Archives 3 (2011) - 101

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VOLUMe 70 - No. 3
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ROMANIAN ARCHIVes OF MICROBIOLOGY AND IMMUNOLOGY
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Copyright
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2011 CANTACUZINO INsTITUTe Bucharest
CONTeNTs
MICROBIOLOGY
97
IMMUNOLOGICAL AsPeCTs IN VIRAL HePATITIs B AND C INFeCTION

Irena Manea, Cristian Nicolae Manea, Nicolae Miron, Victor Cristea
101
CORReLATION OF ANTI-Helicobacter pylori CagA IgG ANTIBODIes WITH ResIsTANCe

TO FIRsT LINe TReATMeNT, BLeeDING GAsTRODUODeNAL ULCeRs AND GAsTRIC CANCeR
Mdlina Ilie, Luminia Dasclu, Carmen Chifiriuc, Marcela Popa,
Gabriel Constantinescu, Coman Tnsescu, Alina Baltac
105
INFLUeNCe OF ORTHODONTIC TReATMeNT ON ORAL sTRePTOCOCCI

Theodor-Cristian Vizitiu, Mihaela Cristina Giuca, ecaterina Ionescu
IMMUNOLOGY
109
DYNAMICs OF eNDOTHeLIAL PROGeNITOR CeLLs

FOLLOWING seVOFLURANe PReCONDITIONING
Mihaela Popescu, Adelina Munteanu, Gheorghia Isvoranu, Laura suciu,
Bogdan Pavel, Bogdan Marinescu, Leon Zagrean
114
A HIGHLY PURIFIeD VeGeTAL FRACTION ABLe

TO MODULATe HMGB1 AND TO ATTeNUATe sePTIC sHOCk IN MICe
Natalia simona Apetrei, Ana Clugru, F. kerek, Minerva Panteli,
I. Rasit, Lidia Cremer, G. szegli, Andreea-Roxana Lupu
124
Is INTeRLeUkIN -17 A PROATHeROGeNIC BIOMARkeR?

Cristina-sorina Ctan, Victor Cristea, Nicolae Miron, Ioana Berindan Neagoe
129
HePATITIs B, C AND D COINFeCTION IN HIV-INFeCTeD PATIeNTs:

PReVALeNCe AND PROGRess
Bogdan Ionescu and Grigore Mihescu
ReVIeW
134
THe GUT MICROBIOTA IN THe MeTAGeNOMICs eRA: sOMeTIMes A FRIeND, sOMeTIMes A FOe
Daniela elena erban
VOLUMe 70
NO. 3
JULY - sePTeMBeR 2011
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INsTRUCTIONs TO AUTHORs
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romanian archives of Microbiology and immunology
IMMUNOLOGICAL AsPeCTs
IN VIRAL HePATITIs B AND C INFeCTION
Irena Manea1*, Cristian Nicolae Manea2, Nicolae Miron3, Victor Cristea3
1immunology
Department, regional institute of Gastroenterology and Hepatology

prof. Dr. octavian Fodor cluj-Napoca, romania; 2UMph iuliu Haieganu, cluj-Napoca, romania;
3immunology Department, UMph iuliu Haieganu, cluj-Napoca, romania
ABsTRACT
Worldwide, viral hepatitis chronic infections are a serious health problem and a very interesting
topic for both clinicians and researchers.
Viral hepatitis has a variety of clinical forms: mild, inactive or severe and with a slow evolution, whose
architectural structure of the hepatic tissue evolves towards cirrhosis or hepatocellular carcinoma.
sometimes, the virally induced hepatic injury evolves spectacularly and rapidly leads to exitus.
The factors that generate this evolution pattern depend on the immune response of the host and
equally on the viral survival and immune surveillance avoidance strategies.
This paper aims to resume new discoveries in the field of immunology of the B and C viral hepatitis infection, from the perspective of the complex interactions between virus and host.
Keywords: immune response, chronic viral hepatitis, cellular adhesion molecules, matrix metalloproteinases
INTRoDUCTIoN
Viral chronic hepatitis is an important health
problem worldwide. Viral aetiology hepatitis has a
variety of clinic forms: mild, inactive or severe and
with a slow evolution, whose architectural structure
of the hepatic tissue evolves towards cirrhosis or hepatocellular carcinoma. sometimes, the virally induced hepatic injury evolves spectacularly and rapidly
leads to exitus [1].
The factors that generate this evolution pattern
depend on the immune response of the host and
equally on the viral survival and immune surveillance avoidance strategies [2].
This paper aims to resume new discoveries in
the field of immunology of the B and C viral hepatitis infection, from the perspective of the complex interactions between virus and host.
The impact of B and C viral infections impact
over the immune response
B and C viruses with hepatic tropism link the
corresponding hepatocitary receptors, are internalized
in the host cell and begin viral replication. The freed
virions infect more and more cells.
Viruses or products of the viral replication are

detected by toll-like receptors (TLR), trans-membrane
protein molecules, expressed especially by antigen
presenting cells (macrophages, dendritic cells, B lymphocytes). These receptors rapidly activate the nonspecific antiviral immune response [3].
TLR signalling induces type I (IFN a and a) interpheron synthesis. Interpherons increase the degree of
expressing of class I MHC genes (Major Histocompatibility Complex) - an important event as cytotoxic T
lymphocytes recognize and destroy only complexes
generated by the viral protein associated with the
class I MHC [3,4]. Also, IFN- a and IFN- b induced by
the viral infection are the strongest activators of the
natural killer (Nk) cells [5].
Human Nk cells are CD3- CD56+ and they do
not carry specific receptors for the antigen. The NkT
lymphocytic system has been recently described it
expresses CD56 (the specific marker for the Nk cells)
at the surface in addition to the TCR antigen receptor.
Numerous Nk and NkT cells have been identified in
the hepatic tissue [6,7].
Both cellular types hold a crucial role in antiviral defence because of their capacity to synthesise
* corresponding author: Irena Manea - Cluj Napoca, str. Porelanului nr. 3, Bloc Corp 1, ap. 40,
tel.: 0751086186; e-mail: irena.manea@gmail.com
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MANEA et al.
IFN-g, which negatively regulates the viral replication

and mediates the recruitment of immune cells to the
center of virally induced tissular injury [7].
studies on murine models with acute B infection
confirmed the contribution of Nk cells to the antiviral immunity through IFN-g and other proinflammatory cytokines synthesis. In consequence, it is not surprising that the infection becomes persistent and
chronic in experimental models that failed to develop
an adequate inflammatory response [8,9,10].
The involvement of Nk and NkT in the defence
against hepatitis C virus (HCV) is limited - this is also
demonstrated by the absence of hepatic inflammation during the first 4-6 weeks from contracting HCV
[11, 12, 13]. An argument of insufficient inflammatory immune response to HCV is that the e2 protein of
the viral structure directly inhibits Nk cells [14, 15].
kupffer cells hepatic macrophages are professional antigen presenting cells (APC) and among the
first elements mobilized as a response to viral infection: they identify, phagocytate and intracytoplasmatically process non-self structures which they present
to T lymphocytes after [5, 7]. Dendritic cells (DC) are
also APCs. After interacting with the antigen, they
migrate to satellite lymphatic ganglions where they
activate T cells that possess adequate lymphocitary
receptors and initiate the specific immune response
[5,7,16].
According to data published up to now, the
adaptive immune response ignores hepatitis B and C
viruses for the next 2 months after infection
[17,18,19,20,21,22].
Late edification of specific cell and humoral
mediated immune response cellular is based on different mechanisms depending on the type of virus
that has been acquired. Intense replication of HCV
and high levels of viral RNA in the first days of contracting the infection escape immune surveillance.
On one hand, this phenomenon is explained by the
immunotolerant hepatic microclimate, which consists
of numerous regulatory T lymphocytes and immunosuppressive cytokines, which limit specific viral eradication [1,2]. In viral hepatitis B infection, DNA and
viral antigens cannot be detected in blood or hepatic
tissue during the first 4-7 weeks from infection. This
is why, in the case of HBV, the late adaptive response
is due to limited quantities of antigen [17, 18].
In acute HCV infection, specific LTc level is
approximately 7% of the total circulating LT CD8+,
while acute HBV infection is associated to a level of
1% LTc. However, in viral C infection, cytotoxic T
lymphocytes show an altered production of IFN-g, a
low level of perforins, a reduced capacity to prolife98
rate and destruction of infected cells [19,20]. All

these functional alterations of the immune response
can be observed in populations with acute infection,
regardless of later evolution, but are transitory only in
cases when immune neutralization occurs [20, 23].
specific CD8+ T lymphocytes remain detectable several years after the resolution of the infection [24].
The development of an inadequate immune
response results in the graduate decline of the adaptive response, by reducing the plasmatic and intrahepatic level of specific CD8+ [20,24], and by the
slow synthesis of specific antibodies [22].
Chronicization of the viral infection is equally
dictated by the immune response of the host and by
viral strategies of escaping immune surveillance. The
recent years bring numerous arguments to undermine the antiviral immune response by hepatic tropism viruses. The extra-hepatic viral reservoir plays a
crucial role in the behaviour of the immune system:
both HCV and HBV have the ability to infect lymphoid cells from peripheral blood and to settle in the
lymphatic ganglions [1, 2].
Cellular adhesion molecules and matrix metalloproteinases biomarkers of the progression and
severity of virally induced hepatic affectation
Recruitment of immune cells to fight the virally
infected hepatocytes is essential for the anti-infection
protection of the host and is the primum movens in
the pathogenesis of chronic hepatic inflammation
[25]. Cellular adhesion molecules and matrix metalloproteinases contribute to this process as well.
Cellular adhesion molecules CAM (integrins,
immunoglobulins, cadherines, molecules similar to
mucin and selectines) have multiple functions: they
mediate and recruit immune cells to the location of
the tissular injury, they perform intracellular communication among subsets of leukocytes and participate
in the intra- and intercellular signalling, both on the
cell-cell level, and on the cell-matrix level. Leukocytes traffic is determined by complex interactions with
intercellular adhesion molecules and endothelial
cells, mediated by an orchestra of chemokines and
cytokines. selectines are cellular adhesion molecules
which facilitate the rolling of leucocytes on the
endothelial surface through their interaction with the
corresponding leukocyte ligand until a firm adhesion,
mediated by integrins, molecules from the representatives of immunoglobulin is established, creating
the irreversible connection between leukocytes to
the vascular endothelium [7].
some CAM show different plasmatic levels depending on the severity and progression of viral hepatic
Immunological aspects in viral hepatitis B and C infection
disease [26]. Immunohistochemical studies showed

