cron-web.org

[A1CR]

http://tinyurl.com/uq2we suggests that CR (combined with n-3
fatty acids)
counter inflammation, and may be involved in the longevity
basis of CR.
Might this apply to humans? In CR Conference notes,
"Posted by: CRONie_A
April 18, 2006 ... "I was one of Luigi Fontana's study
subjects. ... We had ... much better inflammatory markers."
Now a paper excerpted below has been made available
indicates that maybe such reduction of inflammation by CR
leads to longer human lives, in that inflammation systems in
the long-lived centenarians is reduced.

Vasto S, Candore G, Balistreri CR, Caruso M, Colonna-Romano
G, Grimaldi MP,
Listi F, Nuzzo D, Lio D, Caruso C.
Inflammatory networks in ageing, age-related diseases and
longevity.
Mech Ageing Dev. 2006 Nov 20; [Epub ahead of print]
PMID: 17118425

Inflammation is considered a response set by the tissues in
response to
injury elicited by trauma or infection. It is a complex
network of molecular
and cellular interactions that facilitates a return to
physiological
homeostasis and tissue repair. The individual response
against infection and
trauma is also determined by gene variability. Ageing is
accompanied by
chronic low-grade inflammation state clearly showed by
2-4-fold increase in
serum levels of inflammatory mediators. A wide range of
factors has been
claimed to contribute to this state; however, the most
important role seems
to be played by the chronic antigenic stress, which affects
immune system
thorough out life with a progressive activation of
macrophages and related
cells. This pro-inflammatory status, interacting with the
genetic
background, potentially triggers the onset of age-related
inflammatory
diseases as atherosclerosis. Thus, the analysis of
polymorphisms of the
genes that are key nodes of the natural immunity response
might clarify the
patho-physiology of age-related inflammatory diseases as
atherosclerosis. On
the other hand, centenarians are characterized by marked
delay or escape
from age-associated diseases that, on average, cause
mortality at earlier
ages. In addition, centenarian offspring have increased
likelihood of
surviving to 100 years and show a reduced prevalence of
age-associated
diseases, as cardiovascular disease (CVD) and less
prevalence of
cardiovascular risk factors. So, genes involved in CVD may
play an opposite
role in human longevity. Thus, the model of centenarians can
be used to
understand the role of these genes in successful and
unsuccessful ageing.
Accordingly, we report the results of several studies in
which the
frequencies of pro-inflammatory alleles were significantly
higher in
patients affected by infarction and lower in centenarians
whereas
age-related controls displayed intermediate values. These
findings point to
a strong relationship between the genetics of inflammation,
successful
ageing and the control of cardiovascular disease at least in
men, in which
these studies were performed. These data are also briefly
discussed in the
light of antagonistic pleiotropy theory and in order to
pursuit a
pharmacogenomics approach.GC-1

... Around the 70s, the industrialized countries showed
progressive decline
in mortality rate (1-2% year) in individuals over 80 years
old. Along with
these data, the number of oldest old people has risen up of
about 20 times
(Vasto and Caruso, 2004 and Candore et al., in press).
Centenarians
represent a cohort of select survivors who have, at least,
markedly delayed
diseases that normally cause mortality in the general
population at earlier
age. So, centenarians may be a human model of disease-free
or at the least,
disease delayed ageing (Franceschi et al., 1995 and
Franceschi and Bonafe,
2003). Alongside our recent findings allow us to suggest
that different
alleles at different genes coding for pro- or
anti-inflammatory molecules
may affect individual life-span expectancy by influencing
the type and
intensity of the immune-inflammatory responses against
environmental
stressors (Caruso et al., 2005, Franceschi et al., 2005 and
Rea et al.,
2006). Moreover, centenarian offspring have a marked
increased likelihood of
surviving up to 100 years and show a reduced prevalence of
age-associated
diseases, in particular related to cardiovascular diseases
and less
prevalence of cardiovascular risk factors (Terry et al.,
2003). So, genes
involved in CVD, the leading worldwide cause of morbidity
and death (Nabel,
2003), should play an opposite role in human longevity.

