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[A1CR]

It appears that the immune system of dogs is improved by CR.
Dogs may be
more closely related to humans than are rodents. Their life
span is
increased by CR and they live lives intermediate in length
compared with
rodents and humans.

Greeley EH, Spitznagel E, Lawler DF, Kealy RD, Segre M.
Modulation of canine immunosenescence by life-long caloric
restriction.
Vet Immunol Immunopathol. 2006 Jun 15;111(3-4):287-99. Epub
2006 Mar 29.
PMID: 16567002

http://tinyurl.com/ychkmo

Caloric restriction (CR) has been shown to retard
immunosenescence and to
extend median and maximum life span in rodent species.
Longitudinal effects
of CR on the canine immune system are presented in this
report. A group of
48 Labrador Retrievers, divided at weaning into weight- and
sex-matched
pairs, were maintained on a diet restriction protocol from
age 8 weeks until
death. Each restricted dog received 75% of the total food
consumed by its
control-fed pair mate. Immune parameters were monitored from
4 to 13 years.
CR retarded age-related declines in both lymphoproliferative
responses and
absolute numbers of lymphocytes and the T, CD4, and CD8-cell
subsets. In
females, CR attenuated the age-related increase in T-cell
percentages and
marginally retarded the age-related increase in memory cell
percentages.
Age-related changes in B-cell percentages and numbers were
augmented by CR.
No direct effect of CR on phagocytic activity of PMN,
antibody production or
NK cell activity, was observed. Lower lymphoproliferative
responses, lower
numbers of lymphocytes, T, CD4 and CD8 cells, lower CD8
percentages and
higher B-cell percentages were all found to be significantly
associated with
a decreased likelihood of survival in these dogs.

... While the precise mechanisms
through which CR prolongs life and retards age-related
physiological decline are not entirely understood,
evidence from a number of species suggests that
the attenuation of age-related changes in immune
function is integral to this process. ...

Immunosenescence is associated with decreased
survivability, presumably due to a decline in an
organism's ability to resist both internal and external
stresses, especially infectious agents and tumors.
Investigation of the effects of CR on aging in
laboratory rodents has generated a large body of
evidence supporting benefits of CR in retarding
immunosenescence (for review see Pahlavani,
2000). Early studies demonstrated positive effects
of CR on ex vivo lymphoproliferative responses and
cell cytolytic activity, while more recent studies have
documented effects on age-related changes in lymphocyte
subset distribution, cytokine production,
apoptosis, cell signaling events and most recently,
gene expression (Pahlavani, 2004; Lee et al., 1999;
Cao et al., 2001). CR has also been found to reduce
tumor incidence in susceptible strains (Sheldon et al.,
1995; Weindruch, 1992), and retard the onset and
reduce the severity of rodent autoimmune diseases
(Fernandes et al., 1976; Friend et al., 1978). Until
recently, the question of whether the benefits of CR
would apply to larger, longer-lived mammals as well
has remained unanswered. In the late 1980s and early
1990s studies evaluating the effects of CR on age-associated
physiological changes in primates were
initiated (Ingram et al., 1990; Kemnitz et al., 1993).
While verification of beneficial effects of CR on
primate life span will not be available for another
decade, to date, the calorically restricted primates
have demonstrated many of the age-related physiological
responses observed in rodents (Lane et al.,
1997; Mattison et al., 2003). Some disparity exists,
however, with regard to immune findings in primates
as compared to rodents. In the Wisconsin colony, 2-4
years of CR of male Rhesus monkeys actually resulted
in depression of lymphoproliferative responses to
mitogens, as well as reduced NK activity and Ab
responses; lymphocyte subset distributions and lymphocyte
counts in peripheral blood were unaffected
(Roecker et al., 1996). A benefit of CR in reducing IL-
6 levels associated with oxidative stress has also been
observed in this colony (Kim et al., 1997). In the NIH
colony, lymphoproliferative responses of Rhesus
males following 7 years of CR were lower than those
of control-fed monkeys in those animals restricted
from an early age, but not in those animals whose
restriction was initiated at age 3-5 years; lymphopenia
was observed in restricted-fed animals (Weindruch
et al., 1997). In this same study CR had a beneficial
effect on the age-related decline in IFN gamma
production in response to PHA, but had no effect on
age-related increases in IL-6 and IL-10 production
(Mascarucci et al., 2002).