that hepatocytes and the epithelium of biliary ducts
are negative for e and P-selectines in patients without
hepatic pathology. The eLIsA test also pleads for a
low seric level of the e and P-selectines in healthy
individuals compared to patients with B or C viral
infection [27].
The immunohistochemistry of hepatocytes and
of the epithelium of biliary ducts is negative for
VCAM-1 in subjects without hepatic affectation. They
also show a zero or very small level of ICAM-1, while
patients with B or C viral infection show the contrary
[27]. ICAM-1 and VCAM-1 expressed at the surface of
cells adequately correlate to the release of soluble
isoforms, sICAM, and sVCAM [28]. These can be
detected and measured in peripheral blood and show
a high plasmatic level in patients with hepatic inflammatory disease [29].
Matrix metalloproteinases (MMP) are proteolytic
enzymes, zinc-dependent endopeptidases. Their activity is negatively regulated by tissular inhibitors of
matrix metalloproteinases (TIMP-1, -2, -3 i -4) and by
angiotensin II [30].
Initially, matrix metalloproteinases have been
recognized only for their capacity to degrade structural molecules of the extracellular matrix (eCM) - collagen, proteoglycans, and glycoproteins. Currently it
is known that MMP contribute to maintaining the balance between MeC catabolism and anabolism. Interpreting the involvement of metalloproteinases in the
anti-infection immune response revealed their large
area of action: they facilitate the recruitment of leukocytes, they negatively / positively regulate cytokines
and chemokynes function, they activate defensines
and modulate cellular behaviour (they are involved
in cellular differentiation and proliferation, angiogenesis and apoptosis) [7, 31].
The aflux of the immune cells from the blood
stream to the location of the viral inflammation implies the proteolysis of the basal membrane. in vitro,
the migration of T lymphocytes and of dendritic cells
is MMP-9 dependent. In murine models, MMPs are
necessary for the transmigration of lymphocytes to
lymphatic nodes. It has been observed that MMP-3
deficient mice associate deficient migration of neutrophils to the inflammatory process. It can be speculated that migration of effector immune cells implies
the activation of matrix metalloproteinases [32].
Recent studies show that MMPs are prognostic
factors in chronic viral hepatic infections [33]. MMPs
1, 2 and -14 correlate to the severity of the hepatic
aggression. MMP-1 plasmatic level is an accurate
indicator of chronic viral hepatic activity. MMP-9 and
plasmatic TIMP-1, and -2 proved to be predictive factors of the degree and progression of fibrosis in C
chronic viral hepatitis [34].
Tissue lesions secondary to inflammation and
hepatic fibrosis can be attributed to the intense synthesis of MMPs and to the decline of TIMP secretion
[33, 34].
CoNClUSIoNS
Hepatitis B and C virus infections are major
causes of morbidity and mortality worldwide.
Currently, it is known that the complex interaction
between virus and host determines the evolution and
severity of hepatic affectation during B and C viral
infections.
Despite remarkable progress in unveiling pathologic mechanisms of inflammation and fibrogenesis,
the limits are still far from reach. Identifying efficient
therapeutic targets is the next step that immunology
must take.
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YJ, Cho M, Yang JM, Kim YB, Park YN,ChoSW.Noninvasive index for predicting significant liver fibrosis:
comparison of diagnostic performances in patients
with chronic hepatitis B and C. Dig Dis Sci.
2011;56(2):555-63.
34. leroy V, Monier F, Bottari S, Trocme C, Sturm N,
Hilleret MN, Morel F, Zarski JP. Circulating matrix
metalloproteinases 1, 2, 9 and their inhibitors TIMP-1
and TIMP-2 as serum markers of liver fibrosis in patients with chronic hepatitis C: comparison with PIIINP
and hyaluronic acid. am J Gastroenterol.
2004;99(2):271-9.
CORReLATION OF ANTI-Helicobacter pylori CagA IgG

ANTIBODIes WITH ResIsTANCe TO FIRsT LINe TReATMeNT,
BLeeDING GAsTRODUODeNAL ULCeRs AND GAsTRIC CANCeR
Mdlina Ilie1*, luminia Dasclu2, Carmen Chifiriuc2, Marcela Popa2,
Gabriel Constantinescu1, Coman Tnsescu3, Alina Baltac4
1emergency
Hospital, Gastroenterology, bucharest, romania;
2University of bucharest, Faculty of biology, Microbiology immunology Department, bucharest, romania;

3colentina clinical Hospital; 4carol Davila UMph, Faculty of Medicine, bucharest, romania
ABsTRACT
Helicobacter pylori was recognized in 1994 as a class I carcinogen by the International Agency for
Research on Cancer (IARC). The prevalence of H. pylori infection varies from 20 to 50% in industrialized countries to over 80% in developing countries. The cagA strains are more virulent than
others, being able to induce morphological changes, vacuolization and degeneration of in vitro
cultured cells. Aim: During this study we investigated the possible correlations between the presence of H. pylori cagA (cytotoxin associated gene antigen)-IgG antibodies and the severity of clinical and endoscopical findings. Methods: Anti-cagA IgG was screened by eLIsA in 104 selected
patients exhibiting resistance to first line therapy for H. pylori, bleeding gastroduodenal ulcers, non
cardia gastric cancer and gastric polyps. Results: A statistically significant association between resistant cases to first line therapy for H. pylori, bleeding gastroduodenal ulcers, non cardia gastric cancer, gastric polyps and cag A Ig G antibodies (p value 0.02 calculated by T-Test) was observed. As
Cag A antibodies titer persist up to four months, their level could be an useful marker in detecting
previous long-term H pylori infection especially in gastric cancer patients. Conclusions: CagA positive H. pylori are virulent strains and the cagA IgG antibodies titer is associated with persistence of
infection after treatment, upper gastroduodenal ulcers or gastric cancer. The presence of these antibodies, associated with positive biopsy for H. pylori, indicates the need of H. pylori treatment.
Keywords: Helicobacter pylori cagA IgG antibodies, non cardia gastric cancer, bleeding gastroduodenal ulcer,
resistance to treatment
INTRoDUCTIoN
Helicobacter pylori is a Gram-negative microaerophilic bacterium that infects nearly half of the
worlds population and in 1994, the International
Agency for Research on Cancer (IARC), a branch of
the World Health Organization (WHO), classified it
as a class I human carcinogen [1]. Marshall and Warren discovered that H. pylori infection may produce
gastritis, peptic ulcer disease (10-20%), distal adenocarcinoma (1-2%) and gastric mucosa-associated lymphoid tissue (MALT) lymphoma (<1%) [2].
The findings that most patients infected with H.
pylori have no complications, other than gastritis, concluded that some strains may be more virulent than
others. Indeed, the severity degree of pathologic modifications was correlated with the strain ability to
undergo morphological changes, vacuolization and

degeneration of in vitro cultured cells [3].
Despite the difficulty of searching in the large
genetic diversity of H. pylori for bacterial pathogenicity factors, several genomic loci enconding virulence
factors, such as the cytotoxin associated gene pathogenicity island (cag PAI), the toxin Vac (vacuoliting)
A and the Bab2 adhesin have been strongly associated with an increased risk of developing gastric cancer and ulcer disease.
The cag pathogenicity island contains 31 genes
and is approximately 40kb and the terminal gene of
this island, caga, is often used as a marker for the entire
cag locus. In comparison with caga-negative (cag)
strains, cag-positive (caga+) strains are associated with
more severe inflammation, higher degrees of atrophic
changes, and a greater chance of progressing to
* corresponding author: Mdlina Ilie, emergency Hospital, Gastroenterology, Bucharest, Romania, email: drmadalina@gmail.com
101
IlIE et al.
gastric adenocarcinoma. The estimated risk has ranged

from an odds ratio of 2 to as high as 28.4. However,
many of the genes adjacent to caga code for a type 4
secretion system (TFss), often viewed as an injector
of bacterial proteins (such as cagA) into host cells. In
the host cells, cagA is phosphorylated by src and c-Abl
kinases, leading directly to growth, migration, and
transformation. Indeed, transgenic expression of H.
pylori cagA induces gastrointestinal (GI) and hematopoietic neoplasms in mice [4, 5]. Infecting gerbils
with mutated strains lacking cagA reduces the severity of gastritis and the development of gastric ulcers,
intestinal metaplasia, and gastric cancer compared
with gerbils infected with the wild-type strain.
In this study we tried to establish the association
between cases of H. pylori resistance to first line therapy, bleeding gastric/duodenal ulcers, gastric polyps
and gastric cancer with the presence of H. pylori
cagA-IgG antibodies, determined by qualitative and
quantitative assay.
METHoDS
We have investigated a number of 104 patients
selected over a period of three months in Floreasca
Clinical emergency Hospital by upper GI endoscopy
(OGD) and by performing cagA-IgG antibodies. The
selection criteria included clinical features: upper GI
bleeding from ulcer, persistent epigastric pain in spite
of Proton Pump Inhibitor administration, alarm symptoms (weight loss, nocturnal pain, dysphagia, anemia, palpable mass, vomiting), resistance to first line
antibiotic therapy for H. pylori (proved by positive
fecal antigen tested one month after treatment) or
endoscopic evidence: pathological findings at
endoscopy (ulcers, polyps, tumors). The selected
cases were further submitted for the assessment of
cagA-IgG antibodies.
Fig 1. Distribution of cases resistant to first line

triple therapy, depending on the presence of anti-H.
pylori cagA-IgG antibodies
102
For this purpose 2-5 ml of serum were taken

from patients and stored at +2-8C for up to five
days after collection. For longer storage periods, samples were kept frozen at 20C for several months.
The method used for testing was eLIsA. The microplates were coated with H. pylori specific cagA
synthetic antigen. In the 1st incubation, the solid phase
was treated with diluted samples and anti cagA-Ag
IgG was captured, if present, by the antigens. After
washing out all the other components of the sample,
in the 2nd incubation bound anti cagA-Ag IgG were
detected by the addition of anti human IgG antibody,
labeled with peroxidase (Horse Radish Peroxidase).
The enzyme captured on the solid phase, acting on
the substrate/chromogen mixture, generated an optical signal that is proportional to the amount of anti
cagA-Ag IgG antibodies present in the sample. IgG in
the sample could be assessed quantitatively by
means of a standard curve calibrated in arbitrary units
per milliliter (Uarb/ml) as no international standard is
available. A positive result was considered more than
5 arbU/ml.
RESUlTS AND DISCUSSIoN

The cagA-IgG antibodies are assessed for determining if a person has this type of strain. These antibodies can persist four years after infection and have
the advantage of detecting retrospective infection; [6]
this fact being particularly useful in gastric cancer
patients for correlating the association of the malignant disease with H. pylori [7]. The disadvantage of
this marker is that the presence of the respective
antibiodies cannot differentiate present from past
infections and it cannot be used for checking the
eradication of H. pylori infection, the rapid urease
test performed from gastric mucosa, urea breath test
or less invasively fecal antigen test being more reliable to achieve this task.
From the cases resistant to first line therapy, 21
patients with gastritis proved to be positive for anti-H.
pylori cagA-IgG antibodies and 4 negative (Fig. 1).
Most of the cagA positive patients exhibited very high
titres of antibodies (>100 arbU/ml).
A number of 35 cases of bleeding ulcers were
cag A positive, 15 (42%) of them being also NsAID
(Nonsteroidal anti-inflammatory drugs) positive.
seven cases of bleeding ulcers were cagA negative, 4
from them being NsAID positive. The cagA positive,
NsAID negative exhibited high titres (>60 arbU/ml)
in comparison with cag A positive, NsAID positive
that had low titres (Fig. 2).
Correlation of anti-Helicobacter pylori cagA IgG antibodies with resistance to first line treatment
Fig. 2. Distribution of bleeding gastroduodenal ulcers in

function of cag positivity and NSAID administration
CagA IgG antibodies can persist up to four years,