4. Aterosclerosis and longevity Atherosclerosis is a
complex vascular
disease that usually begins in the first decade of life and
is now
recognized as an inflammatory disease (Lusis, 2000).
Fatty-streak lesions in
the large arteries are initiated by the accumulation of
monocytes in
subendothelial spaces where they develop into lipid-laden
macrophages,
otherwise known as foam cells. Complex interactions between
the resident
endothelial cells and smooth muscle cells and the
infiltrating monocytes and
T lymphocytes determine the progression of fatty streaks
into vascular
lesions that block the normal blood flow and ultimately rupture
atherosclerotic plaque, leading to either myocardial
infarction or stroke
(Nabel, 2003). Inflammatory mechanisms play a central role
in mediating all
phases of atherosclerosis, from initial recruitment of
circulating
leucocytes to the arterial wall to eventual rupture of the
unstable plaque.
Signs of inflammation accompany the earliest accumulation of
lipid within
the arterial wall. Circulating leucocytes do not adhere to
the normal
endothelium, but shortly after the initiation of an
atherogenic diet, the
diseased endothelium begins to express adhesion molecules
that selectively
bind to circulating leucocytes. Once adherent to the
endothelium,
inflammatory cells migrate into the subendothelial space
(Blake and Ridker,
2002). Here the leucocytes contribute to the local
inflammatory response,
with macrophages expressing receptors for modified
lipoproteins and
macrophage colony stimulating factor combining with
different chemokines to
augment the differentiation of blood-derived monocytes into
macrophage foam
cells (Qiao et al., 1997).

The T-cell activation leads to expression of
interferon-gamma and
lymphotoxin, which further amplify the pro-inflammatory
state. Macrophages,
endothelial cells and smooth muscles cells produce the
pleiotropic cytokine
tumor necrosis factor-a (TNF-alphalpha) (Barath et al.,
1990), which along
with interferon-gamma and interleukin (IL)-1 stimulates
smooth muscle cell
production of IL-6 (Sanceau et al., 1995). As the
atherosclerotic lesion
matures, the accumulation of foam cells leads to the
formation of a lipid
pool, rich in pro-thrombotic tissue factor. Smooth muscle
cells produce
collagen, which contributes to the strength of the fibrous
cap, shielding
the circulating blood from the pro-thrombotic lipid pool.
The synthesis and
breakdown of collagen in the fibrous cap is dynamically
mediated by
inflammatory signals (Libby, 2001).

Actually, inflammation is a key component of atherosclerosis
(Table 1) and
genes coding for inflammatory or anti-inflammatory molecules
are, therefore,
good candidates for the risk of developing atherosclerosis.
Accordingly,
common gene polymorphisms regulating high inflammatory
molecules production
have been associated with atherosclerosis (Andreotti et al.,
2002, Stephens
and Humphries, 2003, Licastro et al., 2005 and Candore et
al., 2006c). We
hypothesised that polymorphisms associated with a positive
control of
inflammation might play a protective role against
atherosclerosis and
promote longevity. If pro-inflammatory genotypes
significantly contribute to
the risk of coronary heart disease, alleles associated to
disease
susceptibility should not be included in the genetic
background favouring
longevity. In our previous reports, we have observed a
significant
distribution of pro/anti-inflammatory genes among male
controls, male
centenarians and male patients affected by acute myocardial
infarction
(AMI). The frequencies of the studied pro-inflammatory
alleles were
significantly lower in centenarians and higher in AMI
patients, i.e. the
number of centenarians with a pro-inflammatory allele was
lower than the
number of AMI patients with the polymorphism. Conversely the
frequencies of
anti-inflammatory alleles were significantly lower in AMI
patients and
higher in oldest old, in both cases age-related controls
presented
intermediate frequency values. Fig. 1 shows the obtained
results (Balistreri
et al., 2004, Caruso et al., 2004, Lio et al., 2004, Listì
et al., 2005,
Listì et al., 2006a, Listì et al., 2006b, Candore et al.,
2006d and Grimaldi
et al., 2006). These results strengthen the hypothesis that
genetic
background protecting against cardiovascular disease is a
relevant component
of the longevity trait at least in men where these studies
have been
performed (Candore et al., 2006a, Candore et al., 2006d,
Candore et al., in
press and Caruso et al., 2005).

