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EUROPEAN JOURNAL OF INFLAMMATION
Vol. 5, no. 2, 57-63 (2007)
REVIEW ARTICLE
INFLAMMATORY RESPONSE IN INFECTIVE ENDOCARDITIS
E. DEVIRI and B.E. GLENVILLE
Department of Cardiothoracic Surgery, Hadassah University Hospital, Jerusalem, Israel
Received January 10, 2007 – Accepted June 8, 2007
Endocarditis remains a devastating disease with a high mortality despite timely diagnosis
and treatment. The mainstays of treatment include appropriate antibiotics and when indicated,
removal of the septic focus. This sounds extremely simple and belies the necessity for a sophisticated
multidisciplinary approach to its treatment, the success of which depends not just on the right antibiotic
at the right dosage via the right portal, but also on a profound understanding of the inflammatory and
infective pathophysiology at work. This review aims at assisting both the clinician and the lab-based
physician in the task.
arthritis, vasculitis, splenomegaly, splenic infarcts
cutaneous and ocular signs cannot be explained
alone by the presence of circulating bacteria (7).
Understanding the inflammatory response
and the interaction between the bacterium, white
blood cells, platelets and endothelial cells and the
role of various cytokines may explain some of the
pathophysiological findings associated with infective
endocarditis. In addition, a better understanding of
the inflammatory response to infective endocarditis
may improve the diagnosis rate (especially in cases
of culture negative endocardi), the follow up of the
activity of the disease, the treatment, and above all
the survival and cure rate.
In 1885 Osler described the clinical course of
infective endocarditis (1). Until the introduction
of antibiotics the course of the disease was mostly
fatal. The introduction of penicillin in 1941 made
the disease treatable. However the mortality was
considerable, mainly due to heart failure secondary
to valve destruction.(2). In 1960 Kay was the first
one to report surgical treatment for a valve lesion
caused by infective endocarditis(3). In the USA
infective endocarditis has an annual incidence of
5-7 cases per 100,000 persons (4). Despite the
progress in diagnosis, antimicrobial treatment and
surgical techniques, the mortality varies from 5%
in uncomplicated cases to 59% in complicated cases
(5), and in 2-31% etiologic diagnoses cannot be
obtained (6).
The pathogenesis of infective endocarditis is not
fully understood. The disease involves a complex
interaction of bacteria, endocardium and blood
components.
Heart failure is sometimes out of proportion to the
valve incompetence. Bacteria of low virulence can
induce severe disease when present in vegetations.
Extracardiac manifestations such as renal lesions,
Animal studies
The vegetation
Data based on experimental endocarditis
Infective endocarditis is characterized by a
combination of systemic and local findings. The
central lesion is the vegetation which is an infected
fibrin platelet thrombus which forms on the surface
of damaged cardiac valves.
It is well recognized that the normal endothelium
Key words: infective endocarditis, blood culture negative endocarditis vegetation, inflammatory markers
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Jerusalem 93104, Israel
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e-mail: [email protected]
1721-727 (2007)
57
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58
E. DEVIRI ET AL.
is refractory to the development of infection and
that pre-existing endocardial lesions are required
for the establishment of experimental endocarditis
(8). A widely used animal model of experimental
bacterial endocarditis is a catheter-induced valve
lesion followed by bacterial colonization proposed
by Garrison and Freedman (9). The preliminary
stage of infective endocarditis is denudation of
the endothelial cell, exposure of the underlying
extracellular matrix and local deposition of platelets
and fibrin. This process leads to the formation of a
non-bacterial thrombotic vegetation (10). In the next
stage, bacteria which are in the blood stream colonize
the sterile vegetation leading to the formation of
an infected vegetation Subsequently, interactions
between bacterial cells, platelets and white blood
cells occur through the activation of an inflammatory
response which leads to the growth or disappearance
of the vegetation.
Procoagulant activity following endothelial cell
injury
Fibrin is the primary constituent of the vegetation
in infective endocarditis, and Tissue Factor (TF)
expression is a major marker of coagulation
activation on infected valves.
The initial trigger to increased procoagulant
activity (PCA) is endothelial cell damage rather
than bacterial infection (11). Exposure of the valve
stromal cell to the blood induced PCA following
interaction with lipopolysaccarides (LPS) and IL-1.
Induction of tissue factor activity (TFA) by bacteria
mainly depends on their ability to internalize within
the endothelial cell (12)
Bacterial colonization within the vegetation
tPMP
Once there is activation of the coagulation
system and the inflammatory response, host defense
mechanisms are activated against the invading
bacteria. Activated platelets are capable of secreting
thrombin that induces platelets microbiocidal
peptides production (tPMP) (13). These peptides
limit the progression of the disease. Staphylococcus
aureus strains resistant to tPMP may grow faster in
the presence of tPMP than susceptible strains, and the
vegetation size rate and progression rate was higher
in rabbits infected by tPMP resistant strains (14-16).
tPMP susceptibility of the infective organism may
affect the entire course of the disease regarding both
local and systemic lesions. Resistance to tPMP was
more prevalent in S. viridans bacteria cultured from
blood of patients with IE than from those cultured
from the blood of bacteremic patients without IE
(17). Thus tPMP may be an important host defense
factor against IE due to certain bacteria.
Bacterial colonization on the vegetation is a
complex process involving interaction between
the infecting organism, endothelial cells, platelets
and monocytes. After the initial response of the
endothelium to injury and platelets to bacterial
invasion, the monocytes play a major role in the
pathogenesis of infective endocarditis. Monocytes
augment the induction of TFA in bacterium infected
endothelial cells (18-19). The mechanism of this
process is partially mediated through the adhesion
of the monocyte to the infected endothelial cell and
by the proinflammatory cytokine IL-1 (18). After
cytolysis of endothelial cells by S. sanguis, S. aureus
or S. epidermidis TFA persists from monocytes
bound to bacteria-infected endothelial cell matrix
(20). Adhesions of monocytes to the vegetation via
fibronectin receptors stimulate monocyte TFA (21).
Later, IL-1 and TNF-alpha induce IL-10 production
which down regulates monocyte TFA (22). From
these data it seems that the growth of the vegetation
and TFA of the monocyte result from a balance
between pro- and anti- inflammatory cytokines.
Bacteria resist host defense by various
mechanisms. Durac and Beeson (23) found that
the majority of S. sanguis strains in the vegetation
are phagocytosed, transported in the blood, and
deposited on the vegetation in adherent monocytes.
The monocyte disappears and viable bacteria
persist in the vegetation. Recently it was found
that monocytes infected by oral streptococci are
transformed to short-lived IL-12 producing mature
dendritic cells (24). These cells live in the vegetation
for a few days as macrophages and can survive at the
site of inflammation and kill microorganisms for a
long period of time.
Other virulence factors related to the vegetation
Fibronectin plays a major role in bacterial
adhesion to the vegetation. It was found that in some
streptococcal and staphylococcal species the ability
Eur. J. Inflamm.
to bind to fibronectin and to other extracellular
matrix proteins on the vegetation increases the
virulence of the disease (8, 25). Toy et al (26) found
that some S. aureus strains can adhere to fibronectin
adhered to solid fibrin thrombi. Adherence ability of
S. aureus to fibronectin was directly related to their
number on damaged heart valves and the virulence
of the disease (27-28). S. mutans colonization was
directly related to sucrose-derived exopolysaccharide
production (29).
Glucosyl transferases (GTFs) secreted by some
strains of S. viridans are important modulators of the
inflammatory response in early stages of the disease.
It was found that levels of IL-6 and tumor necrosis
factor alpha secreted from endothelial cells of mice
infected by S. viridans strains were directly related
to their GTFs expression (30). During the chronic
stage of the disease, IL-6 production persists, but at
that stage it may also originate from the monocytes
(30). Another study showed that GTF was capable of
inducing the expression of adhesion molecules and
the release of IL-6 and plays an important role in the
transition from neutrophil to monocyte recruitment
in the early stages of IE (31). Aggregation substances
(AS) and enterococcal binding substances (EBS)
secreted by E. faecalis are essential for the
development of vegetation. Rabbits infected by
strains lacking AS and EBS did not develop cardiac
vegetations, those infected by strains able to produce
either AS or EBS developed small vegetations
and those that were infected by both AS and EBS
producing bacteria produced large vegetations (32).
The presence of bacteria within the vegetation
depends on monocytes, platelets and endothelial
function and the ability of the invading organism
to resist phagocytosis by the polymorphonuclear
cell (PMN). Young et al. (33) found that S. gordonii
strains that resist phagocytosis by PMN were found
within the vegetation.
Infective endocarditis is characterized by
vegetation formation which is the local inflammatory
response to the disease. The formation of the lesion is
a complex process that starts on damaged endothelial
cells which induce TFA and activates platelets,
leading to the formation of a sterile vegetation.
Bacterial adherences to the vegetation promote a
cascade of interactions between endothelial cells,
platelets, PMNs, monocytes and infecting organisms.
59
The growth of the vegetation is determined by the
ability of the organism to adhere to the vegetation
and to resist various host defense mechanisms, and
also by the inflammatory response of the host.
Systemic reaction
In addition to the local reaction within the site
of the endothelial injury, IE is characterized by
a spectrum of systemic reactions involving the
heart, distant organs such as the spleen, kidneys,
skin and eyes as well as the cardiovascular system.
The degree of heart and distant organ involvement
might be determined in the early stages of the
disease mediated by tPMP. In a rabbit experimental
model Kupferwasser et al (14) showed that in tPMP
resistant bacteria strains there was a higher degree of
perivalvar damage, aortic regurgitation, and degree
of impairment of left ventricular function as well
as a higher degree of renal infarcts. AS and EBS
(aggregation substance and enterococcus binding
substance) secreted by E. faecalis modulated the
systemic reaction and the virulence of the disease. A
rabbit model with bacteria lacking these substances
did not cause clinical signs of the disease, whilst
bacteria, having both substances, caused 100%
lethal disease associated with cardiac and lung cell
destruction, splenomegaly and minimal pericardial
inflammation. Animals infected with one substance
producing bacteria developed an intense pericardial
reaction and a relatively low mortality rate of
16% (32). It was hypothesized that AS and EBS
secreting strains produced factors that prevented
pericardial inflammation, either through destruction
of inflammatory cells or by preventing PMN influx
into the site of infection (32). The resistance of S.
gordonii to PMN killing determines the virulence of
the infection. (33).
Clinical applications
Infective endocarditis is a serious disease
associated with significant morbidity and mortality.
The diagnosis, especially in culture negative
endocarditis or in cases where antimicrobial
treatment had been started before diagnostic tests,
might be impossible to achieve. Other cases may
have a complicated course associated with severe
complications such as thromboembolic events
resulting in an increased mortality. Monitoring of
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E. DEVIRI ET AL.
the disease activity might be important to prevent
disease exacerbation and other complications. A
better understanding of the inflammatory process
may enable an earlier diagnosis, anticipation of a
complicated course and a better monitoring of the
disease activity which might lead to better results.
Diagnosis
Early diagnosis of IE is very important, as the
earlier the diagnosis is made and proper treatment
started, the higher the rate of survival and the lower
the rate of complications. Diagnosis of blood culture
negative endocarditis (BCNE) is very difficult and
not always possible. BCNE occurs in 2.5-31% of
IE cases (6). The Duke criteria perform poorly in
these patients (34). In such cases a proper antibiotic
treatment may be delayed, and inflammatory markers
may contribute to the diagnosis of IE.
Rheumatoid factor (RF) is considered as a minor
sign among the Duke criteria for the diagnosis of IE.
Comparison has been made of C-reactive protein,
sedimentation rate, rheumatoid factor (RF) and
TNF levels in patients with definite IE and rejected
IE (according to Duke criteria). It showed that only
RF was significantly higher in definite IE patients
(35). In 427 cases of Q fever endocarditis systematic
RF examination enabled the diagnosis of definite
endocarditis in 8 out of 427 cases (6). Other studies
showed that IL6 and IL2-R levels were elevated both
in patients with culture positive and culture negative
endocarditis (36-37). It was proposed to include
IL-6 and IL 2-R as a diagnostic criterion in IE (37).
TNF, CRP, WBC, IL-1 and ESR were not found to
contribute to the diagnosis of IE (35-37).
From these data it seems that inflammatory
markers play a secondary role in the diagnosis of IE.
However in BCNE the role of inflammatory markers
in the diagnosis may be more important. Inclusion
of IL-6 or IL-2R as minor criteria should be further
investigated.
Pathogenesis
Q fever endocarditis is a challenging form of IE to
treat. Coxiella brunetii is an intracellular pathogen;
vegetations are rare and blood cultures are often
negative. This frequently leads to a delayed diagnosis
and treatment which results in a poor prognosis.
During the early stage of the infection it was
found that monocytes and peripheral mononuclear
blood cells produce higher levels of TNF alpha
and IL-1 beta (38). Ongoing Q fever endocarditis
is associated with a specific defective killing of C.
burnetii which does not exist in uninfected patients
or in patients who were recently cured of the disease
(39). While survival of the pathogen within the
infected monocyte is mediated by TNF (39), it was
found on the other hand that TNF with interferon
gamma (IFN-gamma) induces apoptosis of infected
monocytes and enhances killing of the pathogen.
(40).
Course
In addition to the value of early diagnosis, it
is very important to monitor the disease activity.
Inaccurate monitoring of the disease activity may
lead to premature completion of antimicrobial
therapy which may increase the risk of disease
exacerbation. On the other hand, unnecessarily
prolonged treatment may expose the patient to
iatrogenic complications. Measuring IL-6 and IL2R levels in IE patients revealed that their levels are
continuously decreasing during antibiotic treatment,
and therefore their level can monitor the activity of
the disease (36-37).
The inflammatory process can also be investigated
locally. IL-8 and TNF alpha were studied in excised
heart valves of IE patients. It was found that IL8 and the greatest amount of inflammation were
seen in patients with short preoperative treatment
course. No such relationships were seen in TNFalpha containing cells (41). It is concluded that IL-8
containing cells in infected heart valves could be
used as a marker of disease activity.
A serious complication of IE is thromboembolism.
Echocardiographic studies to correlate the size and
morphology of the vegetation with thromboembolic
events were controversial, therefore other possible
mechanisms other than just the presence of
vegetation may influence the occurrence of this
complication (42). Korkmaz et al (43) found that
levels of p-selectin and E- selectin were highly
significant rates in patients with thromboembolic
events. Kupferwasser et al. (44) found that
embolic events occurred more frequently among
patients with elevated antiphospholipid antibodies
(APA) levels. They concluded that APA levels in
61
Eur. J. Inflamm.
patients with IE are related to increased risk for
major embolic events. Buyubasyk et al (45) found
that concentration of prothrombin fragment 1+2
(PF1+2), thrombin-antithrombin III complex (TAT),
plasminogen activator inhibitor 1 (PAI-1), beta
thromboglobulin (beta-TG), and platelet factor 4
(PF-4) were all higher in comparison with patients
without thromboembolic complications. From these
data it seems that thromboembolic complications in
IE patients is not merely related to the vegetation but
is rather a complication of a hypercoagulable state
which results from the inflammatory process.
Summary
IE is a severe disease associated with significant
morbidity and mortality despite the advances
in antibiotic treatment and surgical techniques.
Understanding the mechanisms of the inflammatory
response may improve the results.
The result of the inflammatory reaction is the
formation of a vegetation that is initially sterile.
Later it becomes infected on an injured valve
endothelium which may lead to valve destruction
followed by a systemic reaction with disturbances
of the coagulation system and damage to the other
organs such as the kidneys, eyes, heart, and spleen.
Even though the diagnosis of IE is based on
positive blood cultures of the causative organisms,
in cases of BCNE or intracellular organisms the
diagnosis may be extremely difficult. In other
cases the diagnosis may be clinically uncertain. A
further understanding of the role of serum cytokine
concentrations may lead to a more accurate and
rapid diagnosis. Assessment of IL-6 and IL-2r could
provide new diagnostic criteria for inflammation.
Assessment of the IL-8 and TNF in valves excised
during the operation for valve replacement can
contribute to the information regarding the disease
activity. (This may be especially important during
replacement of an undiagnosed infected valve for
other reason than IE)
In cases of intracellular infecting organisms a Q
fever eradication of the infective organisms may be
very difficult, mainly because of defective killing of
the organisms by the monocytes. Since IF-gamma
induces apoptosis of C. burnetii infected monocytes
and enhances bacterial killing, it may be useful as an
adjuvant treatment of C. burnetii infected patients.
In cases of defective ability of the monocytes to
kill intracellular C. burnetii which is mediated by
TNF-alpha, probably interference with TNF activity
(by using anti-TNF antibodies) may improve the
monocyte ability to eliminate the infecting organism.
Therapeutic strategies based on cytokine antagonists
may limit the putative monocyte traffic and thus
enhance the efficacy of antibiotic treatment. Further
investigations on the various serum cytokines can
give more information about the disease activity and
may predict a complicated course of the disease.
IE patients with embolic events have increased
E- and P- selectin, PF1+2, ASA, TAT, beta TG,
PF4 and PAI-1 levels. Determination of these
molecules may provide a marker to identify patients
in high thromboembolic risk from IE, and probably
anticoagulant treatment may help reduce the risk..
We hope that in the future manipulations on the
inflammatory reaction will limit valve destruction
and remote organ damage, will improve bacterial
elimination by the host, enhance the efficacy of
antibiotic therapy, will reduce the need for valve
operations and reduce the morbidity and mortality
associated with the disease.
