04-O1
WO 02/030256 PCT/IBO1/02351
44
Godfrey, D.I, Izon, D.J., Tucek, C.L., Wilson, T.J. and Boyd, R.L. 1990. The
phenotypic heterogeneity of mouse thymic stromal cells. Immunol. 70:66.
Godfrey, D. I, and Zlotnik, A. 1993. Control points in early T-cell
development. Immunol.
Today 14:547.
Hirokawa, K. 1998. Immunity and Ageing. In Principles and Practice of
Geriatric Medicine.
M. Pathy, ed. John Wiley and Sons Ltd.
Hirokawa, K. and Makinodan, T. 1975. Thymic involution: the effect on T cell
differentiation.
J. Immunol. 114:1659.
Hirokawa, K., Utsuyama M., Kasai, M., Kurashima, C., Ishijima, S. and Zeng, Y.-
X. 1994.
Understanding the mechanism of the age-change of thymic function to promote T
cell
differentiation. Immunology Letters 40:269.
Hobbs, M.V., Weigle, W.O., Noonan, D.J., Torbett, B.E., McEvilly, R.J., Koch,
R.J.,
Cardenas, G.J. and Ernst, D.N. 1993. Patterns of cytokine gene expression by
CD4+ T cells
from young and old mice. J. Immunol. 150:3602.
Homo-Delarche, R. and Dardenne, M. 1991. The neuroendocrine-immune axis.
Seminars in
Immunopathology.
Huiskamp, R., Davids, J.A.G. and Vos, O. 1983. Short- and long- term effects
of whole body
irradiation with fission neutrons or x-rays on the thymus in CBA mice.
Radiation Research
95:370.
Kendall, M.D. 1988. Anatomical and physiological factors influencing the
thymic
microenvironment. In Thymus Update I, Vol. 1. M. D. Kendall, and M. A. Ritter,
eds.
Harwood Academic Publishers, p. 27.
CA 02462758 2004-04-O1
WO 02/030256 PCT/IBO1/02351
Kurashima, C, Utsuyama, M., Kasai, M., Ishijima, S.A., Konno, A. and Hirokawa,
A. 1995.
The role of thymus in the aging of Th cell subpopulations and age-associated
alteration of
cytokine production by these cells. Int. Immunol. 7:97.
5
Mackall, C.L. et. al. 1995. Age, thymopoiesis and CD4+ T-lymphocyte
regeneration after
intensive chemotherapy. New England J. Med. 332:143.
Mackall, C.L. and Gress, R.E. 1997. Thymic aging and T-cell regeneration.
10 Immunol. Rev. 160:91.
Penit, C. and Ezine, S. 1989. Cell proliferation and thymocyte subset
reconstitution in
sublethally irradiated mice: compared kinetics of endogenous and
intrathymically transferred
progenitors. Proc. Natl. Acad. Sci, U.S.A. 86:5547.
Penit, C., Lucas, B., Vasseur, F., Rieker, T. and Boyd, R.L. 1996. Thymic
medulla epithelial
cells acquire specific markers by post-mitotic maturation. Dev. Immunol. 5:25.
Plosker, G.L. and Brogden, R.N. 1994. Leuprorelin. A review of its
pharmacology and
therapeutic use in prostatic cancer, endometriosis and other sex hormone-
related disorders.
Drugs 48:930.
Randle-Barrett, E.S. and Boyd, R.L. 1994. Thymic microenviror~rnent and
lymphoid
responses to sublethal irradiation. Dev. Immunol. 4:1.
Scollay, R.G., Butcher, E.C. and Weissman, LL. 1980. Thymus cell migration.
Quantitative
aspects of cellular traffic from the thymus to the periphery in mice. Eur. J.
Immunol. 10:210.
Shortman, K., Egerton, M., Spangrude, G.J. and Scollay, R. 1990. The
generation and fate of
thymocytes. Seminars in Immuno. 2:3.
CA 02462758 2004-04-O1
WO 02/030256 PCT/IBO1/02351
46
Starzl, T.E., Demetris, A.J., Murase, N., Ricardi, C. and Truce, M. 1992. Cell
migration,
chimerism, and graft acceptance. Lancet 339:1579.
Suda, T., and Zlotnik, A. 1991. IL-7 maintains the T cell precursor potential
of CD3-CD4-
CD8- thymocytes. J. Immunol. 146:3068.
Timm, J.A. and Thoman, M.L. 1999. Maturation of CD4+ lymphocytes in the aged
microenviroment results in a memory-enriched population. J. Immunol. 162:711.
Thomson, A.W. and Lu, L. 1999. Are dendritic cells the key to liver
transplant? Immunology
Today 20:20.
Tosi, R., Kraft, R., Luzi, P., Cintorino, M., Fankhause, G., Hess, M.W. and
Cottier, H. 1982.
Involution pattern of the human thymus. 1. Size of the cortical area as a
function of age.
Clin. Exp. Immunol. 47:497.
van Ewijk, W., Rouse, R.V. and Weissman, LL. 1980. Distribution of H-2
microenvironments in the mouse thymus. J. Histochem. Cytochem. 28:1089.
von Freeden-Jef&y, U., Vieira, P., Lucian, L.A., McNeil, T., Burdach, E.G. and
Murray, R.
1995. Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a
nonredundant
cytokine. J. Exp. Med. 181:1 S 19.
Wiles, M.V., Ruiz, P. and Imhof, B.A. 1992. Interleukin-7 expression during
mouse thymus
development. Eur. J. Immunol. 22:1037.
Zlotnik, A. and Moore, T.A. 1995. Cytokine production and requirements during
T-cell
development. Curr. Opin.Immuno1.7:206.