5. Inflammatory nodes Network investigation has emerged as
a powerful
approach to appreciate complex occurrence and organization
in biological
systems. The "in silico" approach to understanding the cell
functional
organization suggests a scale-free network and the presence
of hub genes
that could receive and direct the activity of many other
genes (Barabasi and
Oltvai, 2004). Recent data confirm the topology of the
network and suggest
that some mediators of immune-inflammatory responses could
be most
responsible for directive interconnections inside the immune
system.
Different mediators as pro- and anti-inflammatory molecules
are involved in
the communication among a large number of cell types. The
analysis of these
mediators can lead to different interpretation of how cells
of the immune
system may communicate with each other: mediators can be
soluble, released
in the intercellular space directly or bound to the cell
surface. The
cellular communication, by different mediators, both at
short range and
across the major body systems have a fundamental role in
regulating the
reaction of the immune system to a possible danger, which
triggers an
integrated response involving both innate and clonotypic
immunity, and which
eventually results in an inflammatory response (Jeong et
al., 2000, Janeway,
2001 and Capri et al., 2006).

In the inflammatory disease under study, this key roles may
be played by
Toll like receptor (TLR)4 that initiates both innate and
clonotypic immunity
to Gram-negative bacteria and to other agents (Kiechl et
al., 2002 and
Schroder and Schumann, 2005) and Cycloxygenases
(COX)/Lipoxygenases (LOX)
enzymes involved in the production of eicosanoids potent
biologically active
arachidonic acid-derived lipid mediators that are intimately
involved in
inflammation (Dubois et al., 1998 and Jala and Haribabu,
2004) (Fig. 2).

Total burden of infection at different sites may affect the
progression of
atherosclerosis, the risk being modulated by host genotype
and chronic
exposure to even moderate levels of inflammatory factors
appears to promote
atherosclerosis (Stoll et al., 2004, Desvarieux et al.,
2005, Caruso et al.,
2005 and Finch and Crimmins, 2004; Crimmins and Finch,
2006). The role of
TLR4 receptor is paradigmatic. Macrophages express receptors
that recognize
molecular patterns foreign to mammalian organisms but
commonly found on
pathogens, and at the same time, important contributing to
the inflammatory
response. In this first line of defence, TLRs recognize
pathogen-associated
molecular patterns. The transmembrane TLR4, that initiates
the innate immune
response to lipopolysaccaride (LPS) of Gram-negative
bacteria, activates the
inflammatory cell via the NF-?B pathway by inducing the
expression of a
variety of cytokines and other molecules crucial to immune
responses
(Schroder and Schumann, 2005 and Arroyo-Espliguero et al.,
2004). TLR4
expression has been described in human atherosclerotic
lesions and the
inflammatory mediators produced through its activation seems
to exert
various atherogenic effects involving the expression of
adhesion molecules
on endothelial cells, proliferation of smooth muscle cells,
activation of
immune cells, stimulation of the acute phase response and
matrix break down.
ASP299GLY (+896A/G) TLR4 single nucleotide polymorphism
(SNP), known to
attenuate receptor signalling (+896G+), seems to determine a
minor risk to
develop carotid atherosclerosis and less intima media
thickness in the
common carotid artery. Furthermore, the subjects with this
mutant genotype
(+896G+) have been claimed to display lower levels of same
pro-inflammatory
cytokines, acute phase reactants and soluble adhesion
molecules, although
the results of in vitro stimulation are discordant (Schroder
and Schumann,
2005, Kiechl et al., 2002, Arroyo-Espliguero et al., 2004,
Balistreri et
al., 2004, Imahara et al., 2005 and van der Graaf et al., 2005).

We demonstrated that +896G+ TLR4 polymorphism shows a
significant lower
frequency in patients affected by AMI with respect to
controls, whereas
centenarians show higher frequency (Balistreri et al.,
2004). This is in
agreement with other our previously discussed data showing
that genetic
background controlling inflammation might play an opposite
role in CVD and
in longevity, people genetically predisposed to a weak
inflammatory
activity, have fewer chances to develop CVD and, therefore,
without any
serious infectious disease complication, have the chance to
live longer.