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molecules, E-selectin and P-selectin, in patients
with infective endocarditis and embolic events. Eur.
Heart J. 22:874.
44. Kupferwasser L.I., G. Hafner, S. Mohr-Kahaly, R.
Erbel, J. Meyer and H. Darius. 1999. The presence
of infection-related antiphospholipid antibodies in
infective endocarditis determines a major risk factor
for embolic events. J. Am. Coll. Cardiol. 33:1365.
45. Buyukasyk N.S., M. Ileri, A. Alper, K. Senen,
R. Atak, I. Hisar, E. Yetkin, H. Turhan and D.
Demirkan. 2004. Increased blood coagulation
and platelet activation in patients with infective
endocarditis and embolic events. Clin. Cardiol. 27:
154.
EUROPEAN JOURNAL OF INFLAMMATION
Vol. 5, no. 2, 65-71 (2007)
NOVEL MUTATION DETECTION OF AN INFLAMMATORY MOLECULE ELASTASE II
GENE ENCODING NEUTROPHIL ELASTASE IN KOSTMANN SYNDROME
G. AHANGARI, Z. CHAVOSHZADEH1, Z. LARI, A. RAMYAR2 and A. FARHOUDI1
Department of Molecular Medicine and Immunology, National Institute for Genetic Engineering
and Biotechnology, Teheran; 1Department of Clinical Immunology, Center for Pediatric Hospital,
Teheran University of Medical Sciences, Teheran;
2
Department of Hematology and Oncology, Center for Pediatric Hospital, Teheran University of
Medical sciences, Tehran, Iran
Received November 7, 2006 - Accepted March 19, 2007
Severe congenital neutropenia (SCN), often referred to as Kostmann syndrome, is a rare immune
deficiency syndrome diagnosed at or soon after birth, characterized by maturation arrest of myeloid
cells at the promyelocyte stage of hematopoiesis. In severe congenital neutropenia due to disorder of
neutrophil production, patients are predisposed to recurrent bacterial infections. Recently, there have
been reports of detected mutations in neutrophil elastase II (ELA2) gene in genomic DNA of severe
congenital neutropenia. In this study we attempted to determine whether there is any mutation in
elastase II gene encoding. Peripheral blood was collected from five patients with severe congenital
neutropenia and 20 healthy individuals. Total RNA was isolated using RNA standard techniques from
fresh separated cells by Polymorphoprep. RNA was analyzed by employing PCR amplification of reverse
transcribed using a total of ten specific primers. We amplified five exons of ELA2 gene separately and
sequenced each exon. Mutational analysis was performed by directed capillary sequencing method. We
found mutations in all severe congenital neutropenia patients and no mutation in 20 healthy individuals.
The most mutations were in exon 4 and no mutation was found in exons 3 and 5.
Severe congenital neutropenia (SCN) is a rare
immune deficiency syndrome diagnosed at or
soon after birth. Congenital neutopenia is a severe
disorder of neutrophil production that brings about
lifelong problems with recurrent infections (1).
Severe congenital neutropenia, often referred to as
Kostmann syndrome, was first described in northern
Sweden as an autosomal-recessive disorder affecting
neutrophil production. Congenital neutropenia and
cyclic neutropenia predispose patients to recurrent
fever, pharyngeal ulcers, sinusitis, and cellulites
and, less frequently, bacterimia and severe deep
tissue infections (2-3). Congenital neutropenia and
cyclic neutropenia occur both sporadically and as
inherited disorders. Severe congenital neutropenia
occurs because of inefficacious granulocyteopoeisis
or maturation arrest in the bone marrow. This
implies that primitive myeloid progenitor cells
may proliferate normally, but are lost during the
differentiation process. These patients, usually
children, are extremely susceptible to bacterial and
fungal infections because their neutrophil counts are
Key words: elastase two, severe congenital neutropenia, polymerase chain reaction, polymorphonueclear, reverse
transcription
Mailing address:
G. Ahangari, MT, Ph.D.,
Department of Molecular Medicine and Immunology,
National Institute for Genetic Engineering and Biotechnology,
P.O. Box: 14155-6343, Teheran, Iran
Tel: ++9821 44580384 Fax: ++9821 44580399
e-mail: [email protected]
1721-727 (2007)
65
Copyright © by BIOLIFE, s.a.s.
This publication and/or article is for individual use only and may not be further
reproduced without written permission from the copyright holder.
Unauthorized reproduction may result in financial and other penalties
66
G. AHANGARI ET AL.
extremely low and generally do not increase with
infections. These children develop life-threatening
neutropenia (4).
Although neutropenia in this disorder has been
attributed to impaired or ineffective neutrophil
production, the molecular and cellular basis for
the disease has remained largely unknown (5).
It was found through positional cloning studies,
that the locus for severe congenital neutropenia
was mapped to chromosome 19p13.3, a region
containing the genes for three neutrophil protease:
azurcidin, proteinase 3, and neutrophil elastase (so
called ELA2) (6). Neutrophil elastase is a potent 218
amino acid protein synthesized by CD34+ precursor
cells during the initial stage in the production of the
primary granules of the neutrophil (7). The cellular
changes due to the mutations of neutrophil elastase
in congenital and cyclic neutropenia are not yet clear
(8). Recently, it has been reported that mutations in
the gene for neutrophil elastase ELA2 seems to be
involved in the etiology of both cyclic and severe
congenital neutropenia (9). As an initial step towards
understanding neutrophil elastase gene expression,
the present study aims at characterizing the ElA2
mutations at RNA level and amino acid changes at
protein level of the ElA2 for better understanding the
role of ElA2 gene encoding in pathophysiology of
severe congenital neutropenia.
MATERIALS AND METHODS
Five severe congenital neutropenia patients and
twenty healthy individuals as control group took part in
this study. All patients and normal subjects were studied
according to the Declaration of Helsinki. Written informed
consent was obtained from each individual. Patients were
referred to the immunodeficiency clinic at the Children’s
Medical Center Hospital affiliated to Tehran University of
Medical Sciences .
Consent for these studies was obtained from the
parents of the patients under the auspices of the human
subjects committee of the Tehran university of medical
Sciences. Diagnoses were assigned at enrollment by
established criteria of the SCNIR.
The diagnosis of severe congenital neutropenia
was based on blood neutrophil counts less than 500/ μl
obtained during the 3 months after birth: a typical pattern
of recurrent fevers, chronic gingivitis and infections
at irregular intervals: a bone marrow aspirate showing
“maturation arrest” at the promyelocyte stage. Healthy
individuals with normal blood counts served as controls.
Peripheral blood samples (6 ml) were collected from
the cubital vein and in tubes containing anticoagulant.
Peripheral polymorphoneuclear cells were isolated by
polymorphoprep (Axis-Shield Poc AS, Oslo, Norway).
The polymorphoneuclear layer was collected and washed
three times in phosphate buffer saline (PBS). The total
mRNA was isolated from lymphocytes by RNA blood
minikit (Roach, Germany), and the amount and purity of
the RNA was determined by spectrophotometry. Elastase
II mRNA expression was determined by RT-PCR. Total
RNA amounting to 1500 ng was reverse transcribed
into first-strand cDNA by using random hexamers and
2-5 units of multiscribe (recombinant moloney murine
leukemia virus) reverse transcriptase in a final volume of
40 μl (10). Primers for exons EL1, EL2, EL3, EL4, EL5
and the whole exon as control were designed using primer
express software to exclude amplification of genomic
DNA and pseudo genes. 75 ng of cDNA was used for PCR
amplification in a final volume of 25 μl with 1 unit of Taq
DNA polymerase (Roach, Germany). PCR was carried
out in a PCR machine (Techneh, USA). For each of the
5 exons and PCR reactions, negative control (water) and
positive control (pooled cDNA) from several healthy
blood donors (peripheral blood polymorphoneuclear)
were used in parallel with every set of experiments. βactin was used to amplify a 140 base pair fragment that
served as an internal standard for cDNA preparation. βactin primer pair was used in the PCR to normalize the
concentration of cDNA. An aliquot of each amplification
reaction was loaded in ethidium bromide stained in1.5%
agarose gel, and electrophoresis performed at 100v for
45 min to confirm the expected size of the amplified
fragment.
Analyses of mutations were performed by capillary
sequencing. Each product of Elastase II gene (Exon
1 to 5) was sequenced by DNA sequencer ABI 3700
capillary system (Applied, Biosystem, USA). Products
were cleaned using the PCR purification kit ( Qiagen,
Germany) and were directly sequenced in both directions
using the same primers. For the complete ELA2 coding
region cDNA was produced by reverse transcription
and it was used as a template for PCR with the specified
primers.
Bidirectional sequencing was performed as above
for each mutation; at least 20 healthy controls were
included.
RESULTS
The aim of this study is to determine mutational
change in different exons of
Elastase II gene at RNA level in patients with
67
Eur. J. Inflamm.
Table I. Results of the mutational analysis of the ELA2 gene in 5 severe congenital neutropenia patients
and 20 healthy individuals by bidirectional capillary sequencing.
Patient
Exon number
RNA changes
1
4
TCT>GTC
2
1
4
Ala 22 Glu
Glu and Leu
3
2
GCG>GGA
Insertion of 5
neucleotide
GCG>GCA
Ala 44Gln
0/20
TGT>TTG
Cys196Leu
0/20
AAT>GAA
Asn45Gln
0/20
4
5
4
2
Predicted amino acid
change
Val169 Ser
Healthy
control
0/20
0/20
severe congenital neutropenia compared to healthy
1 to 5) was sequenced by DNA sequencer ABI 3700
individuals. In order to test this hypothesis, we
capillary system. For each mutation found, at least
examined mutation detection of the different elastase
20 healthy controls were included. Fig. 2 presents
II exons (ELA1-5). The experiments were performed
a sample of capillary sequencing electropherogram
using peripheral blood polymorphonuclear cells
analysis of mutation of normal allele of Elastase
Table
Results
of the
mutational
analysis
of the of
ELA2 II
gene
in 5 4severe
RNAI.with
highly
sensitive
methods.
The results
exon
in congenital
healthy individuals. Capillary
neutropenia
patients
and 20 healthy
individuals
by bidirectional
the present
study revealed
that all different
Elastase
sequencecapillary
analysissequencing.
of mutation by electropherogram
II gene exons (ELA1-5) were expressed in peripheral
documented in the same allele of Elastase II exon
blood polymorphonuclear cells in 20 healthy
4 mutation ( C to A) in SCN (Fig. 2). Mutational
individuals. RT- PCR wasthen carried out on five
analyses was performed by directed capillary
samples from SCN patients. Fig. 1 shows agarose gel
sequencing method. We found mutations in different
electrophoresis of RT-PCR products amplifications in
exons of Elastase ll gene encoding in all severe
SCN patients. Elastase II gene specific amplification
congenital neutropenia patients compared to no
products were different in size of exon1,2 (211bp),
mutation in 20 healthy individuals.
exon 3 (146bp), exon 4 (247bp) and exon 5 (176
Table I presents the results of mutational analyses
bp). Moreover, we designed primers to amplify
in SCN by bidirectional capillary sequencing. The
whole exon as a positive control (735bp). Elastase
mutations were found in high frequency in exon 4
II gene in peripheral blood polymorphonuclear was
and 2 respectively but no mutation was found in
expressed on 20 normal individuals and five SCN
exon 3. All patients had different heterozygous base
diseases. The results presented here provide direct
substitution changes at RNA and at protein level
evidence that human polymorphonuclear express
(TableI). Fig. 2 is a schematic diagram showing the
ELA2 gene. The specificities of the obtained PCR
location of mutations in the ELA2 gene in cyclic
products for the respective ELA2 fragments were
neutropenia. It is thus most likely that a novel 50%
confirmed by capillary sequencing analysis. The
frequency of polymorphism in exon 2 occurs (GCG
PCR products were tested by blasting against
44-NCBI, GCA44-Iranian) in the Iranian population
the entire human genome (NCBI-national center
which had not been reported previously. Moreover,
for biotechnology information 2006) to exclude
only one coding sequence change in the gene for
sequencing at unwarranted sites ( 11).
neutrophil elastase was observed in 20 controls from
Mutational analysis was carried out by capillary
normal individuals, and this change is presumed to
sequencing. Each product of Elastase II gene(Exon
represent a polymorphism, because of its appearance
68
G. AHANGARI ET AL.
Fig. 2. Mutational analysis of Elastase II exon 4 gene by
using capillary DNA sequencing in healthy individuals
(A) and in one severe cyclic neutropenia patient (B),
where the mutation C to A is present.
Fig. 1. Agarose gel electrophoresis of RT-PCR elastase
II gene, Exon1,2 (211bp), Exon 3 (146bp), Exon 4
(247bp), Exon 5 (176bp), All exon as control positive
(735bp) specific amplification products in severe cyclic
neutropenia patient.
also in cyclic neutropenia. Fig. 2 shows a schematic
diagram indicating the location of polymorphism in
the ELA2 exon 2 in healthy and severe congenital
neutropenia.
DISCUSSION
This report describes the occurrence of mutations
of the gene encoding neutrophil elastase in five patients
with severe congenital neutropenia in comparison to
20 healthy individuals. A previous study suggested
that the pathophysiology of congenital neutropenia
may be related to mutations of elastase II gene (1213). The findings reported here suggest a primary
role for mutations of neutrophil elastase in causing
most cases of SCN. This protease is normally
synthesized and packaged in promyelocytes at an
early stage in neutrophil development (14-15).
Neutrophil Elastase has many recognized and
possible substrates, including coagulation proteins,
growth factors, and intracellular signaling molecules
(16-17). It is normally synthesized in the developing
neutrophil as a proenzyme but is stored in the
primary granules in its active form ready with full
enzymatic activity when released from the granules,
normally at sites of inflammation (18-19). In the
present study, a mutation in the ELA2 gene was
detected in all apparently SCN. None of the mutation
was found in the 20 healthy controls. These results
are compatible with those published previously
showing that mutations in ELA2 is the cause of
cyclic neutropenia (20). The current study is the first
report which indicates that mutations occur at RNA
level of ELA2 in patients with SCN. The results
presented in this study confirm the high frequency
of heterozygous mutations in the neutrophil elastase
gene in SCN (21). Moreover, we found new
mutations and 50% frequency polymorphism in
SCN. These data further strengthen the argument
for the role of neutrophil elastase mutations in the
pathogenesis of SCN. It should be noted that the
patients with clinical manifestation of the disease
with ELA2 mutations could actually have autosomal
recessive disease.
The pathogenicity of mutations in ELA2 remains
a unknown (22). Neutrophil elastase has no known
role in myelopoiesis. Interestingly, heterozygous
and homozygous ELA2 knockout mice have normal
neutrophil counts, and only the latter has a higher
69
polymorphism
Eur. J. Inflamm.
EXON1
EXON2
EXON3
EXON4
EXON5
Fig. 3. Schematic diagram showing the location of mutations in the ELAII gene in severe congenital neutropenia.
demonstrable susceptibility to infection (16-17).
Experiments in which the mutant enzyme has been
transfected into cell lines (rat basophilic cells RBL-1)
and murine myeloblasts (32D) have shown reduced
levels of elastase activity in some patients (23).
However, other investigators have reported reduced
elastase activity as a more general phenomenon in
congenital neutopenia (24). At present it is rather
difficult to arrive at a definite conclusion that ELA2
mutation is the only factor which contributes to
SCN. It appears likely that pathogenesis of this
disease is due to an alteration of function in the
mutant protein, allowing the enzyme to bind to a
novel substrate involved in myelopoisis changing its
binding to an intracellular inhibitor. An alternative
explanation may be related to changes in intracellular
packaging. Recent evidence demonstrates increased
susceptibility to apoptosis of myloid precursors in
severe congenital neutropenia (25), however, the
link between this and mutant neutrophil elastase
has not been established. This report describes
the occurrence of mutations of the gene encoding
neutrophil elastase in five patients with SCN.
Mutations in the gene for neutrophil elastase ELA2
were reported to be involved in the etiology of both
congenital and cyclic neutropenia (20, 26-28). The
mutations in SCN which were found in the present
study mostly showed amino acid exchange at protein
level. This particular mutational change has not
previously been reported. This was not found in the
20 healthy donors tested, thus a polymorphic change
is excluded (28). There are results reported by others
which corroborate our hypothesis: (I) a small group of
cyclic neutropenia patients revealed no or silent ELA2
mutations; (II) some single ELA2 mutations have
been described to occur in SCN as well as in CN and
(III) rarely Kostmann’s syndrome, but most probably
also some of the sporadic CN cases are of autosomal
recessive inheritance (27, 29-30).
This is in contrast to an autosomal dominant
pattern of inheritance with heterozygous ELA2
mutations as a single cause for the disease. All ELA2
mutations which so far have been reported for CN
and CYN were found to be heterozygous and the
evaluated patients were characterized by one ELA2
mutation in one allele (20, 26-27, 30).
Moreover, it has been recently reported that by
using a positional cloning approach and candidate gene
evaluation, a recurrent homozygous germ line mutation
in HAX1 in three pedigrees were identified. HAX1
encodes the mitochondrial protein HAX1, which has been
assigned functions in signal transduction and cytoskeletal
control. It has been suggested that HAX1 is a regulator of
myeloid homeostasis and underlines the significance of
genetic control of apoptosis in neutrophil development.