being present also in patients with non cardia gastric
cancer and past infection of H. pylori, proving the
implication of H. pylori in the pathogenesis of noncardia gastric cancer. Also, another drawback is that
in many cases biopsy for H. pylori in gastric cancer
patients is negative (rapid urease test or histology)
because of glandular atrophy and intestinal metaplasia, which are considered precursors of gastric cancer. These are environments in which H. pylori might
disappear. Moreover, the distribution of atrophy and
intestinal metaplasia is uneven, and that might also
affect the sensitivity of the various biopsy sites. In these
cases anti-cagA IgG antibiodies presence proves the
infection with H. pylori. The most appropriate biopsy
site for H. pylori detection in gastric cancer patients,
according to the previous studies, is from upper body
greater curvature, where atrophy or intestinal metaplasia is relatively uncommon (1%-2%). Atrophy and
intestinal metaplasia initiate from the antrum, due to
chronic H. pylori infection, and extend to the corpus
along the lesser curvature [7]. This is in contrast with
non-ulcer dyspepsia or peptic ulcer patients where
the antral biopsy specimens had excellent sensitivity
and specificity for H. pylori (over 90%).
From the cases with non cardia gastric cancer,
25 cases were cag A positive and 5 cagA negative
(Fig. 3). All cases comprised the four categories of
Borrmann classification (Figs. 4-5).
Previous studies have shown that the antibodies
made routinely anti H. pylori Ig G can persist maximum two years, so cag A antibodies are the best
method to date for detecting previous long-term H.
pylori infection.
Fig. 3. Predominance of anti-cagA IgG positive in

gastric cancer cases
Fig. 4. Images of vegetant-ulcerative with spontaneous bleeding tumor (type II in Borrmann classification)
In our study, out of the total number of patients
with cagA-IgG antibodies, 17 were positive for the
rapid urease test (RUT), showing active infection and
8 negative, proving a past infection. so the presence
of cagA-IgG antibodies is important in demonstrating
retrospective infection with H. pylori in non cardial
gastric cancer. The positive cagA and present infection of H. pylori (RUT positive) exhibited high titres
of antibodies in comparison with past infection of H.
pylori with low titres (5-40 arbU/ml).
From the 7 cases of gastric polyps, 5 were positive for anti-cagA IgG, the highest titres being registered for cases with multiple polyps.
The p-value calculated by T-Test regarding the
association between the anti-cagA IgG and different
clinical or bacteriological parameters, i.e.: resistant
cases to first line therapy for H. pylori, bleeding gastro-duodenal ulcers, non cardia gastric cancer and
gastric polyps was 0.02, which is statistically significant (Fig. 6).
103
IlIE et al.
Fig. 5. Images of diffuse infiltrative tumor (type IV in

Borrmann classification)
The presence of the cagA IgG antibodies, with

positive biopsy for H. pylori indicates the need for
treating H. pylori with checking eradication one
month after treatment (by fecal antigen or urea breath
test).There are studies referring the potential of preventing gastric cancer by H. pylori eradication-screening and treating all positive patients [4]. The results
of these studies remain to be established, but the
presence of cagA antibodies strengthens the necessity of giving therapy if the infection with H. pylori is
present (non-invasively tested by fecal antigen test or
urea breath test). For resistant cases with H. pylori
present one month after treatment (when tested by
urea breath test or fecal antigen), culture and antibiotic susceptibility assay should be done [9].
CoNClUSIoNS
CagA positive H. pylori are virulent strains,
which showed to be significantly associated with
resistance to the first line of antibiotics for H. pylori,
bleeding gastroduodenal ulcers, noncardia gastric
cancer and gastric polyps. The highest titers ( more
than 100 arbU/ml) of cagA IgG antibodies were registered in cases resistant to first line therapy of
H.pylori, in bleeding gastroduodenal ulcers cagA
positive, NsAID negative and non cardia gastric cancer present or recent infection. A high percentage
(42%) of the cagA positive, bleeding cases were also
NsAID positive, pointing out that in gastroduodenal
bleeding ulcers associated with NsAID, H. pylori
should be also tested, the summing of these two factors increasing the risk of hemorrhage.
104
Fig. 6. Relation between selected cases (resistance

cases to first line therapy for H. pylori, bleeding gastroduodenal ulcers, noncardia gastric cancer and
gastric polyps) and cagA IgG antibodies
REFERENCES
1. lee Y.C., liou J.M., Wu M.S., Wu C.Y., lin J.T., Review:
eradication of Helicobacter pylori To Prevent Gastroduodenal Diseases: Hitting More Than One Bird With
the same stone, the advGastroenterol; 2008,111-120.
2. Bauer B., Meyer F.T., Review Article The Human
Gastric Pathogen Helicobacter pylori and its Association
with Gastric Cancer and Ulcer Disease; Hindawi
Publishing Corporation Ulcers, 2011, 23 pg.
3. Wu H.A., Crabtree J., Bernstein l., Forman D., Role of
H. Pylori caga strains and risk of adenocarcinoma of the
stomach and esophagus, int. J. cancer, 2002: 103,
815821.
4. Malfertheiner P., Sipponen P., Naumann M., Moayyedi
P., Mgraud F.; Helicobacter pylori eradication Has the
Potential to Prevent Gastric Cancer: A state-of-the-Art
Critique; the american Journal of Gastroenterology,
2005, 2100-2115.
5. sleisenger and Fordtrans Gastrointestinal and Liver
Disease, 9th edition, 2010, chapter:etiology and pathogenesis of gastric cancer.
6. lu C.Y., Kuo C.H., lo Y.C., Chuang H.Y., Yang Y.C.,
Wu I.C., Yu F.J., lee Y.C., The best method of detecting
prior Helicobacter pylori infection, World J Gastroenterol; 2005, 11(36):5672-5676.
7. Kim C.G., Choi I.J., lee J.Y., Cho S.J., Nam B.H., Kook
M.C, Hong E.K., Biopsy site for Detecting Helicobacter
Pylori Infection in Patients with Gastric Cancer, J Gastroenterol Hepatol, 2009, 469-474.
8. Papatheodoridis G., Papadelli D., Cholongitas E.,
Vassilopoulos D., effect of Helicobacter pylori infection
on the risk of upper gastrointestinal bleeding in users of
nonsteroidal anti-inflammatory drugs, the american
Journal of Medicine, 2004, Pag. 601-605.
9. Ilie M., Popa M., Chifiriuc M.C., Baltac A., Constantinescu G., Tnsescu C., Helicobacter pylori cultivation
from gastric biopsies and susceptibility to antibiotics
used in empirical therapy, romanian archives of Microbiology and immunology, 2011, 70(2):60-64.
INFLUeNCe OF ORTHODONTIC TReATMeNT ON ORAL sTRePTOCOCCI

Theodor-Cristian Vizitiu1*, Mihaela Cristina Giuca2, Ecaterina Ionescu1
1Department
of orthodontics and Dento-Facial orthopedics, Faculty of Dental Medicine,

carol Davila University of Medicine and pharmacy, bucharest, romania;
2cantacuzino National institute of research-Development for Microbiology and immunology, bucharest, romania
ABsTRACT
objective of this study is to evaluate the changes of the oral microbial flora, concentrating on the
oral streptococci, after the first 3 and 6 months of orthodontic treatment.
Materials and methods. 40 patients, aged 7-17, that presented for orthodontic treatment between
April and september 2010 in the Department of Orthodontics and Dento-Facial Orthopedics of
Carol Davila University of Medicine and Pharmacy, Bucharest have been selected. According to the
protocol, coronary and subgingival plaque was collected from the dental surface before starting
any orthodontic treatment (T0), 3 months after wearing orthodontic appliances (T1) and 6 months
after wearing orthodontic appliances (T2). The samples were studied in Cantacuzino National
Institute of Research-Development for Microbiology and Immunology [isolation on Columbia agar
with 5% sheep blood, identification on morphotinctorial, growth and biochemical characteristics
using API 20 sTReP (BioMerieux)]. Bacterial concentration (colony-forming units/sample =
CFU/sample) for the aerobic and anaerobic flora was calculated by the serial dilution method of
counting bacteria.
Results. 106 strains of oral streptococci were isolated from dental plaque, belonging to 6 species
(Streptococcus mitis, Streptococcus oralis, Streptococcus mutans, Streptococcus salivarius, Streptococcus sanguis and Streptococcus acidominimus), 37 strains of oral streptococci in patients from
group I (T0), 40 strains from group II (T1) and 29 strains of oral streptococci from group III (T2).
After 3 months (T1) the aerobic bacteria percentage, detected at a concentration between 105 and
106, increased from 30 to 38.2%. The percentage of patients with a bacterial concentration higher
than 106 CFU/sample increased from 5% to 8.8%. The samples colected at T2 (patients examined
after 6 months of orthodonic treatment) presented a lower bacterial concentration, as compared to
group II (T1). The most common isolated species of streptococci were S. salivarius, S. oralis and
S. mutans (37.5%, 22.5% and 10%), whose frequency increased after 3 months of treatment to
41.14%, 32.3% and respectively 14.4%, returning after 6 months of treatment at values similar to
those recorded before beginning the orthodontic treatment.
Conclusions. Presence of orthodontic appliances may produce a transitory increase of bacterial
concentration (CFU/sample) and isolation rate of oral streptococci, returning to the level prior to
the application of these devices after a time interval of several months.
Keywords: bacterial concentration, oral microbial flora, orthodontic appliances, S. mutans, S. salivarius, S. oralis.
INTRoDUCTIoN
Oral streptococci are most commonly involved
in the oral infections etiopathogeny. some of them
are initiators of the cavity process, producing the demineralization of dental enamel. Many studies indicated an increased prevalence of detection of these
organisms in combination with other bacteria, or as
single etiological agents in pyogenic oral and maxillofacial infections: dentoalveolar abscesses, peri-
coronitis, sialadenitis, sinusitis, osteitis and osteomyelitis of the jaw, infected jaw tumors (e.g. cysts)
and other facial infections.
The presence of orthodontic appliances impedes
the maintenance of proper oral hygiene and result in
plaque accumulation, thus creating proper conditions for increasing bacterial concentration and consequently for the development of the oral streptococci
of medical interest [1, 2, 3, 4].
* corresponding author: Theodor-Cristian Vizitiu, e-mail: theodorvizitiu@yahoo.com
105
VIZITIU et al.
Aim
The aim of this study was to evaluate the
changes of the oral microbial flora, concentrating on
the oral streptococci, after the first 3 and 6 months of
orthodontic treatment.
MATERIAlS AND METHoDS
Three groups of patients have been studied. The
patients presented for orthodontic treatment between
April and september 2010 in the Department of Orthodontics and Dento-Facial Orthopedics of Carol
Davila University of Medicine and Pharmacy,
Bucharest. The parents of the patients (all minor)
were informed on the study and signed their consent.
All patients were instructed on the appropriate oral
prophylaxis in accordance with their individual risk
factors.
Group I was represented by 40 patients (17 males
and 23 females) before starting any orthodontic treatment (T0).
Group II was formed by 34 of these 40 patients, 14
males and 20 females, 3 months after wearing
orthodontic appliances (T1).
Group III was formed by 31 of the 34 patients included in group II, 13 males and 18 females, 6
months after wearing orthodontic appliances
(T2) (Fig.1).
The patients were aged 7-17 for all groups, with
an average of 11.6 for group I, 11.3 for group II and
11.4 for group III.
According to the protocol, coronary and subgingival plaque was collected from the dental surface
with a sterile curette followed by the rapid transfer of
the sample in Amies transport. The samples were
transported to Cantacuzino National Institute of
Research-Development for Microbiology and Immunology, registered and processed according to the
protocols of the National Reference Centers for Bacterial Respiratory Infections and Anaerobic Infections.
Bacterial concentration (colony-forming units /
sample = CFU/sample) for the aerobic and anaerobic
flora was calculated by the serial dilution method of
counting bacteria [5]. 0.1 ml from each diluted sample (10-3, 10-4, 10-5, 10-6) were plated on 2 agar
growth medium plates. The colony-forming units /
sample number was established using the formula:
CFU/sample number = number of colonies counted
on a plate x dilution factor (103, 104, 105, 106) x 10
(correction factor for 1ml).
Isolation of bacteria was performed on growth
media: Todd Hewitt broth, Columbia agar with 5%
sheep blood. After 24-48 hours of incubation at 37oC
in CO2 atmosphere, the developed colonies were
morphologically examined. each colony type was
106
subcultivated on unselective growth media to obtain