The present study was initiated in 1987 with the
goal of investigating the effect of CR on the incidence
and severity of canine hip dysplasia. Evaluation of the
dogs at 2 years of age revealed marked benefits of the
diet in retarding orthopedic disease (Kealy et al.,
1992). At that point, the study was extended and
expanded with the intention of determining the effects
of CR on life span as well as on a number of age-associated
physiological parameters. CR, from 8
weeks of age until death, was found to extend the
median life span of the dogs by 15%, from 11.2 years
in the controls to 13 years in the restricted group
(Kealy et al., 2002).

A battery of immunological parameters known to
undergo age-related changes in other species were
identified and adapted for dogs; repeated measurements
of these parameters were collected from age 4
years until death. Analysis of age-related changes in
immunological parameters in the control-fed dogs
from age 4-11 years have been previously reported
(Greeley et al., 2001). The relationships between
immune findings and diet group, age, gender and
ultimate life span are reported herein.

... Forty-eight Labrador Retrievers (30 females, 18
males) from a total of seven litters ... were divided at
weaning into
litter-, weight- and gender-matched pairs and randomly
assigned to a feeding group. ... from
age 8 weeks until death, each restricted-fed dog
received 75% of the total food consumed by its
control-fed pair mate on the previous day. At 3.25
years, the diet was adjusted from a growth formula
suitable for younger dogs to an adult formula. At the
same time, the ad libitum feeding protocol for the
''control-fed'' dogs was modified to prevent obesity
and maintain an ideal body weight for each dog based
on skeletal size; the restricted-fed dogs continued to
receive 75% of the control-fed diet (Kealy et al.,
1997). The feeding regimen resulted in a reduction in
mean body weight of 26% and a significant extension
of median life span in the restricted-fed dogs (Kealy
et al., 2002). ...

3. Results
3.1. Lymphoproliferative responses to mitogens
An age-related decline in the maximum responses to
all three mitogens was detected in both diet groups
(p < 0.001; Fig. 1A), with the restricted-fed dogs
demonstrating a significantly slower rate of decline
compared to control-fed dogs (age × diet Con A:
p < 0.001;PHA: p < 0.01;PWM: p = 0.08). While CR
significantly retarded the rate of decline of
lymphoproliferative
responses to Con A in both females and
males, (age × diet, p < 0.01 for females; p < 0.05 for
males), the pattern of diet-related effects in the two
genders differed as seen in Fig. 1B. In females, the
restricted-fed dogs had responses to Con A equal to or
exceeding those of the control-fed dogs at all time
points (p = 0.01); a similar pattern was seen for PHA
(p < 0.05) and PWM (p = 0.06). In males, the pattern
was somewhat different: at 4-7 years, the control-fed
dogs actually had higher responses to Con A than
restricted-fed dogs with no measurable differences
between the two diet groups from 8 years onward.

3.2. White blood cell and lymphocyte subset
analysis
Commencing at age 7 years, total WBC and
differential counts were monitored. While the
numbers of WBC and PMN were not affected by age
or diet (data not shown), the absolute numbers of
lymphocytes and all subsets of lymphocytes declined
with age (all p < 0.001; Table 3). Restricted-fed
dogs had lower numbers of B cells than their control-fed
pair mates (p = 0.02). Analysis of rate of change
over time reveals that for numbers of total
lymphocytes, T cells and CD8 cells, the control-fed
dogs demonstrated significant age-related
declines over time (age × diet, p </=0.001 for each
cell type) while the restricted-fed dogs demonstrated
no decline. For CD4, the decline over time in both
diet groups was significant, but the rate of decline for
the control-fed dogs was of significantly greater
magnitude (age × diet, p < 0.001).