ELA2 was also sequenced, associated with cyclic and
congenital neutropenia in all affected individuals with
HAX1 mutations. However, no affected individual was
found with mutations in both ELA2 and HAX1. Our
findings confirm by these studies that suggesting ELA2
and HAX1 define two mutually exclusive groups of
individuals with SCN (31).
ACKNOWLEDGEMENTS
We thank the families for kindly providing
materials. We are indebted to professor D. Dale from
Washington University for his helpful scientific
70
G. AHANGARI ET AL.
suggestions. The authors gratefully acknowledge
Professor M. B. Eslami for his critical reading of the
article and the financial support of (o-146) Tehran
University of Medical Science and National Institute
for Genetic Engineering & Biotechnology.
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EUROPEAN JOURNAL OF INFLAMMATION
Vol. 5, no. 2, 73-81 (2007)
SERUM AMYLOID A ACTIVATION OF INFLAMMATORY AND ADHESION MOLECULES
IN HUMAN CORONARY ARTERY AND UMBILICAL VEIN ENDOTHELIAL CELLS
K. LAKOTA, K. MRAK-POLJŠAK, B. ROZMAN, T. KVEDER, M. TOMŠIČ
and S. SODIN-SEMRL
University Medical Centre, Division of Internal Medicine, Department of Rheumatology, Ljubljana, Slovenia
Received December 22, 2006 – Accepted June 11, 2007
Inflammation is considered to be the driving force leading to atherogenic and atherosclerotic
mechanisms. Increased levels of SAA predict the risk of coronary artery disease and even mortality from
cardiovascular disease in humans. Recent animal and human studies have indicated that SAA plays a
causal role in atherogenesis, although it is largely unclear how this occurs. The objectives of this study
are to understand the role of SAA in activating possible atherogenic inflammatory responses in human
coronary artery endothelial cells (HCAEC) and to compare them with human umbilical vein endothelial
cells (HUVEC). Our hypothesis is that vein and artery endothelial cells have different expression
patterns and levels, leading to differential inflammatory responses. HUVEC and HCAEC were grown
in order to analyze the effects of SAA on endothelial expression of pro-inflammatory cytokines, such
as IL-6, chemokines, such as IL-8, and adhesion molecules (s-ICAM, s-VCAM, E-selectin) by reverse
transcription-PCR and ELISAs. We compared the dose responses of SAA between HUVEC and
HCAEC. SAA activated both HUVEC and HCAEC pro-inflammatory factors in a dose-dependent
manner. In comparison however, HCAEC showed a strikingly greater sensitivity to SAA, with a higher
level of expression of all pro-inflammatory markers at much lower concentrations of SAA, and their
much greater stimulation at higher SAA concentrations. SAA also generated a dose-dependent positive
feedback response on its own mRNA expression in HCAEC as compared to HUVEC. In summary,
there are distinct significant differences in the levels of inflammatory markers and adhesion molecules
between HUVEC and HCAEC SAA induced dose responses that could potentially account for HCAEC
greater susceptibility to inflammation and atherogenesis.
Inflammation is known to be a major driving
force in atherogenesis and in the initiation of
coronary plaque formation (1). Cohort studies have
reported that acute inflammatory parameters [such
as C-reactive protein (CRP) or serum amyloid A
(SAA)], cellular adhesion molecules, cytokines and
chemokines are all elevated among patients at risk
for future coronary occlusion (2-4). Increased SAA
levels predict the risk of coronary artery disease in
humans (5) and multivariate studies have identified
SAA as an independent predictor of mortality in
acute myocardial infarction patients (6). Recent
animal and human studies indicate that SAA plays a
causal role in atherogenesis (7), however it is largely
unclear how the induced SAA play this role.
The endothelium is heterogeneous due to its
Key words: serum amyloid A, endothelial cells, interleukin-6, adhesion molecules
Mailing address: Dr Snezna Sodin-Semrl,
Department of Rheumatology,
University Medical Centre,
Vodnikova 62,
SI-1000 Ljubljana, Slovenia
Tel: ++ 386 1 522-5596 Fax: + 386 1 522-5598
e-mail: [email protected]
1721-727 (2007)
73
Copyright © by BIOLIFE, s.a.s.
This publication and/or article is for individual use only and may not be further
reproduced without written permission from the copyright holder.
Unauthorized reproduction may result in financial and other penalties
74
K. LAKOTA ET AL.
vascular bed and tissue type origin. Different
endothelial cell phenotypes exhibit differential
responses to changes in their environment and their
underlying molecular mechanisms and signaling (8).
Thus, endothelial cells derived from the umbilical
vein have specific and intrinsic expression patterns
of inflammatory molecules as compared to coronary
arterial endothelial cells.
Widespread expression of SAA has been
reported in histologically normal human tissues
(9). Endothelial cells in atherosclerotic lesions of
human coronary and carotid arteries have been
found to express SAA mRNA, in addition to other
cells such as smooth muscle cells, macrophagederived “foam cells,” adventitial macrophages,
and adipocytes (10). SAA has also been found to
be present in atherosclerotic plaques (11), and was
locally increased at the site of ruptured plaques in
acute myocardial infarction in comparison to CRP
(12). These findings enhance the postulation that
SAA is involved in atherogenesis, atherosclerosis
and thrombogenesis.
Human SAA belongs to a multigene family
comprising of induced SAA1 and SAA2 isoforms,
jointly named acute phase SAA (A-SAA1/2), and the
constitutively expressed SAA4 (C-SAA) (13). SAA3
gene is not expressed in humans (14). Multiple events
can trigger the stimulation of A-SAA synthesis in
the liver. The expression of SAA is most potently
regulated by the pro-inflammatory cytokines IL1, TNF-α and IL-6 and their synergistic actions.
During inflammation, elevated SAA production is
primarily due to its increased transcription (15) and
stability of mRNA (16). Studies of the mammalian
SAA gene promoter indicate involvement of NF-κB,
AP-2, NF-IL-6, SAF, SP-1, YYI and CEB/P factors
in the transcriptional regulation of SAA (17), which
can be cell (18) and stimuli specific (19).
A-SAA is a prominent positive acute phase
reactant (20) which itself has been shown to
stimulate pro-inflammatory cytokines, such as
IL-1, TNF-α, IL-6, chemokines, such as IL-8,
the inflammatory regulator NF-κB as well as
cyclooxygenases in epithelial cells, fibroblast-like
synoviocytes and neutrophils (21-23). A-SAA can
induce extracellular matrix enzymes, such as matrix
metalloproteinases (MMPs) (24-25), collagenase and
stromelysin, which are important in repair processes
after tissue injury (26), but following their prolonged
production these enzymes can play degradative
roles. A-SAA has potent chemoattractant activity
for, among other human cell types, monocytes (27),
phagocytes (28), T cells (29) and mast cells (30), the
in vivo consequence of which is their recruitment to
inflammatory sites. Little however is known about
the role of A-SAA in activating human endothelial
cells.
The objectives of this study are to investigate
the role of acute phase SAA in stimulating
possible atherogenic inflammatory responses in
the endothelium and to compare these responses
between two different human endothelial cells. We
used primary human umbilical vein endothelial cells
(HUVEC) and human coronary artery endothelial
cells (HCAEC) in order to determine SAA dependent
expression of the pro-inflammatory marker IL-6,
chemokine IL-8 and adhesion molecules (ICAM,
VCAM and E-selectin) and to find out whether ASAA itself is expressed in these endothelial cells.
MATERIALS AND METHODS
Materials
The lyophilized recombinant human SAA (endotoxin
tested) was purchased from CytoLab (Peprotech/
Asia), spun down and reconstituted according to the
manufacturer’s instructions. The recombinant SAA
used is a hybrid of SAA1 and SAA2, corresponding to
SAA1α, except for the Nt methionine and a substitution
of asparagine to aspartic acid at position 60 and arginine
to histidine at position 71, which are known to be present
in SAA2β. Human recombinant IL-1β was obtained from
Invitrogen Europe (KemoMed, SI). Aliquots of both
reagents were stored at -20°C until used.
Cell culture
Human umbilical vein endothelial cells (HUVEC)
passage 1 and human coronary artery endothelial cells
(HCAEC) passage 3 were purchased from Cambrex
BioScience, Belgium. Cells were grown under standard
5% CO2 humidified atmosphere at 37ºC in endothelium
growth medium (EGM-2), supplemented with singlequots
EGM-2-MV (HCAEC with 5% fetal bovine serum) and
singlequots EGM-2 (HUVEC with 2% fetal bovine
serum). Cells were routinely passaged with Trypsin/
EDTA, grown in 6 well plates (TPP, Switzerland) and
were used for experiments at low passages between 3 and
6. Two hours prior to each experiment cell cultures were
75
Eur. J. Inflamm.
serum starved and fresh serum-free medium was delivered
again immediately prior to treatments.
Measurement of human IL-6, ICAM, VCAM and Eselectin proteins
HUVEC and HCAEC supernatants were collected 24
h after starting cell culture treatments and spun for 3 min
at 5,000 rpm, aliquoted and stored at -20oC. Samples were
thawed and allowed to reach room temperature and, when
necessary, diluted in the standard diluent buffer provided
by the ELISA manufacturers. The concentrations of
human released IL-6, soluble ICAM-1, soluble VCAM-1
and E-selectin were measured in duplicate using ELISA
kits (BioSource, Belgium) following the manufacturer’s
instructions. Statistical analysis was performed using
analysis of variance, from the mean + SD of three
separate experiments with *p<0,05; **p<0,01 compared
to basal levels of cell expression.
RNA isolation, reverse transcription, and polymerase
chain reaction (PCR) analysis
Total RNA was extracted using RNAgents. Total
RNA Isolation System (Promega, Europe) following the
manufacturer’s instructions. RNA purity and amount were
estimated by spectrophotometry. 1 μg of total RNA was
transcribed into cDNA by Reverse Transcription System
(avian myeloblastosis virus, Promega, Europe) and PCR
(with PCR MasterMix, Promega, Europe) was performed
for β-actin, IL-6, IL-8, IL-8R, SAA1/2, ICAM-1, VCAM1 and E-selectin. The specific sets of primers, conditions
and generated fragment sizes are listed in Table I. β-actin
was used in order to compare equal loading, normalize
the data and establish consistency of the PCR reactions.
A negative PCR control reaction was run alongside all
reactions. The PCR products were loaded onto 4% agarose
E-gels (Invitrogen, Europe), electrophoresed and viewed
under UV illumination.
RESULTS
SAA dose dependent stimulation of IL-6 in
HUVEC and HCAEC
To determine whether and to what extent SAA
increases the inflammatory response in HUVEC
and HCAEC, released protein levels of IL-6 of
supernatants were measured by ELISA (Fig. 1, panel
A) following a 24 h period of incubation with SAA,
and isolated the RNA and performed RT-PCR (Fig.
1, panels B and C).
Firstly, confluent cultures of normal human
HUVEC and HCAEC were incubated with SAA in
varying concentrations from 0-2000 nM. We then
examined the effect of SAA increasing doses on IL6 protein levels (Fig. 1 panel A) and on IL-6 mRNA
expression (Fig. 1 panels B and C) and compared
them in HUVEC and HCAEC. The results showed
that HCAEC strikingly elevated both IL-6 released
protein and mRNA expression following SAA
stimulation. HCAEC already exhibited a significant
increase of IL-6 protein following stimulation with
the lowest SAA concentration of 10 nM (p<0.05)
and in contrast there was no such significant increase
of IL-6 found in HUVEC at this concentration. At
the highest SAA concentration of 2000 nM, IL-6
released protein levels on HCAEC were 4 fold higher
than on HUVEC. These significant differences were
not only confirmed by the IL-6 mRNA expression
levels following RT-PCR in HUVEC (Fig. 1, panel
B-middle) and HCAEC (Fig. 1, panel C-middle), but
Table I. PCR primers and conditions.
Table I. PCR primers and conditions.
Name
�-actin
IL-6
IL-8
SAA1/2
ICAM-1
VCAM-1
E-selectin
Primer sequence (all 5' to 3')
s: ATCTGGCACCACACCTTCTACAATGAGCTGCG
as: CGTCATACTCCTGCTTGCTGATCCACATCTGC
s: ATGAACTCCTTCTCCACAAGCGC
as: GAAGAGCCCTCAGGCTGGACTG
s: CAAGGAGTGCTAAAGAACT
as: AACCCTCTGCACCCAGTT
s: CGAAGCTTCTTTTCGTTCCTT
as: CAGGCCAGCAGGTCGGAAGTG
s: GGCAAGAACCTTACCCTA
as: CATTCAGCGTCACCTTGG
s: GGATTCTGTGCCCACAGTA
as: CCTGGCTCAAGCATGTCATA
s: CTCTGATAGAAGAAGCCAAGAAC
as: ACTTGAGTCCACTGAAGCCAGG
Ta
Cycle #
Fragm. size
59°C
25
838 bp
51°C
30
628 bp
35°C
35
187 bp
50°C
30
303 bp
56°C
30
646 bp
60°C
30
1002 bp
58°C
30
255 bp
Ta, temperature
of annealing;
Cycle #, number
of#,cycles
used
in PCR;
Fragm.
size
Ta, temperature
of annealing
Cycle
number
of cycles
used in
PCR size, fragment
Fragm.
size, fragment size
76
K. LAKOTA ET AL.
A.
A.
IL-6 ELISA of supernatants
**
**
IL-6 ELISA of supernatants
**
2500
2500
**
(pg/ml)
IL-6IL-6
(pg/ml)
2000
2000
**
**
1500
**
1000
SAA 100nM
SAA 500nM
**
SAA 500nM
SAA 1000nM
** **
500
SAA 1000nM
SAA 2000nM
*
** **
0
HUVEC
0
B.
HCAEC
HCAEC
C.
HUVEC
1
2
3
4
SAA 2000nM
*
HUVEC
5
6
-
+
800bp
HCAEC
�-actin
2652bp
M
1
2
3
4
5
6
-
+
800bp
IL-6
2652bp
M
1
2
3
4
5
-
6
+
�838bp
IL-6
2652bp
M
1
2
3
4
5
6
-
+
800bp
800bp
IL-8
200bp
SAA 0nM
SAA 10nM
SAA 10nM
SAA 100nM
**
** **** **
500
M
SAA 0nM
*
1000
2652bp
**
**
1500
�-actin
**
**
**
**
**
M
1
2
3
4
5
6
-
150bp
+
�628 bp
IL-8
200bp
150bp
M
1
2
3
4
5
6
-
+
�187 bp
Fig. 1. Expression of IL-6 with increasing concentrations of SAA.
Fig. 1.and
Expression
IL-6 with
concentrations
SAA. + SD of three separate experiments)
A: IL-6 ELISA from HUVEC
HCAECoftreated
withincreasing
increasing
doses of SAAof(mean
A: IL-6 ELISA of supernatants from HUVEC and HCAEC treated with different doses of SAA
*p<0,05; **p<0,01 compared
with basal levels of expression (analysis of variance) B: representative experiment (from
(mean + SD of three separate experiments) *p<0,05; **p<0,01 compared with basal levels of
two separate experiments
performed
in duplicate)
of β-actin,
IL-6, andexperiment
IL-8 PCR(from
fromtwo
HUVEC
(concentrations of SAA:
expression (analysis
of variance)
B: representative
separate
Lane 1) Background control,
0nM
SAA,
2)
10nM,
3)
100nM,
4)
500nM,
5)
1000nM,
6)
2000nM)
C: representative
experiments performed in duplicate) of �-actin, IL-6, and IL-8 PCR from HUVEC
experiment (from three (concentrations
separate experiments)
of β-actin,
IL-6, and
IL-8 PCR
of SAA: Lane
of SAA: Lane
1) Background
control,
0nM from
SAA, HCAEC
2) 10nM, (concentrations
3) 100nM, 4)
500nM,4)
5)500nM,
1000nM,5)
6)1000nM,
2000nM) C:
experiment
separate
1) 0nM, 2) 10nM, 3) 100nM,
6) representative
2000nM); Bottom
panel(from
photothree
of IL-8
RT-PCR on HUVEC was
experiments)
�-actin,
IL-6,rest
andPCR
IL-8 photos.
PCR from HCAEC (concentrations of SAA: Lane 1)
taken at a 2,5 times longer
exposureofthan
all the
0nM,
2) 10nM,IL-1β
3) 100nM,
4) 500nM, 5) 1000nM, 6) 2000nM); Bottom panel photo of IL-8
- negative PCR reaction,
+ positive
control.
RT-PCR on HUVEC was taken at a 2,5 times longer exposure than all the rest PCR photos.
- negative PCR reaction, + positive IL-1� control.
showed even greater differences (larger than 20 fold
as measured by densitometry-data not shown) than
was found at the protein levels. A PBS background
control was always included in the experiments
(lane 1, 0nM SAA), IL-1β was used (1 ng/ml) as a
positive control and a PCR negative control was run
with all reactions. Taken together, there were striking
and significant differences on both IL-6 expression
levels (mRNA and protein) between HUVEC and
HCAEC in response to SAA with HCAEC giving a
much higher response to all SAA concentrations.
In order to determine the effect of SAA
increasing doses on chemokine expression, IL-8
mRNA levels were also compared in both HUVEC
(Fig. 1, panel B-bottom) and HCAEC (Fig. 1, panel
C-bottom). Similarly to IL-6 mRNA expression
levels, IL-8 expression in HCAEC showed SAA
dose dependency starting already at 10 nM. In
HUVEC IL-8 mRNA was not shown to be expressed
until SAA concentrations of 1000 nM and greater.