pure cultures. Reference strains were used as control:
S. mutans ATCC 35668 and S. salivarius ATCC 13419
(American Type Culture Collection).
Isolated bacterial colonies were identified based
on their morphotinctorial characteristics (Gram
stained smears), growth characteristics on media and
also underwent biochemical tests. The species were
identified using API 20 sTReP for streptococci.
The identified streptococci strains were preserved by freezing, for further investigation by molecular diagnosis (PCR) [6].
Following the sex distribution of patients, the predominance of female gender can be observed in all 3
groups (Fig. 1). Regarding the age, most patients were
classified in age groups 7-9 and 10-12 years (Fig. 2).
Table 1 presents the results obtained for aerobic
bacteria counted in samples collected from the three
studied groups. An increased aerobic CFU/sample
number was observed after 3 months of wearing orthodontic appliances.
The aerobic bacteria percentage detected with a
concentration between 105 and 106, increased after
3 months from 30 to 38.2%. The percentage of patients with a bacterial concentration higher than 106
CFU/sample increased from 5% to 8.8%. The results
concerning the aerobic CFU/sample number are in
concordance with the results published by most other
authors [1, 2, 7, 8] who sustain that the presence of
orthodontic appliances may produce an increase of
the oral bacterial concentration in the first months.
The samples colected from group III (patients
examined after 6 months of orthodonic treatment)
presented a lower bacterial concentration, as compared to group II and the values obtained for the risk
concentrations ( 105 and 106) were smaller
Fig. 1. Sex distribution of the patients
Influence of orthodontic treatment on oral streptococci
Fig. 2. Age distribution of the patients

even when compared to group I, represented by patients before wearing any orthodontic appliance.
106 strains of oral streptococci were isolated
from dental plaque, belonging to 6 species (Streptococcus mitis, Streptococcus oralis, Streptococcus mutans, Streptococcus salivarius, Streptococcus sanguis
and Streptococcus acidominimus), 37 strains of oral
streptococci in patients from group I, 40 strains from
group II and 29 strains of oral streptococci from
group III. These results are presented in Table 2.
The most common isolated species of streptococci were S. salivarius, S. oralis and S. mutans (37.5%,
22.5% and 10%), whose frequency increased after 3

months of treatment to 41.14%, 32.3% and respectively 14.4%, returning after 6 months of treatment at
values similar to those recorded before beginning the
orthodontic treatment.
Streptococcus mutans is an aerobic, facultative
anaerobic bacterium, frequently isolated from oral
flora. All species of S. mutans proved cariogenic in
experiments on animals and are considered the major
cause of tooth decay in humans. In the studied patients, an increased isolation rate has been observed
after three months, from 10% to 14.4%, followed by
Table 1 - Total bacteria concentration

Group I
CFU/sample no.
Group II
Group III
No.
No.
No.
22,5
5,5
25,8
17
42,5
16
47,5
13
41,9
12
30
13
38,2
29
10
8,8
3,3
Total
40
<10
4
10 <10
104 < 105

5
10 < 10
6
34
31
- Isolated oral streptococci strains

Table 2 - Isolated oral streptococci strains
Species
Group I (40) (patients)
Group II (34) (patients)
Group III (31) (patients)
No.
No.
No.
S. oralis
22,5
11
32,3
25,8
S. mutans
10
14,4
9,6
S. sanguis
12,5
11,7
12,9
S. salivarius
15
37,5
14
41,14
10
32,2
S. mitis
7,5
14,4
9,6
S. acidominimus
2,5
2,9
3,2
Total
37
40
29
107
VIZITIU et al.
decreasing to 9.6% after 6 months of wearing orthodontic appliances.

S. oralis and S. mitis are part of normal oral flora,
but were isolated from bacteraemia and septic shock
in immunocompromised patients and may cause endocarditis. In this study, S. oralis was isolated at a rate
of 22.5% before treatment, that increased to 32.3%
after 3 months of treatment and after 6 months returned
to values close to the initial ones (25.8 %). S. mitis, present at a rate of 7.5% before treatment, increased to
14.4% after 3 months of treatment and was found at a
rate of 9.6% after 6 months of orthodontic treatment.
S. sanguis is found in normal flora of the oral
cavity; its presence is inhibiting the growth of other
streptococci, such as S. mutans. It is frequently found
in periapical infections and has been isolated in cases
of severe facial cellulitis complicated by regional
thrombophlebitis and sepsis. In this study, S. sanguis
recorded almost constant values in the three groups
of patients (12.5% for group I, 11.7% for group II and
12.9% for group III).
S. salivarius is the principal commensal that colonizes the oral cavity and the oropharynx in the first
hours after birth. It is considered an opportunistic pathogen, rarely involved in septicemia in people with
neutropenia. The values obtained after 6 months were
below the level registered before treatment.
similar data were reported by other studies [9, 10]
that sustain that the presence of orthodontic appliances may produce a transitory increase of the isolation rate of oral streptococci, returning to the level
prior to the application of it after a time interval of
several months.
S. acidominimus is considered not to be significant in human pathology and one strain was isolated
from the same patient, in every phase of the study.
CoNClUSIoNS
Bacterial concentration (CFU/sample) was higher
in group II (patients after 3 months of wearing orthodontic appliances) compared to group I (represented
by patients before wearing orthodontic appliances).
In group III (after 6 months of orthodontic treatment) the values of bacterial concentration were lower than the ones recorded in group II. This suggests
a transitory increase in the total bacteria count, in the
first 3 months of orthodontic treatment.
Oral streptococci isolation rate increased during
the first 3 months of orthodontic treatment and then
decreased after 6 months, reaching values close to
those recorded before treatment.
No increase in the isolation rate has been observed after 6 months of orthodontic treatment, as com108
pared to the beginning of the treatment, for the main

dental pathogen, S. mutans.
ACKNoWlEDGEMENT. This paper is supported by the sectoral Operational Programme Human
Resources Development (sOP HRD), financed from
the european social Fund and by the Romanian Government under the contract number POsDRU/6/1.5/s/s17
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8. Aamda Anne, Schereie l, Arneberg Pal, Krogstad olaf.
effect of orthodontic treatment on prevalence of
Streptococcus mutans in plaque and saliva, european
Journal of oral Sciences 1984. 92(3): 2112179.
9. Ristic M, Vlahovic Svabic M, Sasic M, Zelic o. effects of
fixed orthodontic appliances on subgingival microflora,
int J Dent Hyg 2008. 6(2):129-36.
10. Faltermeier A, Brgers R, Rosentritt M. Bacterial adhesion of Streptococcus mutans to esthetic bracket materials american Journal of orthodontics & Dentofacial
orthopedics 2008. 133(4): 99-103.
DYNAMICs OF eNDOTHeLIAL PROGeNITOR CeLLs

FOLLOWING seVOFLURANe PReCONDITIONING
Mihaela Popescu1, Adelina Munteanu1, Gheorghia Isvoranu2, laura Suciu2,
Bogdan Pavel1, Bogdan Marinescu2, leon Zagrean1*
1physiology
Department, carol Davila UMph, bucharest, romania; 2Victor babe NirD, bucharest, romania
ABsTRACT
There is relevant evidence concerning the involvement of endothelial progenitor cells in neovascularization and wound healing. In this study we investigated the effects of sevoflurane, a volatile
anesthetic with proven cardioprotective virtues, on the mobilization of bone marrow mononuclear
cells with endothelial progenitor markers (CD 34+, flk-1+), an event that may account for the protective effects of delayed anesthetic preconditioning. Male Wistar rats were treated with a mixture
of air and sevoflurane (1 MAC) in cycles of 5 minutes, alternating with 5-minutes wash-out periods
(the preconditioned group), or ventilated for 30 minutes with room air (control group). Following
flow cytometry and immunofluorescence measurements, a considerable increase in circulating
CD34+, flk-1+ and CD34+/flk-1+ cells was observed in the preconditioned group beginning at
12 hours after treatment, with a peak value at 24 hours after sevoflurane administration. These cells
are a potential source of myocardial regeneration in the context of perioperative or periprocedural
ischemia in patients with coronary artery disease.
Keywords: endothelial progenitor cells, anesthetic preconditioning, sevoflurane.
INTRoDUCTIoN
Myocardial preconditioning is an endogenous
phenomenon that increases the hearts ability to tolerate a longer period of possibly lethal ischemia.
Preconditioning can be induced by short periods of
ischemia (ischemic preconditioning) or through a
number of pharmacological agents that mimic the
effects of ischemia, such as volatile anesthetics (anesthetic preconditioning). exposure to volatile anesthetics triggers an adaptive response similar to that
achieved by ischemic preconditioning. Both anesthetic and ischemic preconditioning exhibit an early
phase, which arises immediately and lasts for 2-3
hours, and a late phase that occurs 12-24 hours after
the preconditioning stimulus and lasts for up to 72
hours. Furthermore, many experimental studies indicate similar mechanisms for ischemic and anesthetic
preconditioning, involving the ATP dependent potassium channels (kATP channels), the mitochondrial permeability transition pore (mPTP), nitric oxide (NO) or
reactive oxygen species (ROs).
Bone marrow-derived mononuclear cells differentiate into endothelial cells in adult animals and in
humans. endothelial progenitor cells characterized
by markers like CD34 and flk-1 play a major role in

maintaining the endothelial function and also in neovascularization. A decrease of circulating CD34-positive cells is associated with cardiovascular disease,
leading to the conclusion that they can be considered
a potential target for diagnostic or for cell replacement therapies.
In a recent study kamota et al. [1] reported
increased mobilization and recruitment of bone marrow stem cells in the late phase of remote ischemic
preconditioning. Also, a number of experimental and
clinical data suggest a possible link between the cardioprotection provided by preconditioning with
volatiles and myocardial tissue regeneration through
stem cells mobilized from the bone marrow and recruited into the damaged tissue. sevoflurane enhances the colony forming capacity of mononuclear cells
in vitro [2] and produces a rise in vascular endothelial growth factor (VeGF) and stromal derived factor
1 alpha (sDF-1 alpha), two potent stimulators of endothelial progenitors mobilization and recruitment
[2-7]. Consequently, in this study we monitored the
dynamics of endothelial progenitor cells after
sevoflurane exposure in the course of three days in
* corresponding author: Leon Zagrean, e-mail: leon.zagrean@gmail.com
109
PoPESCU et al.
order to demonstrate an increase in endothelial progenitor cells mobilization after anesthetic preconditioning.
MATERIAl AND METHoDS
Male Wistar rats weighing 300-400 g were provided by Victor Babes National Institute of Pathology, Bucharest. All the procedures were approved
by the ethics Committee of Carol Davila UMPh.
Anesthetic preconditioning
The rats were randomly assigned into two study
groups (n = 30): the preconditioned group, where the
animals were intubated and ventilated with a mixture
of air and sevoflurane (1 MAC) in cycles of 5 minutes,
alternating with 5 minutes wash-out periods, and the
control group, where the rats were intubated and
ventilated for 30 minutes with room air.
Sample collection and mononuclear cell handling
The animals were sacrificed at baseline, 12hrs,
24 hrs, 48 hrs and 72 hrs after the preconditioning
stimulus and blood samples were collected. Mononuclear cells were separated from the whole blood
by gradient centrifugation.
Monitoring circulating CD34 and flk-1 positive
mononuclear cells by flow citometry
Cells were stained with a primary antibody anti
CD34 (mouse anti-rat monoclonal IgG1, santa Cruz)
followed by a second antibody (biotinylated goat
anti-mouse Ig and streptavidin-Pe, BD Pharmigen) or
with a primary antibody anti flk-1 (rabbit anti-rat,
Abcam) followed by a second antibody (F(ab)2 fragment goat anti-rabbit IgG, Invitrogen). For each sample, cells were both single and double stained. Respective isotype controls were used as negative controls. Flow cytometry was performed with a FACs
Canto II (Beckton Dickinson) and a BD FACsDiva
software was used for analysis.
Monitoring circulating CD34 and flk-1 positive
mononuclear cells by immunofluorescence
Cells were stained with a primary antibody anti
CD34 (mouse anti-rat monoclonal IgG1, santa Cruz)
followed by a second antibody (goat anti-mouse Alexa
Fluor 488) or with a primary antibody anti flk-1 (rabbit
anti-rat, Abcam) followed by a second antibody (goat
anti-rabbit Alexa Fluor 594). We used the cytospin
method for fitting the cell suspension onto the glass
slides. A Nikon eclipse e300 microscope was used and
at least 15 separate fields were analysed.
110