In order to gain insight in the significance of these data
and the relevance
of TLR4 as hub, the levels of pro-inflammatory cytokines
IL-6 and
TNF-alphalpha and anti-inflammatory cytokine IL-10 were
determined by ELISA
in supernatants from +896G+ to +896G- cells from healthy
donors after
stimulation with LPS from Escherichia Coli (Fig. 3; Candore
et al., 2006e
and unpublished observations).

These cytokines have been shown to be involved in
atherosclerosis and
reciprocally in longevity (Caruso et al., 2005, Candore et
al., 2006c and
Rea et al., 2006). IL-6 stimulates endothelial activation,
vascular smooth
muscle cell proliferation and leukocyte recruitment, all of
which lead to
plaque growth or instability. IL-6 is involved in impaired
lipid metabolism
and in the production of triglycerides, which is an
important cardiovascular
disease risk factor. IL-6 decreases lipoprotein lipase
activity and
monomeric lipoprotein lipase levels in plasma contributing
to increases in
macrophage uptake of lipids. In fatty streaks and in the
atheromatous cap
and shoulder regions, macrophage foam cells and vascular
smooth muscle cells
express IL-6 suggesting a role for this cytokine in the
progression of
atherosclerosis (Candore et al., 2006c; Stephens and
Humphries, 2003). Major
effects of TNF-alphalpha on the cardiovascular system
include increased
expression of adhesion molecules and major
histocompatibility complex
proteins, release of endothelial cytokines and nitric oxide,
enhanced
vascular permeability, reduced lipoprotein lipase activity,
increased
hepatic fatty acid synthesis, involvement in obesity-related
insulin
resistance and prothrombotic effects. (Candore et al., 2006c
and Andreotti
et al., 2002). The principal function of IL-10 appears to be
to limit and
ultimately terminate the inflammatory signal. Several lines
of evidence
indicate an involvement of IL-10 in the development of
atherosclerosis.
IL-10 can limit the progression of experimental
atherosclerosis and has
several anti-atherogenic effects including inhibition of
adhesion of
LDL-activated monocytes to endothelium and down-regulation
of fibrinogen
biosynthesis. IL-10 is detectable in human atherosclerotic
plaques and has
been claimed to plays a regulatory role in the progression of
atherosclerotic human CVD (Candore et al., 2006c and Lio and
Caruso, 2006).

The results show that TLR4 mutant carriers produce lower
level of IL-6 and
TNF-alpha and higher level of IL-10. Thus, +896G+ TLR4 SNP
seems to be
associated with reduced risk to develop atherosclerosis
complications,
likely because it lowers the pro-inflammatory signal in the
monocytes and
higher the production of anti-inflammatory cytokines
instead. These data
suggest that the intensity of the genetically determined
inflammatory
response against pathogens might exert a major role in
determining the
magnitude of atherogenesis and subsequent clinical outcomes.
Taking all
these informations together, it is tempting to consider TLR4
as the link
between infection and the development of acute coronary
events. This can be
due to remote signalling by inflammatory mediators that
activate immune
cells in the atherosclerotic plaques through TLR4 activation
(Schroder and
Schumann, 2005, Arroyo-Espliguero et al., 2004, Balistreri
et al., 2004 and
Stoll et al., 2004). Besides, these results, showing the
strong effect of
TLR4 SNP on cytokine production, might also explain at least
in part why
most studies on association between IL-6, IL-10 and
TNF-alpha SNPs and
atherosclerosis are inconclusive (Candore et al., 2006c).