IL-8 receptor mRNA expression however, did not
correlate to increasing doses of SAA and stayed
around background levels in both HUVEC and
HCAEC (data not shown).
Expression of adhesion molecules (s-ICAM-1, sVCAM-1, E-selectin) with increasing concentrations
of SAA
The response of both cell types to SAA was further
examined with influence on adhesion molecule
77
Eur. J. Inflamm.
A.
B.
2652bp
sICAM-1 ELISA of supernatants
800bp
sICAM-1 (ng/ml)
**
30
25
**
**
**
3
4
5
6
-
+
SAA 100nM
2000bp
1
2
3
4
5
6
-
+
1
2
3
4
5
6
-
+
�838 bp
VCAM-1
2652bp
M
1
2
3
4
5
6
-
+
�1002 bp
800bp
ICAM-1
2652bp
800bp
HCAEC
�-actin
M
SAA 0nM
M
1
2
3
4
5
6
-
+
ICAM-1
M
800bp
1
2
3
4
5
6
-
1
2
3
4
5
6
-
+
�646 bp
SAA 500nM
15
*
SAA 1000nM
SAA 2000nM
**
0
HUVEC
HCAEC
sVCAM-1 ELISA of supernatants
70
**
60
sVCAM-1 (ng/ml)
2
800bp
SAA 10nM
**
20
SAA 0nM
**
50
**
40
**
30
20
1
800bp
M
35
5
M
VCAM-1
40
10
C.
HUVEC
�-actin
**
**
10
**
SAA 10nM
SAA 100nM
SAA 500nM
SAA 1000nM
SAA 2000nM
*
0
HUVEC
HCAEC
expression. The results indicate that correlation of all
tested adhesion molecules (sVCAM-1, sICAM-1 and
E-selectin) and SAA induction (500 nM and above)
was significant. In contrast to IL-6 the response of
adhesion molecules at the protein levels was similar
on both cell types (Fig. 2, panel A), however with
a higher response in HUVEC than in HCAEC. Eselectin ELISA was measured but not shown due
to 70% of results being below the detection limit of
the kit and thus quantitation was uncertain. At lower
doses of SAA the mean sVCAM-1 protein levels in
HCAEC supernatants were higher than in HUVEC,
but at concentrations of 1000 nM and 2000 nM the
levels of sVCAM-1 rapidly increased in HUVEC.
sICAM-1 protein levels remained higher on HUVEC
following stimulation with all SAA concentrations.
However, the difference between HUVEC and
HCAEC is not as striking as compared to IL-6
(at 2000 nM SAA, sICAM-1 is 1.5 fold higher on
HUVEC, while IL-6 is 4 fold higher on HCAEC).
The SAA dose dependent response of all adhesion
molecules was also confirmed at the mRNA levels
(Fig. 2, panels B and C).
E-selecin
350bp
250bp
M
1
2
3
4
5
6
-
+
E-selecin
350bp
M
250bp
+
�255 bp
Fig.
2. Expression of adhesion molecules with increasing
Fig. 2. Expression of adhesion molecules with increasing concentrations of SAA.
A: sICAM-1 and sVCAM-1
concentrations
of ELISA
SAA.of supernatants from HUVEC and HCAEC treated with
different doses of SAA (mean + SD of three separate experiments) *p<0,05; **p<0,01
A:
sICAM-1
and
sVCAM-1
ELISA
of supernatants
from
compared with basal levels of expression (analysis
of variance)
B: representative experiment
(from two separate
experiments
performed)
of VCAM-1,
ICAM-1, E-selectin
and
�-actin
HUVEC
and
HCAEC
treated
with
different
doses
of
SAAPCR
from HUVEC (concentrations of SAA: Lane 1) 0nM, 2) 10nM, 3) 100nM, 4) 500nM, 5)
1000nM, 6)+
2000nM)
experiment (from
three separate experiments
(mean
SD C:ofrepresentative
three separate
experiments)
*p<0,05;
performed) of VCAM-1, ICAM-1, E-selectin and �-actin PCR from HCAEC (concentrations of
**p<0,01
compared
with
basal
levels
of
expression
SAA: Lane 1) 0nM, 2) 10nM, 3) 100nM, 4) 500nM, 5) 1000nM, 6) 2000nM). - negative PCR
reaction, + positive
control.
(analysis
of IL-1�
variance)
B: representative experiment
(from two separate experiments performed) of VCAM1, ICAM-1, E-selectin and β-actin PCR from HUVEC
(concentrations of SAA: Lane 1) 0nM, 2) 10nM, 3) 100nM,
4) 500nM, 5) 1000nM, 6) 2000nM) C: representative
experiment (from three separate experiments performed)
of VCAM-1, ICAM-1, E-selectin and β-actin PCR from
HCAEC (concentrations of SAA: Lane 1) 0nM, 2) 10nM,
3) 100nM, 4) 500nM, 5) 1000nM, 6) 2000nM). - negative
PCR reaction, + positive IL-1β control.
Expression of SAA mRNA levels as positive
feedback to its own stimulation
In order to determine whether SAA is expressed
in both HUVEC and HCAEC and whether its
own expression is dose dependently induced, we
performed RT-PCR. Following a 24h incubation of
HUVEC and HCAEC with increasing doses of SAA
resulted in a dose dependent SAA mRNA expression,
but only in HCAEC (Fig. 3, panel A). SAA mRNA
expression was slightly detected even at background
control levels in HCAEC, but was not detected at
all in HUVEC, even with the highest concentration
(2000 nM) of SAA used (Fig. 3, panel B).
DISCUSSION
In the past 10 years there has been an increasing
body of reports strengthening the postulation
that inflammation not only provides the baseline
for future atherogenetic processes leading to
atherosclerosis but is also a prerequisite at different
stages of plaque build-up and/or plaque rupture, the
latter providing a gateway to thrombosis (1-2).
78
K. LAKOTA ET AL.
A.
�-actin
2652bp
M
800bp
300bp
1
2
3
4
5
6
-
+
M
1
2
3
4
5
-
+
HCAEC
�-actin
2000bp
800bp
SAA
400bp
B.
HUVEC
M
1
2
3
4
5
6
-
+
�838 bp
SAA
400bp
M
1
2
3
4
5
6
-
+
�303 bp
200bp
Fig. 3. Expression
of SAA with increasing
concentrations
of concentrations
SAA.
Fig. 3. Expression
of SAA with
increasing
of SAA.
A: RT-PCR
representative
experiment
(from
three
separate
experiments
performed)experiments
of β-actin andperformed)
SAA PCR fromofHUVEC
A: RT-PCR representative experiment (from three separate
�-actin
(concentrations of SAA: Lane 1) 0nM, 2) 10nM, 3) 100nM, 4) 500nM, 5) 1000nM, 6) 2000nM) B: RT-PCR representative
and SAA PCR from HUVEC (concentrations of SAA: Lane 1) 0nM, 2) 10nM, 3) 100nM, 4)
experiment (from three separate experiments performed) of β-actin and SAA PCR from HCAEC (concentrations of
500nM,
6) 2000nM)
B: 5)
RT-PCR
experiment
(from three
separate
SAA: Lane
1) 0nM,5)
2) 1000nM,
10nM, 3) 100nM,
4) 500nM,
1000nM,representative
6) 2000nM). - negative
PCR reaction,
+ positive
IL-1β
experiments
performed)
of
�-actin
and
SAA
PCR
from
HCAEC
(concentrations
of
SAA:
Lane
control.
1) 0nM, 2) 10nM, 3) 100nM, 4) 500nM, 5) 1000nM, 6) 2000nM). - negative PCR reaction, +
positive IL-1� control.
Many human vascular diseases are restricted to
specific vessel types, and endothelial cells derived
from these vessels can elicit completely different
responses. For example, the susceptibility of arterial
and vein vessels to atherosclerosis is different as is
the contribution of platelets to the pathogenesis of
arterial and venous thrombosis (31). There is a lack
of reports describing the possible mechanisms of
SAA in arterial and venous thrombosis, even though
antibodies against SAA have been described, and
association was found between deep vein thrombosis
and anti-SAA antibodies (32). Previously our group
has indicated that SAA could serve as a good
predictor of progression from a non-inflammatory
thrombotic condition to an inflammatory one (33).
To date there have been a limited number of
reports indicating SAA expression in either human
artery and/or vein endothelial cells. A-SAA protein
was identified in the supernatants of primary
synoviocyte cultures, and its expression co-localized
with vascular endothelial cells (34). Next, the same
group showed that A-SAA mRNA and its receptor
FPRL1 mRNA were present in endothelial cells
isolated from the synovial tissue of patients with RA
and other categories of inflammatory arthritis (35). In
human microvascular endothelial cells A-SAA was
shown to increase adhesion molecule expression,
endothelial cell tube formation and migration and
induced NF-κB translocation (36).
SAA expression in endothelial cells lining blood
vessels was first reported in 1994 by Meek et al.
(12), which was later confirmed, and studies further
expanded on human aortic smooth muscle cells (37)
which were found to express both the acute and
constitutive isoforms of SAA. HUVEC was not
found to express any detectable levels of SAA (37),
which we can also confirm with our current data
(Fig. 3, panel A).
We report in this study the following novel
observations: first is the exclusive SAA mRNA
expression in HCAEC versus HUVEC and its own
positive feedback (Fig. 3B versus Fig. 3A); second
is the striking difference between IL-6 levels of
HCAEC and HUVEC, with the coronary artery cells
having a ˜4 fold higher induction of IL-6 released
protein (Fig. 1A) and a ˜20 fold higher induction of
IL-6 mRNA expression (Fig. 1B and C, densitometry
not shown); and third is that in HUVEC the level of
adhesion molecule expression is slightly greater than
in HCAEC (Fig. 2A). These observations lead to the
speculation that HCAEC are more responsive to ASAA in inducing pro-inflammatory IL-6 and IL-8
than adhesion molecules in comparison to HUVEC
and that different receptors and/or signaling
pathways may be activated. To date there has been
only one other report in literature describing SAA
induced IL-6 and IL-8 protein secreted levels in
HCAEC presented as a comparison to its main theme
adipokine/adipocyte study, which however, did not
show a dose dependent response or any confirmation
Eur. J. Inflamm.
at the mRNA expression levels (38).
Our current data provide an indication of greater
susceptibility of coronary artery endothelial cells to
inflammatory stimuli and a confirmation of the potential
role which SAA plays in cardiovascular diseases.
In the future we plan to expand our studies
to include a more complete study of A-SAAreceptor signaling and the inhibitory mechanisms
of SAA induced proinflammatory effects in human
endothelial cells.
7.
8.
9.
ACKNOWLEDGEMENTS
We thank all members of the Laboratory for
Immunology, Department of Rheumatology,
University Medical Centre, Ljubljana for their
cooperation during the execution of our work.
Support for this study was obtained by the Marie
Curie International Reintegration Grant MC-IRG #
028414 to S. S-S.
10.
11.
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EUROPEAN JOURNAL OF INFLAMMATION
Vol. 5, no. 2, 83-88 (2007)
MODULATION OF GRO-α AND TNF-α PRODUCTION BY PERIPHERAL BLOOD
MONONUCLEAR CELLS TREATED WITH KILLED HELICOBACTER PYLORI
G. STASSI, A. CASCIO1, C. IARIA1, D. GAZZARA, G.B. COSTA, D. IANNELLO and A. ARENA
Unit of Microbiology, Department of Surgical Science, Policlinico Universitario; 1Section of
Infectious Diseases, Department of Human Pathology, Policlinico Universitario, Messina, Italy
Received January 16, 2007 – Accepted May 18, 2007
GRO-alpha seems to play an important role in recruiting and activating neutrophils during
Helicobacter pylori infection. In the present study, we examined how treatment with killed H. pylori
or/and live H. pylori may differentially influence the in vitro GRO-alpha and TNF-alpha release by
peripheral blood mononuclear cells (PBMC). The amounts of TNF-alpha and GRO-alpha produced by
PBMC after stimulation with live H. pylori were higher than those produced after stimulation with a
combination of killed and live H. pylori and the latter were higher than those produced after stimulation
with killed H. pylori. In conclusion, the treatment of peripheral blood mononuclear cells with killed
H. pylori down-regulates the production of GRO-alpha. Taken together, our data demonstrate that
treatment with killed H. pylori could represent an innovative approach during gastric infection
supported by H. pylori.
H. pylori is the most common bacterial pathogen
involved in human gastrointestinal pathology.
Gastric colonization by H. pylori, a minimally
invasive gram-negative bacterium, is the major cause
of chronic active gastritis and is often associated
with both duodenal and gastric ulceration, as well as
gastric carcinoma and mucosa-associated lymphoid
tissue lymphoma (1-3). The local inflammation
induced by H. pylori is characterized by infiltration
of neutrophils, lymphocytes, plasma cells and
monocytes in the gastric mucosa and by the local
production of cytokines and chemokines (4-5).
Among these, GRO-α, a CXC chemokine, seems
to play an important role in recruiting and activating
neutrophils in the gastric mucosa. In addition,
TNF- α has been demonstrated to be important for
the up-regulation of GRO-α production and that at
high concentrations, it can by itself injure the gastric
mucosa and may be responsible for severe pathology
(6-7). GRO-α possesses potent neutrophilstimulating activity by inducing chemotaxis, shape
change, a rise in intracellular free calcium levels,
exocytosis, and the respiratory burst in these cells
(8). GRO-α was initially isolated and characterized
by its growth stimulatory activity on malignant
melanoma cells (9). Moreover, this peptide can
regulate endothelial cell proliferation, stimulating
angiogenesis (10).
In a previous study, carried out in an in vitro
model (11), we demonstrated that the immunological
disorders determined by H. pylori infection could be
related to a shift from a Th-2 to a Th-1 type cytokine
profile. In light of these results, in the present study
we analyze the direct effect of H. pylori (live or
Key words: GRO-α, TNF-α, Helicobacter pylori, PBMC
Mailing address: Prof. Giovanna Stassi,
Unit of Microbiology,
Policlinico Universitario G. Martino,
Via C.Valeria 1,
98125 Messina, Italy
Tel: ++39 0902213305 Fax: ++39 0902213302
e-mail: [email protected]
1721-727 (2007)
83
Copyright © by BIOLIFE, s.a.s.
This publication and/or article is for individual use only and may not be further
reproduced without written permission from the copyright holder.
Unauthorized reproduction may result in financial and other penalties
84
G. STASSI ET AL.
gentamicin-killed) on human PBMC in order to
evaluate the in vitro production of TNF-α and of
GRO-α which seems to represent a key factor in
the clinical outcome of inflammatory diseases and
malignancies including gastric carcinoma.
MATERIALS AND METHODS
PBMC were obtained from healthy, H. pyloriseronegative donors, after centrifugation of heparinized
venous blood over Ficoll-Hypaque gradient (Pharmacia,
Milan, Italy) (12). PBMC were then washed twice in
RPMI 1640 medium and cultured in 24 well plates
(Corning, Bibby srl, Milan, Italy) at a concentration
of 2x106 (colony-forming unit) CFU/mL per well in
RPMI 1640 medium. PBMC were cultured at 37°C in
5% CO2 atmosphere, in RPMI 1640 (Biochrom KG
Seromed, Milan, Italy) supplemented with 50 mM 2mercaptoethanol, 1 mM pyruvate, 1 mM non-essential
aminoacids, 1 mM HEPES and 5% fetal calf serum (FCS)
(Biochrom KG Seromed, Milan, Italy). Culture media and
reagents tested for the presence of endotoxin by E-Toxate
kit (Sigma, Milan, Italy) were found to contain <10 pg of
endotoxin per mL.
Supernatants from PBMC in different experimental
conditions were collected and analysed for the presence
of TNF-α and GRO-α by an immunoenzymatic
method: human TNF-α Quantikine immunoassay and
human GRO-α Quantikine immunoassay, (all from
R&D Systems, Milan, Italy); the limit of detection was
respectively 4.4 pg/mL for TNF-α and 10 pg/mL for
GRO-α. Monoclonal anti-human TNF-α antiboby (ND50
for this lot of anti-human TNF-α antibody was 0.015-0.06
µg /mL in the presence of 0.25 ng/mL of human TNF-α)
(R&D Systems, Milan, Italy). Monoclonal anti-human
GRO-α antibody (ND50 for this lot of anti-human GROα antibody was 1 µg /mL in the presence of 6 ng/mL of
rhGRO-α) (R&D Systems, Milan, Italy).
H. pylori isolated from the antral mucosa and
associated duodenal ulcer was plated on Skirrow’s agar
and incubated at 37°C in a microaerophilic environment
for 5 days, harvested and diluted in sterile phosphatebuffered saline (PBS) (pH 7.2).
The concentration of bacteria was estimated by
measuring the adsorbance of the suspension and comparing
the value to a standard curve. The standard curve was
generated by measuring the adsorbance of an array of
serially diluted samples before quantifying the number
of viable bacteria in each sample by a colony assay. After
centrifugation at 2,500 x g for 15 min, bacteria were
re-suspended in PBS to a range of concentrations from
105 to 109 CFU/mL, diluted in PBS. The motility of the
organisms was confirmed by phase-contrast microscopy
prior to use. For the experiments with killed bacteria, H.
pylori was treated with gentamicin 4 mg/mL (Seromed,
Milan, Italy) for 45 min at 4°C, washed, and diluted in
PBS to the same concentrations as the live bacteria (13).