Flow cytometry. Anesthetic preconditioning induced an increase in the number of circulating
CD34+, flk-1+ and CD34+/flk-1+ cells at 24 hrs
after sevoflurane exposure (Fig. 1).
With respect to the CD 34+ cells, starting with
12 hrs from baseline there was a rising trend in the
number of cells but without statistical signifficance
(P = 0.062). Conversely, at 24 hrs after the sevoflurane exposure a 7-fold increase in the number of cells
was induced (P = 0.031) (Fig. 1). From 24 hrs
onward the number of circulating CD34+ cells progressively decreases towards the baseline levels. For
the flk-1+ cells the same trend was recorded, with a
small increase at 12 hrs and a significant rise at 24 hrs
(P< 0.0001). The dynamics of CD34+/ flk-1+ cells
had a similar pattern (a 2.3-fold increase at 24 hrs
with a P< 0.0001).
Immunofluorescence. The same hypothesis was
verified through another method, that yielded similar
results (Fig. 2). This was only a qualitative estimation
of the circulating endothelial progenitors, not as precise as the quantitative technique previously used.
However, it revealed the same increase in number
for all cell types studied (CD34+, flk-1+,
CD34+/flk-1+). The boost in number occurred at the
same timelines as observed for the previous method.
At baseline there were virtually no CD34+ and
CD34+/flk-1+ cells, and very few flk-1+ cells. Between 12 hrs and 24 hrs the values increased (Fig. 3),
whereas from 24 hrs onwards a progressive decrease
was recorded.
Other studies tried to prove similar effects of
sevoflurane on endothelial progenitor cell mobilization and recruitment, using different preconditioning
protocols [2] and in different experimental settings.
They only reported an increased colony forming
capacity induced by sevoflurane, and higher VeGF
mRNA levels in CD133+/CD34+ cord blood cells.
This was one of the incentives that drove us to
choose a different protocol, one that was proved to
be superior even in clinical studies [8].
The CD34+/ flk-1+ population can be considered an endothelial progenitor cell population. This
behavior of circulating endothelial progenitors cells
matches the one reported after ischemic preconditioning [1], suggesting the existence of yet another
mechanism of endothelial protection provided by
both anesthetic and ischemic preconditioning. Also,
the signifficant rise in the number of cells at 24 hours
fits the second window of preconditioning, allowing
Dynamics of endothelial progenitor cells following sevoflurane preconditioning
Fig. 1. Endothelial progenitor cell dynamic in the

peripheral blood after anesthetic preconditioning
(APC). Flow cytometry performed on peripheral
blood mononuclear cells at several timelines after
APC. (A) CD34+ cells level increases significantly
24 hrs after APC, as do flk-1+ cells (B) and
CD34+/flk-1+ cells (C) anesthetic preconditioning
Fig. 2. CD34+ and flk-1+ cells in the peripheral blood 12 h after exposure to sevoflurane. Immunofluorescence
(cytospin technique) performed on peripheral blood mononuclear cells, depicting single stained CD34+ cells (A, B),
flk-1+ cells (C, D). In (A) and (C) phase contrast and DAPI are merged with the initial images (B) and (D)
111
PoPESCU et al.
Fig. 3. CD34+/flk-1+ cell, 24 hrs after APC. Immunofluorescence (cytospin technique) performed on peripheral blood mononuclear cells. (A) flk-1+ cell, (B) CD34+, (C) DAPI stained nucleus of the same cell, (D)
merged image of the previous three, depicting the CD34+/flk-1+ cell
us to assume that this could be another way through

which delayed preconditioning plays its part in shielding the heart from ischemia/reperfusion injury.
Therefore, the hypothesis that anesthetic preconditioning is able to mobilize in the peripheral circulation an increased number of endothelial progenitor
cells was confirmed, requiring now further investigation of the impact of our finding on the ischemic
heart subjected to anesthetic preconditioning.
The exact mechanisms through which endothelial progenitor stem cells sustain the myocardial recovery after an ischemic insult is still up for discussion.
A considerable number of experimental studies report
that bone marrow stem cells mobilized through ischemic preconditioning are able to differentiate into
endothelial cells or myocytes [9, 10, 11], but they act
mainly in a paracrine manner [10, 12].
The fact that sevoflurane is able to increase the
circulating number of endothelial progenitor cells is
of major clinical importance, as these cells represent
an endogenous pool of regenerative cells, both endothelial and myocardial. Anesthetic preconditioning
could be extended, with favourable results, to stenting
procedures, where implementation of this protocol
112
could favorize early healing and endothelization, as

endothelial cell capture by anti CD34 antibody covered stents was already tested, and with promising
results [13].
Anesthetic preconditioning could be used as
support for stem cell therapy, both by increasing the
viability of injected cells and by supplementing the
number of stem cells from an endogenous source.
Considering the results of this study, it would be
of major clinical importance to test whether the
effects of late anesthetic preconditioning on endothelial progenitor stem cells contributes to protecting the
endothelium and the myocardium against the deleterious effect of ischemia and reperfusion induced by
surgical procedures.
ACKNoWlEDGEMENTS
This work was supported by the sectorial Operational Programme Human Resources Development
(sOPHRD) financed from the european social Fund,
and by the Romanian Government under the contract
number POsDRU/6/1.5/s/17, A european dimension
of doctoral studies.
Dynamics of endothelial progenitor cells following sevoflurane preconditioning
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C, Antke C, Sorg RV, Kogler G, Wernet P, Muller HW,
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113
A HIGHLY PURIFIeD VeGeTAL FRACTION ABLe

TO MODULATe HMGB1 AND TO ATTeNUATe sePTIC sHOCk IN MICe
Natalia Simona Apetrei1, Ana Clugru1, F. Kerek2, Minerva Panteli3,
I. Rasit3, lidia Cremer1, G. Szegli1, Andreea-Roxana lupu1*
1cantacuzino
National institute of research - Development in Microbiology and immunology, bucharest, romania;

2DoNatur GmbH, Martinsried, Germany;
3National institute for chemical-pharmaceutical research and Development, bucharest, romania
ABsTRACT
High-mobility group box protein 1 (HMGB1) is an intracellular protein that may be released actively
from monocytes and macrophages or passively from necrotic or damaged cells. Its inhibition in animal experiments, even in the late phase of septic shock, significantly enhanced the survival rate of
rodents. The aim of our study was to investigate the effect of a vegetal fraction isolated and highly
purified from Helleborus purpurascens regarding the modulation of HMGB1 release either from
tumor cells or human blood mononuclear cells. Our results showed that the vegetal fraction was
able to down-regulate the release of HMGB1 from activated human blood mononuclear cells
(PBMCs) and tumor cells. By combining the purified fraction with Cyclophosphamide the release
of HMGB1 from tumor cells was strongly decreased. This synergism was not noticed when the vegetal product was associated with Doxorubicin. We also studied the effect of the purified fraction
in mice with septic shock induced by cecal ligation and puncture (CLP) method. The tested vegetal product increased significantly the survival rate of animals compared to the mice not treated
with it. Our data suggest that the purified vegetal fraction may modulate inflammation by downregulating the HMGB1, which can also explain its efficacy in septic shock in mice.
Keywords: vegetal fraction, Helleborus purpurascens, HMGB1, cecal ligation and puncture
INTRoDUCTIoN
HMGB1 (High-Mobility Group Box protein 1), a
member of the high-mobility group protein superfamily, is an abundant nuclear and cytoplasmic protein present in mammalian cells. Although residing
predominantly in the nucleus of quiescent macrophages, HMGB1 can be actively secreted in response
to exogenous and endogenous inflammatory stimuli,
such as endotoxins, TNF-a, IL-1 [1, 2]. In addition to
active release from macrophages/monocytes, HMGB1
can be passively released by necrotic or damaged
cells when the integrity of cytoplasmic membranes is
broken [3, 4]. The secretion into the extracellular
environment revealed a cytokine activity of HMGB1
and mediates inflammatory responses in endotoxemia, arthritis and sepsis [5]. Once released, HMGB1
can bind to cell-surface receptors (e.g., the receptor
for advanced glycation end products (RAGe), Toll-like
receptors (TLR) 2 and 4) and mediate various cellular

responses including chemotactic cell movement and
release of proinflammatory cytokines (e.g. TNF, IL-1)
[6, 7]. Taken together, these observations characterize
HMGB1 as a non-classical proinflammatory cytokine.
HMGB1 was isolated in the early 1970s in calf
thymus as an abundant chromosomal protein [8]. The
name derives from its rapid mobility on electrophoresis gels. The N-terminus is rich in positively charged lysines, and the C-terminus is rich in negatively
charged aspartic and glutamic acids [9]. HMGB1 contains two DNA-binding domains, termed HMG boxes, which confer functionality to the protein. signal
transduction of HMGB1 occurs in part through the
receptor for advanced glycation end-products (RAGe),
expressed on monocytes/macrophages, endothelial
cells, neurons and smooth-muscle cells [10]. The
interaction of HMGB1 and RAGe may be important
in tumor formation, because blocking this interaction
* corresponding author: Andreea Roxana Lupu, Tel./Fax: +40 21 3069298. e-mail address: ldreea@yahoo.com
114
A highly purified vegetal fraction able to modulate HMGB1 and to attenuate septic shock in mice
with anti-RAGe antibodies decreases the growth and

metastasis of both implanted and spontaneous
tumors in mice [11, 12].
extracellular HMGB1 have an important role in
malignant diseases, in the promotion of cell migration and metastasis. Many studies described the overexpression and cytoplasmic localization of HMGB1
in some tumor cells [13, 14]. in vitro studies have
shown that through quantitative determination of
HMGB1 concentrations it is possible to differentiate
between pathological cell death (necrosis) and physiological cell death (apoptosis); in necrosis, much
higher HMGB1 values are observed [15, 16].
HMGB1 plays an important role in the late
phase of septic shock [17]. sepsis is the most important cause of death among hospitalized patients, with
mortality rates from 30% to 70%. sepsis is the result of
an acute and systemic immune response to a variety
of factors, in particular to bacterial infection. This
response leads to the activation of a number of host
mediator systems, including the cytokine, leukocyte,
and hemostatic networks [18]. several experimental
animal models which attempt to mimic pathophysiological changes typically seen in septic patients have
been developed to study the underlying mechanisms
of sepsis and the associated systemic inflammatory
response [19]. Cecal ligation and puncture (CLP) in
rodents has become the most widely used model for
experimental sepsis and is currently considered as
the standard in sepsis research [20, 21].
Though circulating HMGB1 is undetectable in
humans, patients with septic or hemorrhagic shock
have significantly elevated levels of HMGB1 in blood
and plasma. The increase of extracellular HMGB1
correlates with the pathogenesis of inflammatory disorders, including septic shock [22]. The literature
data show that the level of circulating HMGB1 can
be 30-fold enhanced in pacients with severe sepsis
comparatively with healthy donors [23, 24].
The objective of our study was to investigate the
effect of a vegetal fraction, MCs-Ab21, isolated and purified from Helleborus purpurascens, regarding the
modulation of HMGB1 release from both sk-OV-3 tumor cells and LPs-activated human blood mononuclear cells (PBMCs). We also studied the effect of MCsAb21 purified vegetal fraction on mice with septic
shock induced by cecal ligation and puncture method.
MCs compounds are described to date as multicomponent fractions isolated from the roots of Helleborus purpurascens. Preliminary data suggested beneficial effects of this novel herbal product, with possible modulatory activity on inflammatory diseases
[25, 26, 27, 28].