Table 3 The effect of caloric restriction on cellularity
============================================================
Cell type Diet---Age (years)
---7 8 9 10 11 12 13
---Number of cells (×10^6/ml blood)
============================================================
Lymphs^a
Restricted 1.63±0.30 1.70±0.30 1.34±0.30 1.44±0.30
1.60±0.30 1.54±0.30
1.52±0.31
Control 2.06±0.30 1.76±0.30 1.42±0.30 1.63±0.30 1.38±0.31
1.42±0.32
B Cells
Restricted 0.13±0.04 0.13±0.04 0.14±0.04 0.10±0.04
0.10±0.04 0.14±0.04
0.07±0.04
Control 0.20±0.04 0.18±0.04 0.16±0.04 0.13±0.04
0.14±0.04 0.15±0.04
T Cells^a
Restricted 1.40±0.30 1.40±0.30 1.15±0.30 1.19±0.30
1.41±0.30 1.31±0.30
1.35±0.30
Control 1.80±0.3 1.45±0.3 1.17±0.3 1.33±0.3 1.20±0.31
1.20±0.32
CD4 Cells^a
Restricted 0.80±0.17 0.74±0.17 0.58±0.17 0.62±0.17
0.61±0.17 0.60±0.17
0.67±0.17
Control 1.00±0.17 0.78±0.17 0.62±0.17 0.69±0.17
0.56±0.17 0.60±0.18
CD8 Cells^a
Restricted 0.53±0.09 0.55±0.09 0.40±0.09 0.44±0.09
0.58±0.09 0.52±0.09
0.47±0.10
Control 0.67±0.09 0.56±0.09 0.41±0.09 0.46±0.09
0.43±0.10 0.44±0.10
============================================================
... mean numbers of cells±S.E.
a A significant effect of CR on the age-related decline
in numbers of
lymphocytes, T, CD4 and CD8 cells was observed (age × diet,
p </=0.001 for
all cell types).

Age-related alterations of lymphocyte subset
percentages in peripheral blood included decreases
in CD4-cell and B-cell percentages (p < 0.001 for
both), with restricted-fed dogs demonstrating lower B-cell
percentages than control-fed dogs (p = 0.04;
Fig. 2). No age-related changes in CD8 T cell
percentages were observed for the 4-13 years period,
although the values for the young reference controls
appeared to be dramatically lower than those of the
test dogs-even at the 4-year time point. Age-related
increases in T-cell percentages were observed overall
(p = 0.05), with females having higher percentages of
T cells than males (p < 0.01). In females, a direct
effect of CR on T-cell percentages was observed, with
restricted-fed females demonstrating lower T-cell
percentages over time compared to the control-fed
females (p < 0.05).
While it was not feasible to monitor canine
memory T cells at the initiation of the study,
subsequent availability of a suitable CD44 reagent
allowed us to examine this putative memory marker as
a function of diet and age using cryopreserved cells
that had been collected throughout the study. Frozen
cells from three age categories (4-6 years, 7-8 years,
and 10-13 years) were evaluated for each diet pair.
Representative staining patterns for lymphocytes from
young, middle-aged and old dogs of both genders are
presented in Fig. 3. The percentages of CD8 memory
cells (as defined by CD44 bright staining) increased
markedly with age; in females, diet restriction was
found to be marginally beneficial in retarding this
increase (Table 4). (A similar pattern was observed for
CD4 cells, but since slightly lower levels of CD4 were
detected on cryopreserved cells in comparison to
freshly stained cells, these data were not included.)
Significant age-related increases in percentages of
CD44hi-stained cells were corroborated by testing
fresh cells from young dogs and the study dogs at age
12 years. (For CD8 cells, the percent of CD44hi in
young dogs was 47.6±2.2 S.E., while that of the 12-
year-old was 72.4±1.9; for CD4, the percent of
CD44hi in young dogs was 15.8±1.6 and that in 12-
year-old was 34.2±1.4.)


Table 4 The effect of age and caloric restriction on the
distribution of
CD8 + CD44hi T cells
========================
Age group (years) Gender---Percentage of CD8 memory cells^a
(least squares
mean±S.E.)
---Restricted
diet Control diet
========================
4-6
Female 64.2±2.7^b 68.1±2.7
Male 66.1±3.3 64.9±3.5
7-8
Female 74.6±2.8^b 82.3±2.8
Male 76.3±3.3 76.7±3.5
10-13
Female 79.6±2.8^b 87.2±3.0
Male 83.7±3.4 85.3±3.7
========================
a On a single test date, cryopreserved cells from each
of the three age
groupings for both the restricted and control dog in a given
diet pair were
tested. Cells were rapidly thawed, fixed with 1%
paraformaldehyde, and
stained with mouse anti-CD44 followed by 1:40 FITC goat
anti-mouse
Ig. Rat MoAbs for CD8 were added, followed by phycoerythrin
(PE)-labeled
goat anti-rat Ig. The percentage of PE-positive cells staining
brightly for the CD44 marker (log FITC) was recorded as the
percentage of
memory cells.
b In females, development of memory cells was marginally
retarded by CR
(p = .07).