The combined treatment was performed by adding
a suspension of gentamicin-killed bacteria (1.2 x 109
killed CFU/ml) to PBMC for 20 hours, and after this
period adding 1.2 x 109 CFU/mL live H. pylori for further
24 hours. After 44 hours supernatants were harvested,
centrifuged and stored at –80°C until cytokine assays.
To determine the effect of different incubation
times on cell viability a colorimetric assay was used as
described by Mosmann (14). The assay is based on the
tetrazolium salt 3-(4,5 dimethylthiazol-2-yl)2,5diphenylt
etrazoliumbromide (MTT), (Sigma, Milan, Italy) a pale
yellow substrate that is cleaved by active mitochondria
to produce a dark blue formazan product. Briefly, PBMC
were seeded in 96-microwell plates at 2x104 per well, then
treated with live or killed H. pylori for 24 or killed + live
H. pylori for 44 hours. MTT diluted in saline solution
was added to the cells and incubated for 4 hours, then
acid propan-2-ol (0.04M HCl in propan-2-ol) (Sigma,
Milan, Italy) was used to solubilize the formed crystals.
The plates were read with a microELISA reader using
a wavelength of 570 nm. Cytotoxicity percentage was
calculated as follows:
1_ [(experiment OD - lysis control OD) / (cell control
OD - lysis control OD)] x 100.
Results are expressed as the means of five experiments
+ standard deviation (SD). Data were analysed by one
way ANOVA and the Student-Newman-Keuls test.
Differences were considered statistically significant for
p value of <0.05.
RESULTS
The effects of different concentrations of live H.
pylori on cytokine release by PBMC are reported in
Fig. 1. The results demonstrate that the production
of GRO- α and TNF- α was dose-dependent. In
particular, a significant differential stimulation
was evident at a concentration of 1x109 CFU/mL.
In a second series of experiments, we examined
how treatment with killed H. pylori or/and live H.
pylori infection may differentially influence the in
vitro cytokine release by PBMC. Results reported
in Fig. 2 show that infection with live H. pylori
triggers PBMC to release marked amounts of GROα compared with those induced by killed H. pylori
(1972 ± 414 pg/mL vs 642 ± 121 pg/mL; p<0.05).
85
Eur. J. Inflamm.
4000
Gro-alpha
3500
TNF-alpha
3000
pg/mL
2500
2000
1500
1000
500
0
10^5
10^7
10^9
H.pylori live (CFU/mL)
Fig.1
Fig. 1. Effects of Helicobacter pylori concentration on GRO-alpha and TNF-alpha production by PBMC. Results represent
the means of five experiments using PBMC of 5 different donors. Data are shown as mean + standard deviation.
3000
2500
GRO-alpha (pg/mL)
2000
1500
1000
500
0
H.p live
H.p live+Ab-TNF
alpha
H.p killed
Fig.2
H.p killed+AbTNF alpha
H.p
killed+H.p.live
H.p killed+H.p
live+ Ab-TNF
alpha
Fig. 2. Production of GRO-alpha by PBMC after treatment with live, killed and killed+live Helicobacter pylori in
presence or not of monoclonal antibodies vs TNF-alpha. Results represent the means of 5 experiments using PBMC of 5
different donors. Data are shown as mean + standard deviation.
The combined treatments induced a down-regulation
of GRO- α production compared to live H. pylori
(1161 ± 185 pg/mL vs 1972 ± 14 pg/mL; p<0.05). In
order to verify whether these results were influenced
by the incubation time and additional treatment, the
cell viability was analyzed by MTT test. It was found
that the different incubation times (24 and 44 hours)
as well as the treatment with killed and live H. pylori
did not significantly influence the cell viability (data
not shown).
86
G. STASSI ET AL.
4000
3500
TNF alpha (pg/mL)
3000
2500
2000
1500
1000
500
0
Fig.3
H.p live
H.p live+Ab-GRO
alpha
H.p killed
H.p killed+AbGROalpha
H.p.killed+H.p.live H.p killed+H.p live+
Ab-GRO alpha
Fig. 3. Production of TNF-alpha by PBMC after treatment with live, killed and killed+live Helicobacter pylori in
presence or not of monoclonal antibodies vs GRO-alpha. Results represent the means of 5 experiments using PBMC of 5
different donors. Data are shown as mean + standard deviation.
Furthermore, as shown in Fig. 3, similar behaviour
was seen for TNF−α. In particular, whereas live H.
pylori infection induced marked levels of TNF- α
compared with killed H. pylori (3285 ± 92 pg/mL
vs 1436 ± 186 pg/mL; p<0.05), the combined effect
(killed + live) resulted in higher levels of TNF- α
compared to the treatment with killed H. pylori
(2317 ± 417 pg/mL vs 1436 ± 186 pg/mL; p<0.05),
but lower when compared with live H. pylori (2317
± 417 pg/mL vs 3285 ± 492 pg/mL; p<0.05).
To verify a possible correlation between TNF- α
and GRO- α we added monoclonal anti-TNF-α or
anti-GRO-α antibodies to PBMC in all experimental
conditions. As shown in Fig. 2 the addition of
monoclonal Ab-TNF- α determined a downregulation of GRO-α production. In particular,
the addition of anti-TNF- α to PBMC respectively
incubated with live, killed or killed+live H. pylori,
decreased the levels of GRO- α produced by these
cells (540 ± 86 pg pg/mL; respectively p<0.05). /mL
vs 1972 ± 414; 180 ± 27 pg/mL vs 642 ± 121; 364±
61 vs 1161 ± 185.
These results demonstrate that the strong increase
in GRO-α expression in PBMC infected with live H.
pylori was, at least in part, dependent on the presence in
supernatants of TNF- α. On the contrary, as shown in
Fig. 3, the addition of monoclonal antibodies anti-GROα did not influence the TNF-α release. This finding
demonstrates that TNF-α production is not supported
by GRO-α. Furthermore, as shown in Fig. 2 and 3, the
combined treatment with killed+live H. pylori produced
the same effect, though to a lesser extent, on TNF-α and
GRO-α release observed with killed H. pylori.
DISCUSSION
Few studies have investigated the expression
of GRO-α and its role in the outcome of the H.
pylori infection. Such knowledge is critical to fully
understanding the relationships of H. pylori infection
with gastric carcinoma.
Evidence for in vivo GRO-α induction in H.
pylori infection has been reported by Suzuki et
al., who demonstrated increased mucosal levels of
this chemokine in infected individuals, as well as
decreased levels after eradication of infection (15). It
is generally agreed that PBMC are important sources
of chemokines and other proinflammatory mediators.
In particular, it was shown that several chemokines
are an essential component of the primary innate
87
Eur. J. Inflamm.
immune response to H. pylori infection (4, 16-17).
In this paper we report our experimental data
that further characterize the chemokine response
induced in PBMC in vitro treated with live or killed
H. pylori. Our results demonstrate that treatment
with live H. pylori triggers PBMC to release marked
amounts of GRO-α compared with those induced by
killed H. pylori In light of the results obtained with
GRO-α and in order to better understand its role
during H. pylori infection, we made a pre-treatment
of PBMC with killed H. pylori ,and after 20 hours
we infected the same cells with live H. pylori. Our
findings demonstrate that the combined treatments
induced a down-regulation of GRO- α production
compared to live H. pylori. Furthermore a similar
behaviour was seen for TNF−α.
To verify a possible correlation between TNF-α
and GRO-α we added monoclonal anti-TNF-α or
anti-GRO-α antibodies to PBMC in all experimental
conditions. The addition of monoclonal Ab-TNF-α
determined a down-regulation of GRO-α production
On the contrary, the addition of monoclonal
antibodies anti-GRO-α did not influence the TNFα release. These results demonstrate that the strong
increase in GRO-α expression in PBMC infected
with live H. pylori was, at least in part, dependent
on the presence in supernatants of TNF-α. On this
basis, we can speculate that TNF-α induced by H.
pylori may exert a pathogenic effect not only by
itself but also by supporting GRO-α production. Our
results are in agreement with those of other Authors
which demonstrate that TNF−α is important for the
up-regulation of GRO-α production by stimulated
human endothelial cells (18).
In previous studies, we reported that treatment of
peripheral blood mononuclear cells with killed H.
pylori promoted the release of anti-inflammatory
cytokines such as IL-10 and IL-4, thus subverting
immunological disorders
accounted for by a
shift from a Th-2 to a Th-1 type cytokine profile
determined by live H. pylori infection (11, 19).
These data indicate that treatment with killed
H. pylori may alter the balance of cytokines in
the environment of cells, preventing potentially
harmful effects of high levels of TNF-a and GROa representative of the deleterious Th1 immune
response usually associated with H. pylori infection.
In fact, these in vitro results demonstrate that pre-
treatment with killed H. pylori plays a protective role
during H. pylori infection decreasing inflammatory
response supported by Th-1 cytokines and
chemokines.
Taken together our results suggest that treatment
of PBMC with killed H. pylori could subvert the
environment of cytokine patterns responsible for
the inflammatory process and marked recruitment
of monocytes and lymphocytes in gastric mucosa
during in vivo H. pylori infection. Therefore, it could
be speculated that treatment with killed H. pylori
could represent an innovative therapeutical approach
during H. pylori gastric infection.
ACKNOWLEDGEMENTS
This work was supported by “Fondi di ricerca
scientifica dell’ Università degli Studi di Messina”.
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M. Miceli and P. Mastroeni. 2003. Treatment
of PBMC with killed Helicobacter pylori subverts
the environment of inflammatory cytokines. New
Microbiol. 26:227.
EUROPEAN JOURNAL OF INFLAMMATION
Vol. 5, no. 2, 89-95 (2007)
TREATMENT OF MILD TO MODERATE PLAQUE PSORIASIS WITH CALCITRIOL
OINTMENT APPLIED WITH OR WITHOUT A DOSING DEVICE
G.A. VENA, N. CASSANO, G. ALESSANDRINI1, D. FAI1, M. GABELLONE1,
P. LIGORI1, C. MALVINDI1, A. MANCINO1, S. PELLÈ1, F. RINALDI1 and M.R. SODO1
2nd Dermatology Clinic, M.I.D.I.M. Department, University of Bari; 1Collegio Salentino di
Dermatologia, Lecce, Italy
Received December 12, 2006 – Accepted May 20, 2007
Results of topical treatments can be influenced by several factors, including accurate dosing based on
the affected skin area. The aim of this open-labelled multicenter study is to evaluate if correct dosing of
calcitriol ointment has an impact on the clinical response of plaque psoriasis. For this purpose, patients
with plaque psoriasis eligible to be treated with calcitriol ointment were randomized to treatment with a
‘standardized dose’ method, using a dosing device (N. 100), or treatment without the device (N. 101), for
12 weeks. Regardless of the use of the dosing device, calcitriol ointment caused a significant reduction of
both the severity of signs and symptoms and the extent of body surface area affected after both 4 weeks
and 12 weeks. Clinical response was not significantly different between the two groups. Local adverse
events occurred in 12 subjects (of whom five treated with the device) and were mild and transient in
most cases.
response in vivo (3-8). These effects are important
for the therapeutic activity of calcitriol ointment
in psoriasis documented by several clinical studies
(9-14).
Results of topical treatments can be conditioned
by several factors, including patient adherence
and accurate dosing. For example, underdosing
may reduce the efficacy of topical medications, as
well as overdosing may augment the risk of side
effects.
In this study we evaluate whether correct dosing
of calcitriol ointment influences the efficacy and
tolerability outcomes in patients with mild to
moderate plaque psoriasis. For this purpose, we
analyse the results obtained with or without the use
of a dosing device.
Vitamin D3 analogues are well-established
topical treatments for psoriasis and are a first-line
approach to mild/moderate chronic plaque psoriasis.
These drugs are thought to inhibit proliferation
and promote differentiation of keratinocytes by
regulating intracellular calcium concentrations.
Calcitriol (1alpha,25-dihydroxyvitamin D3) is
the physiological active form of vitamin D3 and
is an essential hormone for calcium homeostasis.
Biological activity of calcitriol occurs through
the binding to nuclear receptors, which are found
in several tissue types and cell lines, including
keratinocytes and fibroblasts in the skin (12). Several studies have shown that calcitriol
modulates not only keratinocyte proliferation and
differentiation but also inflammation and immune
Key words: plaque psoriasis, calcitriol, vitamin D3 analogue, topical treatment, dosing device
Mailing address: Prof. G.A. Vena, M.D.
Department of Internal Medicine, Immunology and
Infectious Diseases,
2nd Dermatology Clinic, University of Bari,
Piazza Giulio Cesare, 11
70124 Bari, Italy Tel/fax: ++39 080 5478 920
e-mail: [email protected]
1721-727 (2007)
89
Copyright © by BIOLIFE, s.a.s.
This publication and/or article is for individual use only and may not be further
reproduced without written permission from the copyright holder.
Unauthorized reproduction may result in financial and other penalties
90
G.A. VENA ET AL.
MATERIALS AND METHODS
The study was designed as an open-labelled
multicenter assessment of the efficacy and tolerance of
calcitriol 3 microg/g ointment (Silkis, Galderma S.p.A.,
Italy) used for 12 weeks by adult patients with mild to
moderate chronic plaque psoriasis, affecting less than
35% of the body surface area (BSA).
The exclusion criteria included: forms of psoriasis
other than plaque-type; concomitant skin diseases;
hypercalcemia or other abnormalities in calcium and
phosphorus metabolism; relevant liver and kidney
disorders; pregnancy and breast feeding; hypersensitivity
to any ingredients of the study products (e.g., both
Silkis ointment and emollients); the use of any drugs
known to affect psoriasis severity and study evaluations;
unwillingness to adhere to the study procedures. Patients
must not have received anti-psoriasis treatments during
a fixed period prior to the start of the study defined as
follows: 12 weeks for biologicals, 4 weeks for other
systemic therapies and phototherapy, 2 weeks for topical
medications and 1 week for emollients or keratolytics.
After oral consent, patients applied calcitriol ointment
twice daily to all skin lesions for 12 weeks, with or without
the aid of a dosing device. In order to avoid potential
selection bias, consecutive patients were alternately
randomized to one of the two treatment methods according
to a 1:1 ratio. The dosing device (Silkidose), patented by
Galderma Laboratories, consisted of a graduated spatula
that allowed the patients to quantify the exact amount
of the ointment to be applied on the basis of the size of
affected skin areas (15). So, patients who belonged to
the ‘standardized-dose’ treatment regimen received the
device and an instruction form reporting the exact number
of doses per administration, whereas the quantity of the
ointment to be applied was left to the discretion of patients
in the ‘unstandardized-dose’ group.
No active anti-psoriasis treatments were permitted
during the study period, with the exception of emollients
(a cream containing 10% urea, Nutraplus, or another
containing 10% urea and 5% lactic acid, Nutraplus
Forte) which were used by patients on an ‘as-needed’
basis.
Evaluations were made at the baseline (W0), at week
4 (W4), and at the end of treatment (W12). At each
visit, both the extent of BSA affected and the severity of
symptoms and signs were evaluated. BSA affected was
reported as the percentage of the total area for each of the
following body sites: head, upper limbs, trunk and lower
limbs. The severity of erythema, scaling and thickness
was graded using a 5-score rating scale (0= absent; 1=
mild; 2= moderate; 3= severe; 4= very severe). The same
scale was used by patients for the assessment of symptoms
(pruritus/burning).
Changes of clinical parameters in each group were
analysed under a statistical point of view by the Wilcoxon
matched-pairs signed-ranks test, whereas differences
between the two groups at each visit were evaluated using
the Mann-Whitney U test (significance for p values <0.05
in both cases).
At the end of treatment, regardless of its duration,
patients and dermatologists gave their independent
opinion about the efficacy of the study treatment, rating it
on the basis of a 4-point scale. Patients were also asked to
score the acceptability of treatment.
Details of adverse events (AEs) were obtained
throughout the study period.
RESULTS
The study was conducted between October 2005
and May 2006. A total of 201 patients (109 males)
aged 18 to 80 years (mean age, 42.2) with chronic
plaque psoriasis were enrolled and received study
treatment. Of these, 100 patients were required
to use the dosing device and 101 patients entered
the ‘unstandardized-dose’ treatment with calcitriol
ointment. At the baseline, clinical parameters did not
significantly differ between the two groups (Table
I) with the exception of BSA affected of the trunk
which was greater in the group of patients who did
not use the dosing device as compared to the other
group. No relevant inter-group differences were
observed in the distribution of the gender of patients
and affected sites nor in the type of emollient used
throughout the study period (Table I).
Eight patients (four patients in each group)
withdrew from the study after W4 and did not undergo
clinical assessment at W12 visit. Three patients were
lost to follow-up; reasons for discontinuation in the
other cases were treatment-related AEs in four cases
(two in each group) and lack of efficacy in another
patient.
Independently of the use of the dosing device,
calcitriol ointment caused a significant improvement
of psoriasis lesions at W4 and W12 and reduced both
the severity of signs and symptoms (Fig. 1), and the
extent of BSA affected (Fig. 2). Clinical response
after both 4 weeks and 12 weeks did not significantly
differ between the two groups.
In general, calcitriol ointment was well tolerated.
No serious AEs were observed. Local AEs such as
irritation and pruritus occurred in 12 subjects (of
91
Eur. J. Inflamm.