Preparation of the purified fraction MCS-Ab21
from Helleborus purpurascens
The investigated fraction from extract batch
He-21 was prepared starting from 1 kg of dried roots
of Helleborus purpurascens, which was extracted
twice with methanol:water (90:10), 16 hrs at room
temperature. Methanol was eliminated by vacuumrotatory evaporator and the concentrated water phase
was extracted twice with a mixture of dichloromethane:hexane (1:2). The defatted watery phase was
then precipitated with absolute ethanol and the precipitate was dissolved in water. This raw product was
fractionated primary in aliquots, on Biotage Horizon
Flash chromatography (HPFC) system. All samples
collected in test tubes were pooled together according to the analytical HPLC data and the resulted
pooled fractions were named MCs-a, MCs-b. Next,
the primary fraction MCs-b was further purified by
repeated High Performance Liquid Chromatography
(HPLC) on preparative scale. Preparative chromatographic process with linear gradient of acetonitrile in
water provided the purified fraction MCs-Ab21 tested
in this study.
Cell line
For evaluating the effect of the purified fraction
MCs-Ab21 on tumor cells, sk-OV-3 human ovarian
cancer cell line (ATCC No. HTB-77) was used.
sk-OV-3 cell line derived from the ascitic fluid
from a 64-year old caucasian female with an ovarian
tumour [29]. sk-OV-3 cells were maintained in culture at 37oC and 5% CO2, in RPMI-1640 culture medium supplemented with 10% FCs (fetal calf serum),
10 IU/mL sodium penicillin-G, 10 mg/mL streptomycin
sulfate, and 2 mM L-glutamine.
Culture of SK-oV-3 tumor cells
1x106 cells/ml were cultured for adherence in
flat-bottom 24-wells plates, in RPMI-1640 medium,
supplemented with 10% FCs, 10 IU/ml sodium penicillin-G, 10 mg/ml streptomycin sulphate and 2 mM
L-glutamine, for 4 hrs. Next, in a first experimental
model, the cells were treated with MCs-Ab21 purified
fraction, used in different doses (5, 10 and 25 mg). In
a second experimental model, sk-OV-3 cells were
treated with cytostatic drugs - Doxorubicin or Cyclophosphamide (10 mg and 20 mg), or a combination of
5 mg MCs-Ab21 and each chemotherapeutic drug
(used in the concentations mentined above).
Next day, the culture supernatants were harvested
and HMGB1 levels were quantified using a specific
eLIsA kit.
115
APETREI et al.
Human peripheral blood mononuclear cells isolation

Peripheral blood mononuclear cells (PBMCs)
were isolated from human whole blood of 5 healthy
donors (who gave their informed consent), using density gradient centrifugation (adapted method of Roos
and DeBoer [30]). The protocol received the approval
of the ethics Committee of Cantacuzino National
Institute of Research-Development for Microbiology
and Immunology. Briefly, heparinized venous blood
from donors was diluted 1:1 with RPMI-1640 medium
(sigma) supplemented with antibiotics (10 IU/mL sodium penicillin-G, 10 mg/mL streptomycin sulphate)
and 20 mM L-glutamine, then was carefully layered
onto Ficoll-Paque Plus (Amersham-Pharmacia) and
centrifuged at 2500 rpm for 40 min at 20oC. After sediment removal (containing polymorphonuclear neutrophils cells and red blood cells), PBMCs in RPMI1640 culture medium, supplemented with antibiotics,
L-glutamine and 2% FCs, were twice centrifuged at
1400 rpm for 15 min. Finally, PBMCs were resuspended in RPMI-1640 medium supplemented with
antibiotics, L-glutamine and 10% FCs, at a final concentration of 1x106 cells/mL. PBMCs viability, measured by trypan blue, exceeded 98%.
Culture of human PBMCs
1x106 PBMCs/ml were cultured in flat-bottom
24-well plates containing RPMI-1640 medium supplemented with 10% FCs, 10 IU/ml sodium penicillin-G, 10 mg/ml streptomycin sulphate and 2 mM
L-glutamine. After 1 h incubation at 37oC and 5% CO2
for adherence, the cells were treated with MCs-Ab21
purified fraction, used in different concentrations
(5 mg, 10 mg, 25 mg); after 1 h, LPs (1 mg) was added,
and the plate was incubated overnight.
The culture supernatants were harvested and
HMGB1 levels were quantified using a commercial
HMGB1 eLIsA kit.
ElISA method for HMGB1 quantification in culture supernatants
HMGB1 levels in the culture supernatants were
quantified using a commercial eLIsA kit, according to
the manufacturers instructions (IBL International
GmbH). HMGB1 eLIsA is a sandwich-enzyme immunoassay for the quantitative determination of
HMGB1 in serum, plasma, cell culture medium.
Briefly, on 96-well microtiter plates coated with purified monoclonal capture antibodies for HMGB1,
recombinant standards and samples were added to
the wells for 20-24 hrs at 37oC. Next, the plate was
incubated with enzyme conjugate and allowed to
116
bind for 2 hrs at 25oC. In the third step, the plate was
incubated with colour solution, 30 min at room temperature and kept protected from light. The reaction
was stopped with stop solution and the absorbance
values were measured at 450 nm within 60 min,
using Thermo Multiskan eX microplate reader. The
absorbance values of the standards were plotted
against their concentration using an automated method (4 Parameter Logistics). The concentration of the
samples was read directly from the standard curve.
samples with concentrations exceeding the highest
standard have been diluted and reassayed with the
use of the dilution factor, by calculation of the concentration from the standard curve.
MTT Viability / Cytotoxicity Assay
Cells viability was tested using MTT colorimetric
method [31] which measures a purple formazan compound produced in living cells only. Briefly, 5x104
sk-OV-3 cells/100 ml were cultured at 37oC and 5%
CO2 humidified atmosphere, in complete RPMI-1640
medium (supplemented with 10% FCs, 10 IU/ml
sodium penicillin-G, 10 mg/ml streptomycin sulphate
and 2 mM L-glutamine), for 3-4 hrs in flat-bottomed
96-well tissue culture plates, for adherence. Then the
cells were treated with Doxorubicin (0.5 mg, 1 mg),
Cyclophosphamide (0.5 mg, 1 mg), or a combination
of 5 mg MCs-Ab21 and each cytostatic drug (used in
the concentations mentined above). each sample was
tested in triplicate. After 24 hrs at 37oC and 5% CO2,
the plates were centrifuged at 800 g, 5 min, and the
supernatants were taken out. 50 ml/well MTT (3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (sigma), 1 mg/ml in saline solution, were added
to each well. After 4 hrs incubation at 37oC and 5%
CO2, the plates were centrifuged 5 min at 800 g and
the supernatants were removed. The reaction was
stopped with 50 ml/well 10% sodium Dodecyl sulfate solution (Bio-Rad). The plates were kept overnight at 37oC and then the absorbance was measured
at 540 nm, using an automated spectrophotometer
(Thermo Multiskan eX). Average values for triplicate
samples were calculated.
Cecal ligation and puncture method
Cecal ligation and puncture (CLP) in rodents
became the most widely used experimental sespis
model, currently considered the standard for sepsis
research. Developed in more than 30 years, the model is considered specific for the polymicrobial induced sepsis in experimental studies on the intimate
mechanisms of sepsis. CLP consists in cecal ligation
below the ileocecal valve after a median laparotomy,
followed by cecal puncture with a needle. since the

cecum is an internal source of bacteria, the puncture
leads to a bacterial peritonitis, as bacteria are moving
from enteric space to the blood flow. Bacteremia activates the inflammatory response, followed by septic
shock with multiple organ failure and death [32,33].
The CLP model in rodents is performed to obtain specific clinical signs of sepsis or septic shock, such as
hypothermia, tachycardia and tachypnea. It is imperative that CLP model should be strictly developed, as
the inflammatory response and survival rates may
vary depending on the severity of septic status. The
CLP must be run as a standard and repeatable protocol to reduce the number of factors influencing the
results. standardization of cecal ligation length is an
argument to establish the reliability and reproducibility of the experimental sepsis severity.
In our experimental protocol, we established standardized procedures to induce acute sepsis in mice
according to CLP model. We used male C57BL/6
mice, 19-21 g weight, bred in the Cantacuzino
NIRDMI sPF Biobase. The mice were hosted in small
groups, in adequate cages. Firstly, the animals were
weighted, to determine the amount of anesthetics to
be used. As anesthetic, CP-ketamine 10%, 100 mg per
kg body weight, intramuscular was used. The anesthetized animal was positioned in dorsal decubitus,
with the legs close to operator and the shaved area
was disinfected with Betadine (100 mg/ml). A midline
incision of the skin was made carefully, without
opening the peritoneal cavity. Then, the incision was
extended and the peritoneal cavity was opened after
white line (linea alba) identification and the intermuscular space dissection. Only the cecum, most frequently located on the left side, was brought to outside the abdominal cavity. Damaging the mesenterial
blood vessels was avoided. The cecal ligation included
about 75% of its length, the ileocecal valve was avoided to preserve the intestinal transit. The experiment
was carried out at the same time of the day.
The cecum was diametrically single punctured
between the ligation and his end, avoiding blood vessels damage. After removing the needle, a small
amount of faeces was squeezed out through penetration holes. The cecum was relocated into the abdominal cavity avoiding spreading faeces onto the surgical incision edge. The abdominal wall was closed in
layers. The animals received prewarmed normal sterile
saline solution (at 37oC, 5 ml per 100 g body weight)
for fluid resuscitation and were placed immediately
back in cages in a temperature-controlled (22oC) and
humidity-controlled (50-60%) room, with 12 hrs light
and dark cycles, with access to water and food.
Before CLP, the animals were hosted 7 days for