3.3. Other immune measurements
No significant effects of CR on NK activity or PMN
phagocytic capacity were detected for the 9-year
interval of testing. In addition, monitoring of antibody
responses to thymus-dependent antigens from age 5-9
years revealed no consistent effect of diet group or
gender (data not shown).

3.4. Survival analysis
When relationships of immune parameters to
survival were examined using Cox proportional
hazards methodology, an increased hazard of death
was found to be significantly associated with: lower
lymphoproliferative responses to PHA (p = 0.05) and
PWM (p = 0.04), with a trend for Con A (p = 0.08).
An increased hazard of death was also associated with
lower cell counts for lymphocytes (p < 0.01), T cells
(p = 0.04), CD4 cells (p = 0.07), and CD8 cells
(p < 0.01). Additional risk factors included lower
CD8 percentages and higher B-cell percentages.
These associations with survival were calculated
independent of the diet group; when diet groupings
were taken into account, the PWM responses and the
cell counts and percentages were still predictive.

4. Discussion
This study examining the effects of CR on
immunosenescence was a segment of a comprehensive
longitudinal study undertaken to examine the effects of
CR on life span and age-related changes in numerous
physiological parameters in the dog. The findings
presented herein indicate that CR retards age-related
changes in a number of immune parameters including
lymphoproliferative responses to mitogens, and
changes in lymphocyte subset distribution and numbers.
Furthermore, several of these immune parameters
positively affected by the diet were also predictive of an
increased probability of survival, independent of diet.
Biannual evaluation of lymphoproliferative
responses to mitogens from age 4-13 years revealed
that CR significantly retarded the rate of age-related
decline in Con A and PHA responses, an effect that
was more pronounced in females than males.

Prevention or retardation of the age-related decline
in in vitro lymphoproliferative responses to mitogens
is one of the earliest and most consistently described
benefits of CR on rodent immunosenescence (Gerbase-
DeLima et al., 1975; Weindruch et al., 1982a,b;
Tian et al., 1995; Goonewardene and Murasko, 1995;
Fernandes et al., 1997). Both Gerbase-DeLima et al.
(1975) and Fernandes et al. (1997) found that CR was
more effective in potentiating the lymphoproliferative
responses of older animals, and, in fact observed
higher responses in the control-fed groups in younger
rodents. A similar pattern of effects was observed in
males in the present study. Whether gender consistently
contributes to the effects observed following CR
is not clear, since most studies have examined only
one gender. Goonewardene and Murasko (1995)
utilized both genders in their rat study, but do not
address the issue of gender effects on lymphoproliferative
responses, although they did find a gender
difference in the affect of CR on life span. In two
ongoing primate studies utilizing males, no beneficial
effect of CR on lymphoproliferative responses of
younger animals has been observed (Weindruch et al.,
1997; Roecker et al., 1996). There may well be gender
differences in the level and/or timing of restriction
needed to attain optimal biological effects at each
stage of life. We recognize that the necessity for
therapeutic ovariohysterectomy or orchidectomy in
the present study may be an additional factor that
could influence diet outcomes.

CR prevented the age-related decline in numbers of
lymphocytes, T cells and CD8 cells and retarded the
rate of decline of CD4 cells, while augmenting the
decline in numbers of B cells with age. Although a
borderline lymphopenia was observed in the restricted
dogs at 7 years, this effect did not persist at later time
points. Lymphopenia associated with caloric restriction
has been observed in mice (Weindruch and
Walford, 1988; Volk et al., 1994; Spaulding et al.,
1997; Chen et al., 1998) and in primates following 7
years of CR (Weindruch et al., 1997), but was not
observed in primates following a shorter term of CR
(Roecker et al., 1996). The reported disparities in the
effects of CR on lymphocyte numbers may relate to
strain and species differences, to the organ chosen for
lymphocyte monitoring, and to the length of restriction
relative to the total life span of the animal.