Table I. General characteristics and baseline clinical parameters. At the baseline, no significant inter-group differences
were observed in the distribution of the gender of patients and clinical parameters with the exception of BSA affected of
the trunk which was greater in the group of patients who did not use the dosing device as compared to the other group
(*p<0.05 – Mann-Whitney U test). The type of emollient used throughout the study period did not differ between the two
groups. BSA affected and severity score are reported as mean value along with standard deviation (SD).
CALCITRIOL WITH
DOSING DEVICE
CALCITRIOL WITHOUT
DOSING DEVICE
Total number of patients (n)
100
101
Gender (n)
- Male
- Female
52
48
57
44
Affected sites (n)
- Head
- Upper limbs
- Lower limbs
- Trunk
47
80
76
58
39
85
82
63
8.2 (15.1)
12.5 (12.4)
11.4 (12.15)
7.9 (11.8)*
4.35 (7.03)
12.1 (11.4)
12.4 (12.3)
12.7 (15.3)*
2.1 (0.7)
2.5 (0.8)
2.0 (0.9)
1.8 (0.8)
2.1 (0.7)
2.4 (0.6)
1.8 (0.75)
1.9 (0.8)
48
52
50
51
% BSA affected: mean (SD)
- Head
- Upper limbs
- Lower limbs
- Trunk
Severity score: mean (SD)
- Erythema
- Scaling
- Thickness
- Symptoms (pruritus/burning)
Emollients used during the study
- Nutraplus� cream
- Nutraplus Forte� cream
whom five treated with the device) and were mild
and transient in all cases but four in whom these
events were responsible for study interruption. The
development of AEs did not appear to be related to
either the type of emollient concomitantly used or
the site of treatment, with only two subjects reporting
local irritation after application to the scalp or the
face.
At the end of treatment period, the dermatologists’
and patients’ global assessment of efficacy was
considered positive in the majority of cases with a
similar rate in the two treatment groups (Table II).
Approximately three-quarters of patients in both
groups judged as good or excellent the acceptability
of treatment.
DISCUSSION
The efficacy, safety and tolerability of calcitriol
ointment for the treatment of plaque psoriasis has
been demonstrated by several studies (8-14, 16-21),
which also showed that calcitriol is well tolerated
even when applied to sensitive skin areas and is
less irritating than calcipotriol. 12Our cumulative
92
G.A. VENA ET AL.
A
100
90
week4
week 12
80
% improvement
70
89
85
80
62
60
50
47
45
44
40
30
29
20
10
0
erythema
B
scaling
thickness
symptoms
100
90
week 4
89
88
week 12
82
80
% improvement
70
65
60
48
50
40
50
53
standardized procedures in dermatology which allow
patients to exactly dose their topical treatment. One
of the best known methods of dosing measurement
consists in the use of fingertip units, originally
created for topical therapy in children (22).
However, in clinical practice, prescription of topical
treatments usually disregards dosing procedures or
at least takes into account only generic instructions
(i.e. application of ‘small quantities’; limit of
application on a pre-determined body surface or
limit of consumption of a fixed amount per day or
per week in accordance with the package insert of
medications).
The cumulative results of our study suggest
that the application of an exact dose of calcitriol
ointment, achieved by the use of a graduated spatula
(Silkidose), gave no advantages compared to the
conventional treatment without the dosing device.
31
30
A
20
10
80
72
week 4
70
0
thickness
60
symptoms
Fig. 1. Change in the severity of signs and symptoms during treatment with calcitriol
Fig.
1.applied
Change
the severity
of device.
signs and symptoms
ointment
with (A)inor without
(B) the dosing
Variation of clinical parameters as compared to baseline in each group: p<0.01 at W4, p<0.001 at W12;
during
treatment
with
calcitriol
ointment
applied
differences of parameters between the two patient subgroups at each visit:
p>0.05. with (A)
or without (B) the dosing device.
Variation of clinical parameters as compared to baseline
in each group: p<0.01 at W4, p<0.001 at W12;
differences of parameters between the two patient
subgroups at each visit: p>0.05.
13
results confirm that calcitriol 3microg/g ointment
is an effective and safe treatment for chronic plaque
psoriasis, even when used in affected areas of the
head (scalp and face).
The primary objective of our study was to
determine whether the use of an ‘exact-dose’
treatment, achieved with the aid of a dosing device
(Silkidose), has an impact on efficacy and/or safety
results.
The problem of accurate dosing is theoretically
important for efficacy and tolerability of topical
medications. The estimation of the adequate
amount of topical treatment is individualized after
the evaluation of the lesional skin areas and the
subsequent calculation of the dose to be applied
according to the BSA affected. There are no
% reduction
scaling
50
50
40
30
28
25
23
19
20
10
0
head
B
upper limbs
lower limbs
80
69
70
trunk
74
week 4
68
week 12
60
% reduction
erythema
69
week 12
63
55
50
40
30
31
24
33
27
20
10
0
head
upper limbs
lower limbs
trunk
Fig. 2. Change in the extent of affected skin areas during treatment with calcitriol
Fig. 2.
Change
the extent
affected
ointment
applied
with (A)in
or without
(B) the of
dosing
device. skin areas during
treatment with calcitriol ointment applied with (A) or
without (B) the dosing device.
Variation of affected BSA as compared to baseline in each
group: p<0.01 at W4, p<0.001 at W12;
differences of values between the two patient subgroups at
each visit: p>0.05.
Variation of affected BSA as compared to baseline in each group: p<0.01 at W4, p<0.001 at W12;
differences of values between the two patient subgroups at each visit: p>0.05.
14
12, efficacy of treatment was independently assessed by dermatologists (D) and patients
(P), and P were asked to judge the acceptability of treatment. Positive ratings were
reported in the majority of cases with a similar frequency in the two treatment groups.
Table II. Overall assessment of efficacy and acceptability at the end treatment. At week
12, efficacy of treatment was independently
assessed by dermatologists (D) and patients
Eur. J. Inflamm.
93
Table II. Overall assessment of efficacy and acceptability at the end treatment. At week
(P), and P were asked toCALCITRIOL
judge the acceptability
of treatment.
Positive
ratings were
CALCITRIOL
WITHOUT
WITH
12, efficacy of treatment was independently assessed by dermatologists
(D)
and patients
reported in the majority of cases
with DEVICE
a similar frequency in the
two treatment
groups.
DEVICE
DOSING
(P),
and
asked
to judge
the acceptability
treatment.
Positive
ratings
were
Table
II. POverall
assessment
of efficacy
and
acceptability
at DOSING
the end
treatment.
At week
Table II. Overall assessment
ofwere
efficacy
and
acceptability
at the
endoftreatment.
At
week
12,
efficacy
of treatment was
reported
in the
majority of
cases
with a similar
frequency
in the two treatment
groups.
12,
efficacy
of
treatment
was
independently
assessed
by
dermatologists
(D)
and
patients
independently assessed by dermatologists (D) and patients (P), and P were asked to judge the acceptability of treatment.
(P), and P were asked to judge the acceptability of treatment. Positive ratings were
Positive ratings were reported in the majority of cases with a similar frequency in the two treatment groups.
reported in the majority ofCALCITRIOL
cases with a similar
frequencyCALCITRIOL
in the two treatment
groups.
WITHOUT
WITH
EFFICACY
D (%)
P (%)
D (%)
P (%)
DOSING DEVICE
DOSING DEVICE
CALCITRIOL WITHOUT
CALCITRIOL WITH
DOSING DEVICE
DOSING DEVICE
CALCITRIOL WITHOUT
CALCITRIOL WITH
Excellent
28
30
23
26
DEVICE
DEVICE
EFFICACY
D DOSING
(%)
P (%)
DDOSING
(%)
P (%)
Good
EFFICACY
D52
(%)
P 46
(%)
D54
(%)
P 49
(%)
Moderate
EFFICACY
Excellent
D16
(%)
28
P 13
(%)
30
D18
(%)
23
P 15
(%)
26
Poor
Excellent
Good
4
28
52
11
30
46
5
23
54
10
26
49
Good
Excellent
Moderate
52
28
16
46
30
13
54
23
18
49
26
15
ACCEPTABILITY
Moderate
Good
Poor
16
52
4
13
46
11
18
54
5
Poor
Moderate
4
16
11
13
5
18
Excellent
Poor
ACCEPTABILITY
4
11
5
P (%)
37
P (%)
P (%)
10
15
30
P (%)
Good
ACCEPTABILITY
P 41
(%)
P 43
(%)
Moderate
ACCEPTABILITY
Excellent
P 15
(%)
37
P 18
(%)
30
Poor
Excellent
Good
7
37
41
9
30
43
Good
Excellent
Moderate
41
37
15
43
30
18
Moderate
Good
15
41
Poorand tolerability results obtained
7
In fact, the efficacy
with the two treatment
methods were similar.
Poor
7
Moderate
15
Although there are no reliable reasons for these
Poor
7
results, some reasonable
speculations can be made.
The risk of underdosing is probably low with an
ointment and can be counterbalanced by the intrinsic
characteristic of the formulation which allows a deep
percutaneous penetration and a great spreadability
(23). Overdosing may be a real problem when
substances with a high irritating potential or an
unfavourable safety profile are applied to the skin.
This is not the case of calcitriol ointment, which is
well tolerated in clinical practice and in experimental
conditions (9-13, 16, 18-20). Moreover, patients with
a chronic skin condition like psoriasis usually have
gained a lot of experience in the management of their
disease with topical therapy.
However, the dosing method in our study was
well accepted by patients. Another consideration
derived from our results is that calcitriol ointment
can be safely combined to background treatment with
15
49
10
10
18
43
urea-based emollients9which can in turn enhance the
activity of drugs in psoriasis
acting as ‘therapeutic
9
18
moisturizers’ (24).
9
ACKNOWLEDGEMENTS
The authors thank Dr Monica Carbonara (Bari,
Italy) for her support in the statistical analysis.
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EUROPEAN JOURNAL OF INFLAMMATION
Vol. 5, no. 2, 97-101 (2007)
CARVEDILOL INCREASES ATRIAL NATRIURETIC PEPTIDE PLASMA LEVELS IN
HYPERTENSIVE PATIENTS
P.C. PAPADOPOULOS, P. KOTRIDIS, B. KOKKAS1, V. KOUTSIMANIS, G. AIDONIDIS,
G. DADOUSH, O. GOULI, M. KARAMOUZIS2, A. KOUYOUMTZIS1, G. SAKANTAMIS,
D. HATSERAS3 and C.L. PAPADOPOULOS
Departments of 2nd Cardiology, 1Pharmacology and 2Biochemistry, Medical School, Aristotle
University of Thessaloniki; 3Department of Cardiology, Medical School, Demokritus University of
Thrace, Greece
Received November 22, 2006 – Accepted May 18, 2007
Twenty five patients with moderate essential hypertension were studied for 30 days in order to
evaluate the medium term effects of carvedilol on atrial natriuretic peptide (ANP) levels. This drug
blocks the β1 and β2 adrenenergic receptors as well as α1 adrenergic receptors. In addition, it has strong
antioxidative and antiproliferative properties. The drug was given orally at a dose of 12.5 mg b.i.d.
Quantitative determination of human ANP was made by radioimmunoassay procedure (RIA). At the
end of this clinical trial, mean plasma levels of ANP had risen during treatment by 21.18% (from 37.60
pg/ml to 45.83 pg/ml) while both systolic (SBP) and diastolic (DBP) blood pressure as well as diameters
of the left cardiac cavities had decreased in a statistically significant way. The ratio ANP/SBP was also
increased by 46.6% in a statistically significant way. These findings support the suggestion that the
increase in plasma ANP following the administration of β-adrenergic blockers to hypertensive patients
is a primary effect of beta blockade and not a mechanical one secondary to a negative inotropic action
on the left ventricle and obviously contributes to the anti-hypertensive action.
Major interest has been focused upon the
investigation of unconventional methods of the
anti-hypertensive action of drugs used for the
treatment of arterial hypertension. A strong motive
for the interest in this particular subject is derived
from the fact that no conventional hemodynamic
effect can provide a satisfactory explanation for
the chronic long-term anti-hypertensive efficacy
of β-adrenergic blockers. The investigation, which
was conducted by the research group of the 2nd
Department of Cardiology with the cooperation of
the Departments of Pharmacology and Biochemistry
at the Medical School of the Aristotle University of
Thessaloniki, resulted in a number of presentations
and publications in Greece and abroad. According
to the investigation, ANP seems to be an important
mediator between the β-adrenergic blockers and
the therapy of arterial hypertension. Since then,
interest has been expanded to investigate other antihypertensive agents and their linkage to ANP and the
results of such investigation have been confirmative
(1-4).
Subsequently, it sounded reasonable to investigate
a possible role of ANP upon the anti-hypertensive
Key words: carvedilol, ANP, hypertension, β-adrenergic blockers
Mailing address:
Prof. B. Kokkas,
3 Vogatsikou st,
Thessaloniki,
54622, Greece
Tel/Fax: ++30 2310240094
e-mail: [email protected]
1721-727 (2007)
97
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This publication and/or article is for individual use only and may not be further
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98
P.C. PAPADOPOULOS ET AL.
Table I. Changes in the parameters studied before and after 30 days treatment with cavedilol 12.5
Table I. Changes in the parameters studied before and after 30 days treatment with cavedilol 12.5 mg daily ( means ±
SD, n: 25). mg daily ( means � SD, n: 25).
Parameters
After
Treatment
128.95�13.3
Units
Changes %
SBP
Before
Treatment
168.33�29.7
mmHg
–23.4
Statistical
Significance
p<0.001
DBP
97.91�15.87
81.25�6.95
mm/Hg
-17.0
p~0.001
LVESD
3.34�0.62
3.20�0.62
Cm
-4.19
p<0.001
LVEDD
5.06�0.57
4.86�0.62
Cm
-3.95
p~0.001
65.94�8.82
%
+3.56
p~0.05
3.523�0.47
Cm
-3.69
p~0.05
msec
-11.79
p~0.01
pg/mL
+ 21.18
LVEF
LAD
P-Waves
63.67�7.53
3.658�0.47
133.33�36.21 117.60�14.64
Duration
ANP
37.60�16.34
45.83�24.54
p>0.1
WT= p<0.01
ANP/SBP
0.236�0.11
0.346�0.20
%
+46.6
p~0.001
SBP, systolic blood pressure; DBP, diastolic blood pressure; LVESD, left ventricular end systolic diameter; LVEDD,
SBP, systolic blood pressure; DBP, diastolic blood pressure; LVESD, left ventricular end systolic
left ventriculardiameter;
end diastolic
diameter;
LVEF,left
ejectionLVEF,left
fracture; ventricular
LAD, left atrial
LVEDD,
left ventricular
endventricular
diastolic diameter;
ejectiondiameter;
fracture; ANP, atrial
natriuretic peptide
concentration);
WT,
Wilcoxon
test. peptide (plasma concentration); WT, Wilcoxon
LAD,(plasma
left atrial
diameter; ANP,
atrial
natriuretic
test.
action of newer agents that combine the properties
of β-adrenergic blockers with several other effects.
This study aims to investigate the mediumterm anti-hypertensive effect of cavedilol in
relation to plasma ANP concentration changes in
patients with moderate uncomplicated essential
hypertension. Carvedilol, is a racemic form made up
of S(-)carvedilol and R(+)carvedilol. Carvedilol has
multiple actions. R(+)carvedilol blocks the β1 and
β2 adrenenergic receptors as well as α1 adrenergic
receptors. The S(-)carvedilol has only α1 inhibiting
properties. In addition, carvedilol has strong
antioxidative and antiproliferative properties (5).
MATERIALS AND METHODS
Twenty five patients (11 males and 14 females) aged
between 33 and 78 years (mean age 53.8 ± 11.5) with
mild to moderate uncomplicated essential hypertension
were enrolled in this study. All patients satisfied the
criteria to be treated with β-adrenergic blocker carvedilol
and had resting heart rate of ≥70/min with normal
atrioventricular conduction. All patients had normal renal
function, as detected by conventional biochemical tests,
or by radioisotopic studies whenever it was necessary.
All patients had normal left atrial and ventricular
dimensions as well as systolic ventricular function as
detected by the echocardiogram. There was no evidence
of ischemia on the surface resting ECG. Whenever it
was necessary, ischemia was ruled out with myocardial
perfusion imaging (SPECT, Thallium-201). The patients
were on no drug treatment before the trial or otherwise
all anti-hypertensive medications were discontinued 2
weeks prior to enrollment in the trial, since they had no
severe hypertension. Patients were adequately informed
regarding the objectives of the trial and written consent
form was signed. After a washout period of 15 days for
those who were receiving anti-hypertensive medications,
as well as behavioral modification for the rest, and if blood
pressure was still persistently higher than 150/90, drug
treatment was initiated. Prior to this, an echocardiogram
ANP/SBP
0,7
P < 0,01
0,6
0,5
Eur. J. Inflamm.
BEFORE
TREATMENT
DURING
TREATMENT
0,4
0,3
ANP/SBP0,2
0,1
0,7
P < 0,01
0
CARVEDILOL
0,6
0,5
BEFORE
TREATMENT
DURING
TREATMENT
0,4
Fig 1.
0,3
0,2
0,1
0
CARVEDILOL
Fig. 1. Changes of ANP/SBP ratio on treatment with
carvedilol 12.5 mg daily for 30 days.
Fig 1.
P < 0,001
180
160
140
167
120
126
P < 0,001
100
BEFORE TREATMENT
DURING TREATMENT
80
168
60
129
40
20
0
CARVEDILOL
SBP
P < 0,001
CARVEDILOL
DBP
180
Fig. 2. Blood pressure changes on treatment with
160
carvedilol 12.5 mg daily for 30 days.