acclimatization, at 22oC temperature, 50-60% humidity, with free access to water and food. each group
was composed of 10 mice:
1st group - positive control PC (without CLP, only surgery) - there should be no mortality;
2nd group - negative control NC (CLP and injected
with normal saline solution, 300 ml/mouse);
rd
3 group - CLP and MCs-Ab21-treated (stock solution
2 mg/ml in normal saline solution, sterile). The
sample was injected in the tail vein, 300 ml
(600 mg) per 20 g body weight: 150 ml (300 mg)
per mouse, 1 hr before CLP, and 150 ml (300 mg)
per mouse, 1 hr after CLP.
The experiment was repeated 3 times using the
protocol described above.
Animal experimental protocols were in accordance with the recommendations of the institutional
animal ethical committee ARsAL - FeLAsA.
Statistical analysis
For eLIsA experiments, the coefficient of variation (CV) was less than 20% and final results represented the average of 3 independent quantifications.
In the case of MTT assay, the CV was less than 20%
and final results represented the average of 3 independent experiments. Differences in survival of the
mice with CLP were analyzed by a kaplan-Meier survival plot and the log-rank statistic.
RESUlTS
The effect of MCs-Ab21 purified fraction on
HMGB1 release both in tumor cells and LPs-activated PBMC cells was investigated by in vitro experimental models. The action of MCs-Ab21 product on
septic shock induced in mice was studied by CLP in
vivo experimental model.
The effect of MCS-Ab21 on HMGB1 release
from SK-oV-3 tumor cells
MCs-Ab21 purified fraction was tested regarding
its effect on HMGB1 release in sk-OV-3 tumor cells,
treated or not with different chemotherapeutic drugs.
The levels of HMGB1 in culture supernatants were
quantified by eLIsA.
The results presented in Fig. 1 showed that MCsAb21 was able to down-regulate the HMGB1 release
from tumor cells. At doses of 5 mg and 10 mg MCsAb21 inhibited HMGB1 production to approximately
50%, comparing with the untreated cells (control).
In another experimental model, 2 combined treatments of MCs-Ab21 (used in unique dose of 5 mg) and
117
APETREI et al.
Fig. 1. The HMGB1 release by SK-oV-3 tumor cells treated with MCS-Ab21.
Cells were treated with different doses of vegetal product (5 and 10 mg). Data
represent the average of 3 parallel experiments
Fig. 2. The HMGB1 release by SK-oV-3 tumor cells treated with Doxorubicin (10 and 20 mg),
Cyclophosphamide (10 and 20 mg) and a combined treatment of MCS-Ab21 (5 mg) and each cytostatic drug (10 and 20 mg). Data represent the average of 3 parallel experiments
Doxorubicin (10 mg and 20 mg), or MCs-Ab21 (5 mg)
and Cyclophosphamide (10 mg and 20 mg) were
applied. The tumor cells were also treated with the
cytostatic drugs alone. The results obtained are presented in Fig. 2.
We also investigated the effect of cytostatics
alone and in combination with MCs-Ab21 extract,
respectively, on the viability of sk-OV-3 tumor cells,
using MTT assay. The results are presented in Fig. 3.
MCs-Ab21 (5 mg) applied together with Doxorubicin (0.5 mg) potentiated HMGB1 release, without
significantly modifying cell viability. When a higher
dose of Doxorubicin was used (1 mg), MCs-Ab21 significantly influenced neither HMGB1 release, nor
cell viability. Comparatively with the effect of 0.5 mg
Doxorubicin alone, association with MCs-Ab21
determined a slow decrease in cell viability. The
118
combination of 1 mg Doxorubicin with MCs-Ab21

determined an increase in the viability of the cells,
compared with Doxorubicin alone.
The cells treatment with 0.5 mg Cyclophosphamide induced a 91% viability of sk-OV-3 cells. At 1 mg,
Cyclophosphamide had no effect on the viability of
tumor cells. MCs-Ab21 (5 mg) applied together with
Cyclophosphamide (0.5 mg), slowly decreased HMGB1
release and did not significantly modify cell viability.
When the Cyclophosphamide dose was 1 mg, the
decrease of HMGB1 release was observed, as well as
the cell viability.
The effect of MCS-Ab21 on HMGB1 release
from lPS-activated PBMCs
MCs-Ab21 purified vegetal fraction was studied
in vitro regarding a possible modulatory activity of
Fig. 3. The effect of Doxorubicin (0.5 mg and 1 mg), Cyclophosphamide (0.5 mg and 1 mg) and combined treatment of MCS-Ab21 (5 mg) and each cytostatic drug (0.5 mg and 1 mg) on SK-oV-3 tumor
cells. Data represent the average of 3 parallel experiments
Fig. 4. The effect of MCS-Ab21 vegetal fraction on the production of HMGB1 by lPS-treated
PBMCs. Cells were incubated with different doses of purified product (5, 10 and 25 mg) and then
treated with lPS (1 mg). Data represent the average for 5 healthy donors
extracellular secretion of HMGB1 from LPs-activated
PBMCs, isolated from the periferal blood of healthy
donors. The levels of HMGB1 in culture supernatants
treated with different doses of MCs-Ab21 were quantified by eLIsA. The results obtained are presented in
Fig. 4.
MCs-Ab21 product was also tested regarding the
effect on the viability of PBMC cells and the results
obtained using the MTT assay are presented in Fig. 5.
Taken together, the results presented in Figs 4
and 5 demontrated that MCs-Ab21 5 mg + LPs 1 mg
strongly decreased HMGB1 release and induced a
slow cell viability increase. In cells treated with LPs,
HMGB1 release increased as increasing dose of
MCs-Ab21, while maintaining however under the
control of MCs-Ab21 (10 mg) and correlating with the

decrease of cell viability by comparing with MCsAb21 5 mg + LPs 1 mg. Cell death could be accompanied by cytoplasmic HMGB1 release, so in culture
supernatants a higher concentration of HMGB1
could be identified.
The effect of MCS-Ab21 on mice with septic
shock induced by ClP
Using an in vivo experimental model, we also
tested the possible inhibitory effect of MCs-Ab21
product on septic shock induced by CLP method in
mice. After the surgical intervention and the treatment with the vegetal product, the mice were continously observed during the first 6 hrs and every 12
119
APETREI et al.
Fig. 5. The effect of MCS-Ab21 vegetal fraction on the viability of lPS-treated PBMC cells.
Cells were incubated with different doses of MCS-Ab21 (5, 10 and 25 mg) and then treated
with lPS (1 mg). Data represent the average for 5 healthy donors
hrs, for the next 5 days. The objective pursued in this
mouse died 96 hrs after CLP. The clinical status
decreased progressively, with the modification of the
experiment was the effects on the survival time. The
characteristics of the resting position, with absence of
results are presented in Tables 1 and 2.
attention for alimentation, water and selfcare, pallor
The survival rate for the animals involved in CLP
of the extremities and hypotermia, lethargic status
experiments is presented in Fig. 6.
with exitus.
survivors. Inobserved
the group of
In
the
positive
control
group
(PC),
all
animals
intervention and the treatment with the vegetal product,
theThere
mice were
werenocontinously
withThe
MCs-Ab21
600 mg/20
g body
survived.
the first
first 36-48
the surgical
- the mice
duringIn the
6 hrs hrs
andafter
every
12 hrs, infor
nexttreated
5 days.
objective
pursued
in weight,
this
tervention,
the
clinical
status
was
altered.
After
that,
deaths
occured
after
36
hrs,
with
the
last
recorded
interventionwas
andthe
theeffects
treatment
with
the vegetal
product,
theare
mice
were continously
observed
experiment
on the
survival
time. The
results
presented
in Tables 1 and
2.
an improvement
regarding
nutritional
andhrs,
motor
48 hrs.
animals pursued
presented in
impaired
during the first
6 hrstheand
every 12
for the death
next after
5 days.
TheAll
objective
this
status was observed. In the negative control group
general condition with the modification of the speciexperiment was the effects
thedistribution
survival time.
Thecases
results
presented
Table 1.onThe
of the
onare
mice
groups in Tables 1 and 2.
(NC), the death hapenned after 24 hrs, and the last
fic rest position, horripilation, lack of food, water and
Table 1. The distribution of the cases on mice groups

Survivors
Mice group Table 1. The distribution of the cases on mice groups
Total
No. of deaths
No.
%
Survivors
Mice group
MCS-Ab21
15
10
5
333%
Total
No. of deaths
No.
%
PC
5
0
5
1000%
MCS-Ab21
15
10
5
333%
NC
14
14
0
0%
PC
5
0
5
1000%
Total
34
24
10
294%
NC
14
14
0
0%
Total
34
24 CLP experiments.
10
294%
Tabel 2. Statistical
analysis for
Table 2. Statistical analysis for ClP experiments
Tabel 2. Statistical
analysis for
CLP experiments.
The log rank test (Mantel-Cox)
is a nonparametric
method
for testing treatment differences in
The log rank test (Mantel-Cox) is a nonparametric method for testing treatment differences in sursurvival between two treatment groups. Breslow (Generalized Wilcoxon) is a nonparametric
vival between two treatment groups. Breslow (Generalized Wilcoxon) is a nonparametric method
The
log for
rankcomparing
test (Mantel-Cox)
is a nonparametric
method for testing
in
method
two survival
distributions.Tarone-Ware
test istreatment
used to differences
compare the
for comparing two survival distributions.Tarone-Ware test is used to compare the overall survival
survival
between
two
treatment
groups.
Breslow
(Generalized
Wilcoxon)
is
a
nonparametric
overall
survival
rates for of
each
covariates of interest.
rates for
each covariates
interest.
method for comparing two survival distributions.Tarone-Ware test is used to compare the
overall survival rates for each covariates of interest.
Degrees
Chi2
Significance
of freedom (df)
Degrees
Log Rank (Mantel-Cox)
14179
2
0001
Significance
Chi2
of freedom (df)
Breslow (Generalized Wilcoxon)
10852
2
0004
Log Rank (Mantel-Cox)
14179
2
0001
Tarone-Ware
12560
2
0002
Breslow (Generalized Wilcoxon)
10852
2
0004
Tarone-Ware
The survival rate
for the animals involved in12560
CLP experiments is2 presented in Fig.0002
6.
120
The survival rate for the animals involved in CLP experiments is presented in Fig. 6.
Legend
PC
NC
MCS-Ab21
Fig. 6. Survival rates after ClP in mice (PC positive control, NC - negative control).
PC group (mice without ClP, only surgery); NC group (mice with ClP and injected with
normal saline solution); MCS-Ab21 group (mice with ClP and treated with MCS-Ab21,
600 mg/20g body weight). Data represent the average of 3 parallel experiments
selfcare interest, pallor of the extremites and
hypothermia in the first 48 hrs. some of them showed
an improvement regarding the behavioral and nutritional status, and survived the observational period.
All these results demonstrated that MCs-Ab21
purified extract (600 mg / 20 g body weight) increased
the mice survival rates (33.3%), by comparing with the
mice with CLP and without treatment with MCs-Ab21.
DISCUSSIoN
In our study, we investigated the effect of MCsAb21, a purified fraction isolated from Helleborus purpurascens, on the modulation of HMGB1 release from
both sk-OV-3 tumor cells and LPs-activated PBMC
cells (in vitro studies). We also studied the action of
MCs-Ab21 on septic shock induced in mice by cecal
ligation and perforation method (in vivo study).
The action of MCs-Ab21 vegetal fraction on the
release of HMGB1 from tumor cells and human
PBMC cells was quantified by eLIsA technique.
MCs-Ab21 applied in doses of 5 mg and 10 mg
was able to decrease significantly the release of
HMGB1 from sk-OV-3 tumor cells (3.6 ng/ml and
3.5 ng/ml respectively, comparing with 6.8 ng/ml for
the control). By combined application of MCs-Ab21
5 mg and different cytostatic drugs (Doxorubicin or
Cyclophosphamide), the results indicated that the
association of MCs-Ab21 (5 mg) with Cyclophosphamide (10 mg and 20 mg) significantly decreased the
release of HMGB1 by tumor cells (0.26 ng/ml and