Age-related changes in B-cell percentages were the
only lymphocyte subset distribution directly altered by
CR, with control dogs demonstrating significantly
higher percentages than CR dogs. This finding can be
viewed as a beneficial effect of CR, since higher B-cell
percentages were found to be associated with
decreased survival potential in this study. While no
significant effect of diet on T-cell percentages was
observed for the males, in females CR prevented the
age-related increase in T-cell percentages, again
suggesting that gender may be an important variable
in evaluating the beneficial effects of CR. Roecker
et al. (1996) found no effect of CR on lymphocyte
subset distributions in male Rhesus monkeys. While
some beneficial effects of CR in retarding age-related
changes in subset distributions in rodents have been
described (Miller, 1997; Chen et al., 1998), a clear
pattern of effects of CR on lymphocyte subset
distribution has not emerged.

A hallmark of aging in all species examined to date
is the age-related shift from a naive to memory
phenotype in both CD4 and CD8 T-cell populations;
CR appears to be extremely beneficial in retarding or
preventing this shift. In F-344xBN rats, CR resulted in
only a minimal shift to the memory phenotype
(CD45RC/Ox-22low) in both CD4 and CD8 lymphocytes
of 30-month old rats restricted from 16 weeks of
age (Fernandes et al., 1997). Similarly in mice, the
percentages of CD4 and CD8 memory cells (CD44hi,
CD4+ and CD44hi, CD8+) in CR old animals were
substantially lower than in the ad lib counterparts
(Chen et al., 1998; Miller, 1997). In the present study,
the percentage of both CD4 and CD8 T cells
expressing high levels of CD44 was found to increase
dramatically with age; CR marginally retarded the rate
of increase in CD44 expression by CD8 cells in
females. While a correlation between CD44hi
expression and memory has not been directly
established in dogs, the parallel findings to those in
rodents are intriguing.

A clear-cut effect of CR on canine NK activity was
not observed in the present study. Previous studies
have failed to establish a predictable relationship
between CR and NK activity; Weindruch et al. (1983)
and Roecker et al. (1996) have reported reduced levels
of NK activity in CR mice and primates respectively.
Riley et al. (1989) found no effect in rats. Gilman-
Sachs et al. (1991) observed increased numbers of NK
cells in CR rats; however, this observation may not
have represented a beneficial effect of the diet, since
an age-related increase in numbers of NK cells was
also observed in these animals.

Establishment of reliable biomarkers of aging
would greatly facilitate evaluation of life-extension
strategies in a timely and cost-effective manner. In the
present study, lower values of lymphoproliferative
responses and lymphocyte, T, CD8 and CD4-cell
numbers were associated with an increased hazard of
death while lower B-cell percentages were predictive
of a decreased hazard of death. Heller and colleagues
monitored an extensive panel of potential biomarkers
over time in a group of heterogeneous mice and found
lower Con A lymphoproliferative responses and
higher natural killer cell activity to be associated
with decreased survival (Heller et al., 1998). In a study
of 102 elderly Swedish individuals, immune parameters
were evaluated and associations with survival
were determined at time intervals thereafter; lower
Con A responses and higher CD8 percentages were
associated with decreased survival (Ferguson et al.,
1995). Miller et al. (1997) found decreased survival
was most closely associated with higher levels of CD4
memory cells. It is interesting to note that a shared
hazard for survival in the three species examined in
these studies is a diminished capacity of lymphocytes
to respond to Con A. The biological mechanism
linking robust lymphoproliferative responses with
increased survival is not immediately apparent, since
in vitro responsiveness to nonspecific activators such
as Con A is dependent on a number of factors. Levels
of cytokine production and cytokine receptor expression,
the relative proportions of CD4 versus CD8 cells,
as well as the naive versus memory cell distributions
of these two subsets all play a role in these responses.
Identification of the true immune biomarker(s) of
aging that is identified by proliferative responses
awaits further characterization.

While the benefits of CR on the canine immune
system observed in the present study are not as
dramatic as those previously described in rodent
systems, a significant role for CR in retarding
immunosenescence in the dog has been demonstrated.
Furthermore, several immune parameters that are both
predictive of survival and enhanced by caloric
restriction have been identified.

This work was supported by Nestle Purina Company ...