140
120
167
126
P < 0,001
99
were measured again in the same sequence three hours
after taking the morning dose. ANP was measured again
in blood samples taken by venipuncture three hours after
the last drug dose administration.
Parallel Control groups consisting of patients
receiving anti-hypertensive drug of another category
were not included in this study because drugs such as
β-adrenergic blockers, ACE inhibitors and calcium entry
blockers have previously been shown to increase ANP
plasma levels (1-4, 6-9).
Blood samples for plasma ANP level determination
were collected in pre-chilled tubes containing EDTA2Na (1.5 mg/ml blood) and aprotinin (500KIU/ml blood),
placed on ice, and immediately centrifuged at 1500 g for
10 min. at 4oC. Plasma was frozen and stored at –70oC
until the assay was performed. Quantitative determination
of human ANP was made by radioimmunoassay procedure
(RIA) combined with an extraction step using reagents
supplied by Nichols Institute Diagnostics B.V (California,
USA). Determination by RIA was performed by using an
ANP (125J) radioimmunoassay (RIA) system according to
the manufacturer’s instructions. Intrassay and interassay
variations were 3.9% and 9.7% respectively. The results
obtained (mean ± SD) were expressed as pg/mL. Normal
values calculated in 30 healthy male individuals, mean
age 51 years (range 30-60), in our laboratory were 35.3
± 9.5 pg/mL (10-12).
The values obtained for each parameter, before and
during treatment, were expressed in the form of mean
±1 standard deviation and the statistical analysis was
carried out using the Students paired t-test as well as
the Wilcoxon challenge because the distribution was not
always regular.
was performed on all patients. Parameters measured were
100
the dimensions of the left ventricle and the left atrium
onTREATMENT
BEFORE
RESULTS
DURING TREATMENT
M-mode
80
Fig 2. echocardiogram in the left parasternal location
using
60 the long axis view with the conventional method.
Results are shown in detail in Table I and Fig.
The values shown in the Tables are mean values of three
40
1
and
2. Mean plasma levels of ANP rose after
to four measurements. Subsequently, P wave duration
20
treatment
by 21.18% (from 37.60 pg/ml to 45.83
was measured using the signal average electrocardiogram
pg/ml) while both systolic blood (SBP) and diastolic
0
(SAECG)
with the patient being in the supine position.
DBP expressed by the
The durationSBP
of the atrial electrogram
blood (DBP) pressure as well as left cardiac cavities
filtered signal in the SAECG indicates the time from
diameters were decreased in a statistically significant
the initiation till the end of repolarization of the atrial
way. The ratio ANP/SBP was also increased by
myocardium. At the end of the measurements, the
46.6% in a statistically significant way.
patients were kept in a supine position for 30 minutes.
A veinpuncture was performed in a peripheral vein to
DISCUSSION
determine the concentration of plasma ANP. Blood
pressure from the right arm was measured using a mercury
From this clinical study, it was found that
sphygmomanometer while the patient was seated.
carvedilol
efficiently reduced the systolic and
All the patients received carvedilol of the Roche
Fig
2.
diastolic blood pressure by 23% and 17% respectively
company at the dosage of 12.5 mg b.i.d. per os. Thirty days
after starting treatment, the above measured parameters
(Table I). Left ventricular dimensions were reduced
168
CARVEDILOL
129
CARVEDILOL
100
P.C. PAPADOPOULOS ET AL.
and ejection fraction was increased very slightly but
in a statistically significant way although the patients
had no signs of cardiac failure. The message derived
from the above observations is that carvedilol did not
exert any negative inotropic effect.
During this trial, a statistically significant
reduction by 12% of atrial electrogram duration was
observed, detected by the SAECG. The main factors
regulating this time are the velocity of conduction
and the length of the pathway. Given the fact that
β-adrenergic stimulation exerts positive dromotropic
effect on the myocardium, its inhibition should
have the tendency to prolong the parameter under
investigation. The reduction of atrial electrogram
duration reflects the shortening of the length of the
pathway, that is a reduction in the dimensions of both
atria. This finding is aided by the echocardiogram
where there was a confirmed reduction in the size of
the left atrium.
The main finding of this trial was that the levels
of plasma ANP showed a tendency to increase by
21.18 % during the treatment with carvedilol. What
was also interesting is the fact that there was a
statistically significant increase of 46.6 % in the ratio
of plasma ANP divided by systolic blood pressure
following the reduction of arterial blood pressure
(Table I, Fig. 1). Systolic blood pressure has been
positively associated with the mechanical stimulus
affecting ANP secretion, therefore a reduction of
plasma ANP concentration was to be expected.
The diverting behavior of ANP and blood pressure
clearly indicated by the variation of A.N.P./ S.B.P.
ratio together with the reduced atrial dimensions
strongly support a primary stimulation for ANP
secretion and not a mechanical one secondary to a
negative inotropic action on the left ventricle.
This conclusion is in accordance with our previous
observations on humans with older β-adrenergic
blockers (1-4) as well as those of other researchers
(6-9, 13-15). Experiments performed in cell cultures
have also demonstrated the adrenergic influence of
ANP secretion by the atria (16). Additionally, recent
articles concerning experimental research on animals
report that carvedilol enhances atrial ANP mRNA
expression and release (17) while potentiating its
action (18).
Although the action of cavedilol on ANP plasma
levels seems to be a direct one, it is not easy to
distinguish if such an increase of ANP plasma levels
is only the result of an increased secretion or whether
additional mechanisms may be. Such a suggestion is
based on reports that spontaneously stroke-prone
hypertensive rats treated with cavedilol showed
increased plasma levels of ANP and decreased
mRNN levels of natriuretic peptide-C receptor
which is responsible for the clearance of ANP (18).
No matter which of the mechanisms is involved,
the principal message from our study is that
carvedilol, a new generation β-adrenergic blocker
classified as having polymorphic actions, exerts
part of its anti-hypertensive action by increasing
ANP levels. It also verifies that ANP contributes
to the anti-hypertensive action of many drugs.
Comparative studies are impossible as all antihypertensive drugs in current use have been found to
increase ANP plasma levels in previous studies (1-4,
6-9, 13-14, 18-21). The use of such control groups
would be inappropriate. The only possible method
was to compare ANP plasma levels before the
treatment with carvedilol and during this treatment
on the same patients.
The present report comprises a proof of the theory
derived from clinical trials that was suggested by C.
Papadopoulos, B. Kokkas et al and first published in
1992 (22).
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EUROPEAN JOURNAL OF INFLAMMATION
Vol. 5, no. 2, 103-110 (2007)
INTRARENAL RESISTIVE INDEX IN PATIENTS WITH TYPE 2 DIABETES MELLITUS
WITH AND WITHOUT MICROALBUMINURIA
M. SPERANDEO, A. VARRIALE, G. D’AMICO, G. SPERANDEO1, M.L. PIATTELLI,
A. DE CATA, A. GRECO, F. PRIGIGALLO, M.A. ANNESE, L. CEDRONE,
and G. VENDEMIALE
Department of Internal Medicine, 1Radiology, IRCCS Casa Sollievo della Sofferenza,
San Giovanni Rotondo, Foggia, Italy
Received December 31, 2006 – Accepted May 25, 2007
Diabetic nephropathy affects a subset of about 30% of patients with type 1 Diabetes Mellitus (DM); it
also develops in a less defined percentage (20-30%) of patients with type 2, after a period of 15-20 years. It
is usually divided into stages. The aim of this study is to assess the usefulness of duplex sonography with
Doppler wave form analysis in the evaluation of early diabetic nephropathy, in order to detect patients
at risk for irreversible renal disease. 262 patients (61 males, 201 females; age range: 48-81 years) with
type 2 diabetes mellitus were studied; 100 healthy volunteers with no evidence of diabetes mellitus (74
females, 26 males; age range: 50-80 years) composed the control group. All of them underwent duplex
Doppler sonography of the kidneys; a scanner with a 3.5 MHz transducer (Toshiba 270 SSA) was used,
Pulsatily Index (P.I.) and Resistive Index (R.I.) of Doppler waveform were obtained at the intrarenal
arteries; the average value of 3 bilateral measurements was taken. Doppler sonography was done by
the same authors without knowledge of the patient group (case or control). Both indexes (PI. and R.I.)
resulted to be higher in patients with DM compared to controls in patients with microalbuminuria: PI.
= 1.49 +/- 0.34 vs. 1.07 +/- 0.06, p< 0.05; R.I. = 0.79 +/- 0.15 vs 0.60 +/- 0.03, p<0.05. Even if our data
have to be confirmed by further studies, they suggest that duplex Doppler sonography may be a useful
complementary test in the evaluation of diabetic nephropathy, especially in the early stages, in order to
identify more patients at risk of developing diabetic nephropathy.
Diabetic nephropathy is the most frequent cause
of chronic renal failure: in Italy and in southern
Europe it accounts for more than 15-20%, while
in northern Europe and in the USA for 30 %; its
prevalence is 30% in type 1 diabetes 10-20 years
after the onset of disease and 20-30 % in type 2
diabetes (1). Considering the greatest incidence of
the latter, it is clear that most of the diabetic patients
with renal failure are of type 2 . Diabetic nephropathy
has various stage of development. Up to 15 years ago
there were no methods capable of identifying patients
with early diabetic nephropathy, at a probable
reversible stage (2). Years later a correlation between
hypertension and glomerular damage was detected
and some indices as nephromegaly, the increased
filtration rate and mycroalbuminuria were found
to be predictive of evolution in overt nephropathy.
Microalbuminuria was fìrstly considered to be a
Key words: resistive index ( R.I.) – diabetes mellitus type 2-microalbuminuria
Mailing Address: Dr M. Sperandeo,
I.R.C.C.S. Ospedale-Casa Sollievo della Sofferenza,
Viale Cappuccini 1,
San Giovanni Rotondo,
71013 (Fg), Italy
Tel: ++0882.410563-410424 Fax: ++0882.410563
e-mail: [email protected]
1721-727 (2007)
103
Copyright © by BIOLIFE, s.a.s.
This publication and/or article is for individual use only and may not be further
reproduced without written permission from the copyright holder.
Unauthorized reproduction may result in financial and other penalties
104
M. SPERANDEO ET AL.
marker of incipient diabetic nephropathy (3-4). Now
it is widely believed to be a sign of early stage of
disease. In the urine from normal subjects is possible
to find a low amount of albumin to a variable rate at
different hours of the day and according to physical
activity. The threshold value is 30 mg/die (5). The
appearance of mycroalbuminuria in a diabetic
patient sometime is concomitant with the onset of
hypertension that critically increases the progression
of albuminuria and the decline of glomerular
filtration (6). In the last 30 years, studies evaluating
renal haemodynamics have dramatically changed,
previously being of an invasive type until the coming
of ultrasonography (7).
Color-Doppler ultrasonography has provided
a haemodynamic evaluation of renal flow in a
totally non-invasive manner. In the kidney, Doppler
ultrasound allows an accurate visualization of
vasculature. By Doppler US it is possible to obtain
evaluations both of qualitative type (presence,
direction and type of flow) and quantitative
(resistivity and pulsatility index as measure of
resistance in the arterious district) (8). In the past,
alterations of renal haemodynamics have been
described in the course of diabetic nephropathy
at the stage of renal failure. Resistive Index (RI)
resulted to be different in normal subjects compared
to diabetic patients and in the latter at different
creatinin levels; it was concluded that RI represents
a predictive index in the advanced stage of diabetic
nephropathy (9).
In diabetic patients there are alterations of
arterial vasculature caused by the microvascular
damage characteristic of the disease. The first
study investigating these alterations was that by
Platt in 1989 (10) after which many other authors
investigated the microvascular alteration in diabetic
nephropathy in order to detect early vascular damage
in type 1 and the type 2 diabetes mellitus (11).
The need of a test that could be simple, safe and
repeatable, at low cost, useful in detecting diabetic
nephropathy at an early stage, has motivated many
investigators continue eco-Doppler study of renal
flow in diabetic nephropathy (12-13).
Microalbuminuria in diabetic patients represent a
marker predictive of renal damage, but recent studies
point out that when microalbuminuria is present it
already implies a structural damage of glomerule in
the context of renal and sistemic vascular damage
(14).
The pre-albuminuric stage of diabetic
nephropathy can not be evaluated by the use
of a laboratory test; when microalbuminuria is
detectable, there are pathological lesions, even if at
an early stage. In the pre-albuminuric stage there are
alterations of zonal hemodynamics with increased
intraglomerular pressure (15).
The aim of this study is to measure these
haemodynamic alterations by echo-color-powerDoppler and to confirm the value of RI as marker of
early incipient nephropathy.
MATERIALS AND METHODS
The study population consisted of 262 patients (61
males, 201 females; age range: 48-81 years) with type
2 diabetes mellitus, who had been recruited from the
Diabetes Outpatient Clinic of our hospital between
January 2000 and December 2005. The control group was
made up of 100 healthy volunteers with no evidence of
diabetes mellitus (74 females, 26 males; age range: 50-80
years). Exclusion criteria for both groups were: current
use of tobacco; current or past disease of the kidneys
or urinary tract (including cysts, kidney stones, solid
tumors, acute or chronic inflammatory diseases); systemic
disease with renal involvement; congestive heart failure;
arterial hypertension; chronic liver disease; pregnancy;
current use of drugs that could affect haemodynamic
parameters (e.g. finasteride, nitrates, calcium antagonists,
ACE inhibitors, aminophylline, theophylline, estrogenprogestin compounds).
Blood glucose (measured in venous plasma),
glycosylated hemoglobin, serum creatinine (all three
measured after a fast of 10 h or more), and 24 h urinary
excretion of albumin (measured by radioimmunoassay)
were measured in all 362 participants. In diabetic
patients, three 24-h urine specimens were assayed, and
the average of the three results was used for analysis; for
control patients, only one 24-h specimen was assayed.
Albumin excretion rates of <30 mg/day were considered
normal; rates of 30-300 mg/day were classified as
microalbuminuria.
Based on the results of these tests, the diabetic
patients were divided into three groups: Group I included
patients with normal albumin excretion and serum
creatinine levels (i.e. <1.3 mg/dL); Group II patients had
microalbuminuria with normal serum creatinine levels;
and Group III presented microalbuminuria and serum
creatinine levels of >1.3 mg/dL.
Moreover, to assess whether this subdivision into three
complications and verify the value of renal RI and PI in identifying early stages of diabetic
nephropathy.
105
Eur. J. Inflamm.
Table
of the two study groups.
Table I. Characteristics
of I.theCharacteristics
two study groups.
p
Diabetic Group
Healthy Controls
(n=262)
(n=100)
No. Females (%)
201 (76.7%)
74 (74.0%)
NS
Mean age (yrs.)
65.5 +/- 6.25
64.4 +/- 6.26
NS
Body Mass Index
25.2 +/- 2.33
25.1 +/- 2.25
NS
Table II. Characteristics of the three groups of type 2 diabetic patients studied.
Group I
Group II
Group III
(n=145)
(n=45)
(n=72)
Mean age
63.0 +/- 4.3
65.5 +/- 5.5
69.5 +/- 4.7
No. females (%)
107 (73.8%)
35 (77.8%)
59 (81.9%)
12.8 +/- 3.3
20.1 +/- 5.3
13.2 +/- 6.4
63.7 +/- 23.6
73.6 +/- 29.5
0.84+/-0.16
0.98+/-0.15
1.85+/-0.28
7.44 +/- 0.49
8.15 +/- 0.70
8.57 +/- 0.72
Years
from 7.4 +/- 2.5
diagnosis
Microalbuminuria
(mg/day)
Serum creatinine
mg/dl
Glycosylated
hemoglobin (%)
Table II. Characteristics of the three groups of type 2 diabetic patients studied.
F 65.5+/-9.5
F 29+/ 5
GFR estimated by F 75.5+/-15.5
Cocroft-Gault
M 103+/-24
M 84+/16
M 39.5+/-7.5
formula (ml/min)
F: female patients
M: male patients
Table III. Resistance and Pulsatility indices in the renal arteries of type-2 diabetics and healthy controls.
Resistance Index
Group I
Group II
Group III
Controls
(n=145)
(n=45)
(n=72)
(n=100)
0.68 +/- 0.04
0.78 +/- 0.05
0.91 +/- 0.08
0.60 +/- 0.03
1.13 +/- 0.12
1.19 +/- 0.26
1.74 +/- 0.27
1.07 +/- 0.06
(I.R.)
Pulsatility Index
(I.P)
REFERENCES
106
M. SPERANDEO ET AL.
Fig. 1. Intrarenal eco colour Doppler in a normal subject, within normal range. Resistive Index (R.I)= 0.63 and Pulsatily
Index (P.I)=1.00
Fig. 2. Intrarenal eco colour Doppler in a diabetic patient without microalbuminuria. Both indexes resulted to be
increased (R.I.= 0.65 and P.I.= 1)
Fig. 3. Intrarenal eco colour Doppler in a diabetic patient with microalbuminuria and Nephropathy. Further increased
R.I. and P.I value ( R:I:=0.86 and P.I.=1.86)
107
Eur. J. Inflamm.
groups according to the level of serum creatinine reflected
the real difference in value of glomerular filtration rate
(GFR), for each patient estimation of GFR was made
by the Cockroft-Gault formula (16). All participants
underwent color and power Doppler studies, which were
performed with a Toshiba 240SSA scanner and a 3.5-MHz
convex probe. The examinations were carried out by two
operators who were blinded to the subject’s group origins.