As regards eicosanoids, COX-1 and COX-2 are the key enzymes
in the
conversion of arachidonic acid to the precursors of
bioactive lipid
mediators, prostaglandin, thromboxane and prostacyclin. COXs
play a key role
in pathophysiological processes of inflammatory diseases and
are the main
target for non-steroidal anti-inflammatory drugs. The COX-2
inducible enzyme
has been shown to be express mostly in central nervous
system, in
circulating blood leukocytes, vascular cells and
inflammatory cells. COX-2
is largely expressed in macrophages that infiltrate
atherosclerotic plaques.
Prostanoids have potent actions on vascular smooth muscular
cells by
controlling besides contractility cholesterol metabolism
(favouring the
formation of foam cells) and proliferation. In particular,
antiproliferative
and antimigration effects may imply the evolution of the
plaque toward a
more vulnerable one, depleted of smooth cells and enriched
in macrophages.
Moreover, increased COX-2 expression in the plaque cells
allows the plaque
to expand through the formation of new blood vessel.
Finally, the
prostaglandin PGE2 is actively involved in metalloproteinase
2 and 9
production which are tightly linked to atherosclerosis and
plaque
instability (Cipollone and Fazia, 2006, Dubois et al., 1998,
Vane et al.,
1998 and Smith et al., 2000).

LOX are enzymes that catalyse the stereospecific insertion
of molecular
oxygen into various positions in arachidonic acid. 5-LOX is
the initial key
enzyme of the leukotriene pathway. Leukotrienes, generated
from arachidonic
acid through the action of 5-LOX have been known for over
two decades and
are implicated in a wide variety of inflammatory disorders.
G-protein-coupled receptors mediate the effects of different
leukotrienes in
distinct cell types. Novel cellular and molecular targets
were recently
discovered for these mediators, with important consequences
for the function
of both clonotypic and innate immunity. LOX can be induced by
pro-inflammatory cytokines and its expression in endothelial
cells is
relatively lower in the basal condition, but it can be
induced by
pro-inflammatory cytokines (Fig. 4). These studies have
outlined crucial new
roles for leukotrienes in the development of atherosclerotic
lesions. In
particular, LTB4 is a chemoactractant for monocytes and
activate the gene
expression in inflammatory cells with a positive loop (Jala
and Haribabu,
2004, Lusis et al., 2004, Dwyer et al., 2004, Lotzer et al.,
2005 and Manev
and Manev, 2006).

Accordingly, recent studies have shown that -765GC and
-1708GA SNPs in the
promoter region of COX-2 gene and 5-LOX genes, respectively,
resulting in a
significant lower promoter activity, were found to be
associated with
reduced risk of severe atherosclerosis (Cipollone and Fazia,
2006 and Dwyer
et al., 2004). Interestingly, circulating plasma levels of
the acute phase
protein C-reactive protein are significantly influenced by
-765G/C genotype
(Papafili et al., 2002). Noteworthy, in our homogeneous
population, the
frequencies of these pro-inflammatory alleles were
significantly higher in
AMI patients and lower in centenarians whereas age-related
controls
displayed intermediate values. Around 10% of patients
analysed, carries both
high pro-inflammatory alleles versus 0% in controls and
centenarians
(unpublished observations).

Besides, on the whole, these data clearly show the central
role of
eicosanoids, formerly thought to act only in acute
inflammation, in the
chronic inflammatory network.

6. Antagonistic pleiotropy and immunosenescence The ageing
of immune
system, immunosenescence, is the consequence of the
continuous attrition
caused by chronic antigenic overload. Immunosenescence and
probably
morbidity and mortality will be accelerated in those
subjects who are
exposed to an extra burden of antigenic load, such as
chronic infections.
Conversely, immunosenescence will be delayed in those
subjects who lived for
most of their life in clean environments in which exposure
to persistent
infections are minimized. Indeed, this is exactly what
happened in the last
century in the most economically developed countries in
which medical cares,
vaccination and hygiene in food, water and house contributed
to reduce the
antigenic load and consequently the rate of
immunosenescence. In other
words, the continuous antigenic challenge could be
responsible for a
progressive pro-inflammatory status, which appears to be a
major
characteristic of the ageing process. The continuous
attrition caused by
clinical and sub-clinical infections, as well as the
continuous exposure to
several types of antigens (microbes, food and allergens) in
the gut, skin,
dental and periodontal tissues, respiratory tract, urinary
tract, is likely
responsible for the chronic activation of both innate and
clonotypic
immunity. Some of the most important characteristics of
clonotypic immunity
in ageing, as the accumulation of memory cells with the age
and the
exhaustion of virgin T-cells, are compatible with this
assumption
(Franceschi et al., 2000b, Pawelec et al., 2002, Pawelec et
al., 2004,
Pawelec et al., 2005 and De Martinis et al., 2005). So,
immunosenescence
fits with the basic assumptions of evolutionary theories of
ageing, such as
antagonistic pleiotropy (Nesse and Williams, 1995). In fact,
the immune
system, by neutralizing infectious agents, plays a
beneficial role until the
time of the reproductions and parental cares. Subsequently
by determining a
chronic inflammation, can play a detrimental role late in
life, in a period
largely not foreseen by evolution (Franceschi et al., 2000b
and Caruso et
al., 2005). So we could conclude that age-related diseases
are "the price we
pay" for an active immune system that defends us in youth
but harms us later
(Wick et al., 2003). In particular, the data coming from the
longevous male
population under study shows that the genetic polymorphisms
responsible for
a low inflammatory response might result in an increased
chance of long
life-span in an environment with a reduced pathogen burden.
Such modern and
healthy environment also permits to obtain a lower grade of
survivable
atherogenic inflammatory response (Crimmins and Finch, 2006).