After standard sonographic examination of the kidneys
and urinary tract, power Doppler was used to determine
the RIs and PIs of the intrarenal arteries in each kidney.
Resistive Index and Pulsatility Index were calculated
according to the following formula:
RI=S-D/S, where S is the height of the systolic peak
and D is the height of the end diastolic trough; PI =
maximum velocity excursion/ mean of the velocity (17).
The mean of three measurements was used for analysis,
as recommended by Platt (18).
SPSS software was used for statistical analysis.
Preliminary testing verified that the distribution of the data
was normal (KS). For this reason, parametric tests were
chosen for inferential analysis of the study variables. The
chi square test was used to evaluate differences between
qualitative variables. Correlation between variables was
assessed with the Pearson correlation coefficient. A P
level of p <0.05 was considered significant.
RESULTS
As shown in Table I, the diabetic and control
groups were not significantly different in terms of
sex, age, or body-mass indices. Based on the results
of enrolment lab work, the 262 diabetic patients
were divided into three subgroups: Group I included
145 patients (38 males, 107 females) with normal
renal function and no signs of microalbuminuria;
Group II included 45 patients (35 males, 10 females)
with microalbuminuria but normal renal function;
and Group III contained 72 patients (13 males,
59 females) with microalbuminuria and renal
insufficiency. The characteristics of these three
subgroups are summarized in Table II
According to the estimation of GFR by the
Cocoroft-Gault formula, the division in the three
groups did not change, as reported in Table II
The mean age of Group I patients was significantly
lower than those of the other two groups (p<0.001).
The three groups were also significantly different
(p<0.001) in terms of the duration of diabetes (years
since diagnosis), and this variable was significantly
correlated with the severity of the disease. There
was no significant difference between the mean
values of microalbuminuria in Groups II and III.
Glycosylated hemoglobin values were frankly
pathological in all three groups, with no significant
inter-group differences.
Fig. 1 and 2 show representative scan results for
a control subject and a diabetic patient from Group
1. As shown in Table III, the mean RIs for the three
groups of diabetic patients were all significantly
different from that of the control group. The RI for
Group I was significantly higher than that of controls
(p <0.001) and significantly lower than those of
Groups II (p<0.001) and III (p<0.0001) (Fig.3). The
difference between the RIs of the latter two groups
was also significant (p<0.0001). The PIs of the three
groups of diabetic patients were also significantly
different and increased with the severity of the
disease (p<0.001). The difference between the PIs
of Group I and the control group was significant
(P<0.05) but less so than that of the RIs.
The RI displayed significant correlation with
the duration of diabetes (p<0.01). Serum creatinine
levels displayed a highly significant correlation
with RI (R2=0.75) and a slightly less significant
correlation with the PI (R2=0.63). Microalbuminuria
levels were not significantly related to either of the
Doppler indices or to serum creatinine levels. In
fact, while the latter three variables (I.R., I.P. and
microalbuminuria) all increased progressively from
Group 1 to Group 3, this trend was not observed
for albumin excretion. The correlation between the
RI and PI was highly significant (p<0.001). The
duration of diabetes was significantly associated
with the haemodynamic parameters and with renal
function (as reflected by serum creatinine).
DISCUSSION
Diabetic nephropathy is an important cause of
mortality in patients with type-1 and type-2 diabetes
mellitus. Although much of the work that has been
done in this field has focused on renal disease in
type 1 diabetes, the pathophysiology of diabetic
nephropathy associated with type 2 disease appears
to be essentially the same. In both cases, the renal
effects of diabetes are related to the macro- and
microvascular complications of altered glucose
metabolism. In fact, the initial stage of diabetic
nephropathy is manifested by functional alterations
108
M. SPERANDEO ET AL.
in renal haemodynamics, which result in glomerular
hyperfiltration and increases in the intraglomerular
pressure (19).
There is currently no reliable laboratory marker
for detection of diabetic nephropathy in this
preclinical phase. The onset of microalbuminuria
(>30 mg albumin / day) is widely considered to
be the first detectable sign of diabetic damage to
the kidney (20). Its appearance reflects structural
damage to the glomerular membrane resulting in
loss of its selective permeability. According to some
investigators (21) microalbuminuria represents
the first step in an inexorable progression to overt
proteinuria and renal failure although this view has
been challenged by others (22).
Intraglomerular pressure can not be directly
measured in humans, but it can be measured
indirectly and in a non-invasive manner by means
of color or power Doppler ultrasound studies of the
renal vasculature (23). This approach provides us
with quantitative indices of renal vascular resistance
based on blood-flow velocities in the intrarenal
arteries (24).
The scope of the present study is to identify
changes in renal vascular resistance associated with
the onset and progression of diabetic nephropathy.
To this end, we performed power Doppler studies
of the intrarenal arteries, with calculation of the RI
and PI, on 262 type-2 diabetic patients, who were
divided into three groups reflecting their renal status.
Compared with 100 healthy non-diabetic controls,
the Group I diabetics, who had normal creatinine
levels and no signs of microalbuminuria, presented
significantly higher renal Ris and Pis. Furthermore,
additional significant increases in both indices were
observed in diabetic patients of Group II, who had
microalbuminuria (mean RI: 0.78+/-0.05), and
Group III, with microalbuminuria and elevated
serum creatinine levels (mean RI: 0.91+/-0.08).
Unlike 24 h albumin excretion rates, which were
not significantly different in patients from Groups III
and III, the RI and PI displayed significantly higher
values in patients whose altered albumin excretion
rates were accompanied by signs of altered renal
function.
Our study indicates that the renal RI and PI
increase progressively with the onset and evolution
of diabetic nephropathy.
The significant differences between the RIs of
all three diabetic groups and those of the healthy
controls indicate that this parameter can also be
used to distinguish normal and diabetic populations,
regardless of the latter’s stage of disease. The
discriminatory power of the IP proved to be slightly
lower than that of the RI, in all probability because
its calculation is based on the relatively inexact
variable, mean blood flow velocity.
These findings suggest that the renal RI might be
used as a marker of early (pre-microalbuminuric)
renal involvement in type 2 diabetes. It is important
to note that the patient and control populations
analyzed in this study were carefully selected to
minimize the risk of confounding effects on renal
vascular resistance due to non-diabetic factors.
Discordant reports have been published on
the time of appearance of laboratory changes in
diabetics (25). Nonetheless, microalbuminuria
seems to be extremely rare during the first five
years of diabetes. Our data confirm this observation
and indicate that the duration of the disease is
directly correlated with the stage of nephropathy.
In fact, the mean number of years from diagnosis
increased significantly from Group I through Group
III. On the average, microalbuminuria developed
approximately seven years after the diabetes was
diagnosed, whereas outright renal failure developed
only when the diabetes had been present for around
20 years. Group III patients were significantly older
than those of Group I but not those of Group II.
These differences do not diminish the value of the RI
as an index of the severity of diabetic nephropathy
although it is clear that intrarenal haemodynamics
are physiologically influenced by age.
Glycosylated hemoglobin levels showed no
correlation with albumin excretion rates or serum
creatinine levels. In fact, there were no significant
differences between the mean HbA1c values
observed in the three groups.
In conclusion, although the mean serum
creatinine levels and albumin excretion rates of
our Group 1 diabetic patients were clearly within
normal limits, the RI for this group was significantly
higher than those of the non-diabetic control group.
This finding suggests that the intrarenal arterial
RI might be used as a reliable predictor of latent
diabetic renal damage. Compared with albuminuria
Eur. J. Inflamm.
levels, the RI also appears to be a more sensitive
index of the progression of diabetic renal disease.
Nonetheless, our findings support the hypothesis
that these Doppler indices can be used as markers
of early-stage diabetic nephropathy. This does
not mean that all of our Group I patients will
go on to develop albuminuria and outright renal
failure, since the onset and progression of diabetic
nephropathy also depends to some extent on genetic
and environmental factors alone. The follow-up of
these patients will be important to determine the
incidence of complications and verify the value of
renal RI and PI in identifying early stages of diabetic
nephropathy.
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EUROPEAN JOURNAL OF INFLAMMATION
Vol. 5, no. 2, 111-114 (2007)
LETTER TO THE EDITOR
DELAYED INTERVAL DELIVERY OF A SECOND TWIN:
A CASE REPORT AND REVIEW OF THE LITERATURE
A. DANIILIDIS, M. MAVROMICHALI1, N. KLEARHOU, T. KARAGIANNIS
and V. KARAGIANNIS
Third department of Obstetrics and Gynecology Hippokratio University Hospital of Thessaloniki;
1
First Department of Neonates Hippokratio University Hospital of Thessaloniki, Greece
Received January 19, 2007 - Accepted May 18, 2007
We report a case of diamniotic, dichorionic pregnancy that presented at 26 weeks with premature
rupture of the first amniotic sac. Nine days later, premature labour and delivery of the first male twin
took place, with death of the first twin. The second twin was left in utero. The management included
combination of tocolytics, antibiotics and cervical cerclage. Caesarean section was performed 48 days
later, at 34 weeks due to breech presentation and contractions. We delivered a live male infant with
apgar scores 4/1 and 7/5 and 1680 gr weight. The infant was discharged home 29 days later.
The implementation of assisted reproduction
during the last ten years has increased the incidence
of multiple pregnancies. In some cases one or more
infants must be born due to intrauterine risks or
stillbirth. According to the relevant literature there
is an absence of unanimity on the best management
for these pregnancies. The aim of this report is to add
our experience to the currently limited literature.
A 31 year-old nullipara woman was admitted to
the hospital at the 26th week of a twin dichorionic,
diamniotic pregnancy after in vitro fertilisation (IVF),
because of premature rupture of the membranes of
the first amniotic sac. The ultrasound examination
revealed cephalic presentation of the first female
fetus, whereas the second male fetus was breech.
Both had normal amniotic fluid index and growth.
Two independent placentas were also visualized.
The patient was treated with bed rest, erythromycin
250 mg three times per day (TID) for 7 days,
atociban iv for 48 hours and 24 mg betamethasone in
two separate doses. Seven days later, the first infant
was born, weighting 780 g, but died seven days later
at the Neonatal Intensive Care Unit (NICU) because
of severe lung prematurity. The contractions ceased
after the delivery of the first fetus. A ligation of the
umbilical cord was performed, as high in the cervix
as possible, in aseptic conditions, and the placenta
was left inside the uterus. A McDonald cervical
cerclage was also performed. During the procedure a
course of iv coamoxyclav 1.2 gr was administered. In
addition, prophylactic ritrodine iv was administered
for 48 hours.
The patient was kept in the hospital for close
monitoring which involved daily auscultation of the
fetal heart and measurement of body temperature,
twice weekly full blood count, CRP and clotting
screen and ultrasound examination for growth and
doppler once weekly. Pregnancy was terminated by
caesarean section 48 days later (34th week) due to
uterine contractions and breech presentation. A male
Key words: twin pregnancies, delayed interval delivery, cervical cerclage
Mailing address: Angelos Daniilidis, MD,
81 A Nimfeou,
56224 Evosmos,
Thessaloniki, Greece
Tel: ++30 6932211395
Fax: ++30 2310 559711
e-mail: [email protected]
1721-727 (2007)
111
Copyright © by BIOLIFE, s.a.s.
This publication and/or article is for individual use only and may not be further
reproduced without written permission from the copyright holder.
Unauthorized reproduction may result in financial and other penalties
112
A. DANIILIDIS ET AL.
infant was born weighing 1,680 g, with Apgar scores
of 4 and 7 at 1 min and 5 min, respectively. Two
placentas were delivered, one of which was small,
fibrous and calcified. The post operative recovery of
the mother was uneventful. The neonate stayed in
the hospital for 29 more days and was dismissed in
excellent condition.
Due to a surge in the availability of assisted
reproductive techniques (ART), the incidence of
multiple pregnancies is increasing. Patients with
multiple pregnancies have an increased risk of
preterm labor and delivery with the associated
high prenatal morbidity and mortality. Obviously,
as the number of multifetal pregnancies increases
(due primarily to the use of assisted reproductive
treatments), a rise in the unexpected birth of one
or more siblings at premature or previable stages
is observed. Delaying delivery of the remaining
fetus(es) in a multifetal pregnancy is feasible in
some cases (1-3).
After the birth of the first fetus delayed delivery
in multiple pregnancies can be successful in selected
cases. There is absence of agreement regarding the best
management of these pregnancies. Each case is a unique
medical situation that must be met with the best possible
solution. The main problem appears to be preterm labor
and preterm prelabor rupture of the membranes, with
one condition often leading to the other. A possible
reason for the premature rupture of the membranes
could be an ascending infection from the vagina or the
cervix into the uterine cavity. Both infection and rupture
of the membranes can lead to uterine contractions and
subsequent delivery. Modern management procedures,
including tocolytics, corticosteroids, antibiotics and
cervical cerclage would appear to be important in
the overall success of treatment in a retained twin.
Although both tocolytics and cerclage appear to
prolong the mean delivery interval, no statistical
difference in survival was found concerning cerclage,
antibiotic therapy, tocolysis and hospitalization (1, 34). The use of prolonged bed rest, cervical cerclage,
tocolysis, antibiotics and corticosteroids compose
complex, frequently debatable issues. It is not clear
whether the management described in this case report
is the most adequate treatment and if all the possibilities
mentioned above are really necessary to increase the
rate of success (5-6). Early tocolytic and antibiotic
therapy may delay delivery and, in combination with
antenatal glucocorticoids to stimulate lung maturation,
may thereby improve the condition of the second
twin (7-8). Tocolysis may be used precautionarily
after the first twin’s birth, or only later during uterus
contractions, but never in the presence of a well
established chorioamnionitis. After premature rupture
of the membranes, the suspicion of an infection could
be raised on the basis of a rise of the temperature, of the
white blood cell count and of the C - Reactive Protein
(CRP). CRP is considered as a good prognostic index
for incipient chorioamnionitis (3). In cases of high CRP,
tocolysis cannot achieve the prolongation of pregnancy.
The role of cervical cerclage remains controversial
(9-10). There is an important publication bias in the
literature due to under-reporting of the failed attempts
of delayed deliveries. Cervical cerclage after the first
delivery is associated with a longer inter-delivery
interval without increasing the risk of intrauterine
infection (9-10). However, the impact of this procedure
on infant survival is unclear. If cervical cerclage is
decided, it is advisable to be carried out in aseptic
conditions, during the first two hours after the birth of
the first fetus, and of course when there is no evidence
of infection. To avoid ascending infection, the cord of
the first born twin should be legated with an absorbable
suture as close to the cervix as possible, under aseptic
conditions. Patients with previous cervical cerclage(s)
during the index pregnancy are less likely to achieve
significant latency intervals (9, 11). Delayed delivery
of the remaining fetus(es) for 2 or more days before
30 weeks of gestation was associated with improved
infant survival (10-11). When a first twin was delivered
at 22 to 23 weeks, delayed delivery of the second
twin was associated with reduced perinatal and infant
mortality of the second twin if the interval was less
than 3 weeks (11-12). Delayed delivery of the second
twin when the first was delivered at >/=24 weeks had
no benefit on mortality (9, 11). Interval delivery of
the fetuses in multiple gestations has been shown to
increase perinatal survival (9-11). Earlier delivery of
the first twin and premature rupture of membranes for
the second twin were significantly related to a longer
interval between deliveries.
In reviewing the literature, in most cases the
outcome of the second twin was favorable, in contrast
to the bad outcome of the first twin. Survival of the
first born was clearly linked to its gestational age and
birth-weight (13). The survival of the second born was
Eur. J. Inflamm.
dependent upon a number of factors, including the
delivery interval between the first and second twin and
the presence of obstetric problems appearing during
the latency period; the longer the interval, the greater
was the chance for surviving (10-11, 14). Likewise, the
absence of significant obstetric problems in the latency
period also improved survival. In cases where the
membranes of the second twin remained intact with no
evidence of ongoing labor or other obstetric risk factors,
a conservative approach could be adopted (4-5, 8).
Vaginal examinations should be avoided; however the
length and dilatation of the cervix should be followed
ultrasonographically (13). There is no evidence that
retention of placenta causes disseminated intravascular
coagulation (11, 15). Even a large placental mass can
be retained in the uterus and produce no demonstrable
clinical symptoms (12, 15). Monitoring should be
carried out and limited to a weekly full blood count,
CRP, prothrombin time and fibrin degradation product
(FDP) (13).
In conclusion, delayed delivery in multifetal
pregnancies can be successful if there are no
contraindications and these pregnancies are managed
in a tertiary perinatal center. Possible risks should
be thoroughly discussed with the patient as well as
possible future problems of the child (16). In highorder multiple pregnancy, delayed interval delivery
is strongly recommended, provided that there are no
signs of chorioamnionitis, fetal distress or maternal
compromise. In multiple pregnancies with imminent
preterm birth, delayed delivery may offer survival
chances and a favorable outcome for the remaining
fetus(es) (11-13). The results gained from pregnancy
prolongation are a clinically significant benefit to the
second-born twin, without significant morbidity in
the mother. Even modest intervals between births
of siblings at critical gestational stages can improve
neonatal survival and decrease neonatal morbidity.
With careful patient selection and thorough patient
counseling, delayed delivery offers a safe and
beneficial management option for selected multiple
gestations.
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