7. Pharmacogenomics Understanding the genetic determination
of the
inflammatory process includes the possibility of developing
valuable
diagnostic tools and new therapeutic approaches in
inflammatory related
diseases. It is, in fact, well recognized that different
patients respond in
different ways to the same drug. These differences are often
greater among
members of a population than they are within the same person
at different
times. The existence of large population differences with small
intra-patient variability is consistent with inheritance as
a determinant of
drug response. It has been estimated that genetics can
account for 20-95% of
variability in drug disposition and effects. Many
non-genetic factors as
age, organ function, concomitant therapy, drug interactions
and the nature
of the disease may influence drug effects. However, there
are numerous
examples in which interindividual differences in drug
response are due to
polymorphisms in genes encoding drug-metabolizing enzymes,
drug transporters
or drug targets. Besides, gene polymorphisms involved in the
biology of the
disease may also determine the response to drugs (Evans and
McLeod, 2003 and
Kalow, 2006).

Clinical observations of inherited differences in drug
effects were first
documented in the 1950s, giving rise to the field of
pharmacogenetics and
later pharmacogenomics. Although the two terms are
synonymous for all
practical purposes, pharmacogenomics studies the effect of a
drug as it
relates to the functions and interactions of all the genes
in the genome,
whereas pharmacogenetics studies the effect of a drug as it
relates to
single or defined sets of genes. In any case, their goal is
to design or
utilize a drug at the right dose for a desired effect,
taking into account
that there is a significant genetic variability both in the
pharmacology and
in the target of the therapy as well as in the
pathophysiology of the
disease (Kalow, 2006 and Cooke, 2006). Current therapies are
the direct
result of understanding the epidemiological, molecular and
genetic basis of
a specific disease with the application to clinical
practice. A major goal
for the next years will be to understand aetiology and
pathogenesis of
disease in the context of genetic variability as well as to
refine the
treatment strategies in the context of genetic variability:
so, we will able
to deliver a true personalized therapy (Evans and McLeod,
2003, Kalow, 2006
and Cooke, 2006). From our studies previously discussed is
visible how
pharmacogenomics might be applied in clinical therapy.
Long-life pathogen
burden is thought to be involved in the pathophysiology of
atherosclerosis.
However, large, high-quality, clinical trials for the
secondary prevention
with antibiotics of coronary heart disease, recently
published have been
convincingly negative (Anderson, 2005). So, it should be
necessary to move
on to new beginnings to explore the possibility that
antibiotics are
effective in chemo-prevention of atherosclerosis
complications. So, only
subjects, carriers of high responder TLR4 polymorphisms,
might be selected
for a clinical trial on antibiotic prophylaxis for the
prevention
atherosclerosis complications. Results from COX and LOX
polymorphisms
striking show that around 10% of patients analysed, carries
both high
pro-inflammatory alleles versus 0% in controls and
centenarians. These
patients should be a feasible responder population for COX,
LOX inhibitor
drugs. Hence, the translation of pharmacogenomics into
clinical practise
will allow bold steps to be taken toward tailored medicine. ...