1.27 ng/ml respectively, comparing with 6.8 ng/ml
for the control). This effect was not observed in the
case of a combined treatment of MCs-Ab21 and
Doxorubicin.
Our results revealed that in the case of human
PBMC activated with LPs, MCs-Ab21 only for a dose
of 5 mg could down-regulate HMGB1 release; for
higher doses of MCs-Ab21, HMGB1 release in culture supernatants increased and correlated with the
decrease of cells viability. This effect could be due to
a cytoplasmic HMGB1 release, followed by the increase of extracellular HMGB1 concentration.
In the experimental CLP model of sepsis induced
in C57BL/6 mice, the animals were treated with
MCs-Ab21 and the survival rate was monitored compared with the control animals. Our results showed
that the survival rate of the mice treated with MCsAb21 increased (33.3%), in comparison with the survival of the mice not treated with MCs-Ab21 (0% survivors).
The data disclosed here support the assumption
that MCs-Ab21 5 mg/ml can modulate inflammation
efficiently by interfering with the proinflammatory
cytokine HMGB1 mechanism which can also explain
the attenuation of septic shock in mice.
These findings suggest further work to disclose
the composition and structure of the complex MCs
substances, to be applied as potential anti-inflammatory agents in cancer and/or sepsis.
121
APETREI et al.
ACKNoWlEDGMENTS
This study was supported by the National Research Program PN II, Grant No. 62052/2008.
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123
Is INTeRLeUkIN -17 A PROATHeROGeNIC BIOMARkeR?

Cristina-Sorina Ctan1*, Victor Cristea1, Nicolae Miron1, Ioana Berindan Neagoe1,2
1iuliu
2ion
Haieganu University of Medicine and pharmacy, cluj-Napoca, romnia;

chiricu cancer institute - comprehensive cancer center, cluj-Napoca, romnia
ABsTRACT
The importance of chronic inflammation in atherogenesis and cytokine involvement in all stages
of atherosclerotic plaque development is now obvious. Our approach of the significant cytokines
involved in atherogenesis or cardiovascular diseases is based on a correlation between clinical
research and experiments on animal models. The contribution of IL-17 in atherogenesis remains
controversial.
In this study we investigated the role of IL-17 in cardiovascular diseases and in atherosclerosis associated with pathological aging. We performed a case-control study, enrolling subjects aged over 65
years in both groups. We included 40 patients with cardiovascular disorders and 10 healthy volunteers. IL-17 levels were measured in the serum of patients and healthy controls, along with
serum total cholesterol and triglycerides.
significantly higher levels of IL-17 were obtained in patients compared to healthy controls
(p<0.001). The level of this biomarker correlated significantly with two biochemical parameters serum total cholesterol and triglycerides (the Pearson coefficient showed statistical significance,
p=0.033, respectively p=0.043). We did not find any correlation between IL-17 and these two
parameters in the control group.
Our study is useful in understanding the physiopathological implications of IL-17 in the atherogenesis process. This could represent a starting point for future studies, including research regarding
the therapeutic potential of IL-17 in pathological aging.
Keywords: cytokines, aging, biomarker, atherosclerosis, IL-17
INTRoDUCTIoN
Atherosclerosis, already reaching an epidemic
proportion, is a progressive disease characterized by
the formation of a plaque consisting of cholesterol,
other lipids, connective-tissue elements and debris
from cellular death (immune or non-immune cells),
in the innermost layer of the artery (the intima) of
medium muscular arteries and large elastic arteries.
Clinical studies have revealed risk factors for this
multifactorial disorder: age, hypercholesterolemia,
genetic predisposition, gender, smoking, hypertension, sedentary life, and other factors [1].
For over a decade, atherosclerosis has been regarded as a chronic inflammatory disease characterized by migration of monocytes and T lymphocytes
to the area of arterial wall injury. Inflammation controls the development and the destabilization of arterial plaque. Adhesion of monocytes and lymphocytes
to the activated endothelial cells and immune mediation are nowadays sustained by numerous animal
experiments and clinical studies. In this inflammatory
disease, immune mechanisms interact with metabolic
risk factors, contributing to the initiation, progression
and activation of lesions in the arterial tree [2].
Atherosclerosis is a dynamic process and the
artery wall is an active site involved in chronic inflammation, innate and adaptive immunity, and production of cytokines, chemokines and growth factors.
Cells (endothelial cells, smooth muscle cells, macrophages, T-lymphocytes, mast cells) involved in formation, developing or progression of atherosclerotic
lesions, are rich sources of cytokines production,
which concur in initiation, maintenance and amplification of inflammation events [3].
The local and systemic effects of cytokines in
atherosclerosis are: oxidation of LDL (low density
lipoprotein)-cholesterol; induction of chemokine
*corresponding author: Cristina-sorina Ctan, Iuliu Haieganu University of Medicine and Pharmacy, Department of Biochemistry,
6 Pasteur street, Cluj-Napoca 400349, Romnia; Phone: 004 0744 450723; Fax: 004 0264 280602; e-mail: kristymed@yahoo.com
124
Il-17 a proatherogenic biomarker?
release; altered endothelial permeability; modulation

of endothelial pro-coagulant activity; induction of
sMCs (vascular smooth muscle cells) migration/proliferation; sMC progenitor differentiation; up-regulation/down-regulation of endothelial-dependent vascular relaxation (through effects of IL-10 and IFN-g on
nitric-oxide synthase-3); activation of adhesion molecule expression; modulation of scavenger receptor
expression; stimulation of MMP (matrix metalloproteinase) expression; stimulation of microparticle
release; modulation of extracellular matrix expression); regulation of neovessel formation; promotion
of intraplaque neovascularization; induction of apoptosis [4].
in vitro studies suggest that IL-17A, the prototypic
family member, activates vascular endothelial cells,
which secrete cytokines that in turn enhance foam
cell formation in macrophages. IL-17 plays an important role in both diet-induced atherosclerotic lesion
development and chlamydia pneumoniae infectionmediated acceleration of atherosclerotic lesions in
the presence of high-fat diet [5]. Other data demonstrate that atherosclerosis-prone conditions induce
the differentiation of IL-17Aproducing T cells. IL-17A
plays a proatherogenic inflammatory role during
atherogenesis by promoting monocyte/macrophage
recruitment into the aortic wall [6].
In this study we analyzed whether IL-17 could
be used as a marker for age-related cardiovascular
disorders, in conjunction with two biochemical parameters: serum cholesterol and triglycerides.
SUBJECTS AND METHoDS
We conducted our study after a case-control
design. The study protocol was approved by the ethics
Committee of Iuliu Haieganu University of Medicine and Pharmacy Cluj Napoca, (according to the
Declaration of Helsinki regarding clinical studies).
Study group I: we included 40 patients with cardiovascular disorders (26 men and 14 women), with
a median age of 73 years (range 66-89).
Control Group II (n=10 healthy volunteers).
We excluded the subjects with coronary disease (history of myocardial infarction, stable and unstable angina, coronary angioplasty or bypass surgery), with
equivalents of coronary risk (diabetes mellitus, peripheral arterial disease, abdominal aortic aneurysm),
with hypertension (blood pressure, BP140/90 mm
Hg or anti-hypertensive therapy), hyperlipidemia (total cholesterol (TC) 240 mg/dl, triglycerides (TG)
200mg/dl). serum levels of TC and TG were determined using standard procedures.
Il-17 measurement
Venous blood samples were collected into 50
tubes without anticoagulant substance from subjects
( jeun, free of acute, inflammatory diseases) The
whole blood (4 ml) was centrifuged for 10 minutes at
3,000 rpm and serum was removed. The quantitative
determination of human IL-17 concentration in
serum was done using a high-sensitivity enzymelinked immunosorbent assay (eLIsA), with the
Quantikine commercially-available kit (R&D
systems). This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for human IL-17 has been precoated onto a microplate. standards, Control, and
samples are pipetted into the wells and human IL-17
molecules from the sample are bound by the immobilized antibody. After washing away any unbound
substances, an enzyme-linked polyclonal antibody
specific for human IL-17 is added to the wells.
Following a wash to remove any unbound antibodyenzyme reagent, a substrate solution is added to the
wells. The enzyme reaction yields a blue product that
turns yellow when the stop solution is added. The
intensity of the color measured is proportional to the
amount of human IL-17 bound in the initial step. The
sample values are then read off the standard curve.
Statistical analysis
The results were analyzed with the students t test
for unpaired groups. All analyses were performed
using the sPss software. Differences were considered
statistically significant if p < 0.05. Because Levenes
test for equality of variances revealed statistically significant differences (p=0.021) of variances between
patients and the control group, we used the t test for
equality of means.
RESUlTS
In the present study we evaluated the involvement of IL-17 in cardiovascular diseases using two
groups: patients group including 40 patients with cardiovascular diseases and a control group including
10 members; subjects in both groups were aged over
65 years. We measured the IL-17, total cholesterol
and triglycerides concentrations in serum. The results
obtained from the quantitative determination of
human IL-17 concentrations in serum are presented
in Fig.1.
significantly higher levels of IL-17 were obtained
in patients, compared to healthy controls (p<0.001).
IL-17 levels correlated significantly with serum values
125
CTAN et al.
of total cholesterol and triglycerides (TG) in the patient group. We found a moderate correlation between IL-17 level and serum cholesterol (Chol). The
Pearson coefficient showed statistical significance
(p=0.033). There was also a moderate correlation
within the same group, between the IL-17 level and
that of serum triglycerides (p=0.043). The results are
presented in Table 1 and Table 2.
Conversely, there was no correlation between
the serum concentration of IL-17 and total cholesterol
and triglycerides
controlCorrelation
group.
IL-17 in thePearson
DISCUSSIoN
elevated concentrations of IL-17 are associated
with the physiopathology of numerous inflammatory
and autoimmune diseases. IL-17 has been detected in
lesions of multiple sclerosis and is believed to have a
role in altering the blood brain barrier [7, 8].
Higher amounts of IL-17 and Th17 cells were
found in the synovium of rheumatoid arthritis
patients, compared to healthy subjects [9]. High
serum levels of both Il-17 and IL-23 were detected in
IL-17
TG
ulcerative
disease [10, 11].
1 colitis and Crohns
.322(*)
Sig. (2-tailed)
0.043
Table 1. Correlation between Il-17 and TG
N
40
IL-17
TG
TG
Pearson Correlation
.322(*)
1
IL-17
Pearson Correlation
1
.322(*)
Sig. (2-tailed)
.043
Sig. (2-tailed)
0.043
N
40
40
N
40
Table
1. Correlation
between IL-17 and TG. .322(*)
TG
Pearson Correlation
1
Correlation
is
significant
at the 0.05 level (2-tailed).
Sig. (2-tailed)
.043 N: number of patients, IL-17:
interleukin-17,
Sig: significance, TG: triglycerides
N
40
40
correlation
is significant
the 0.05 level (2-tailed).
Table 1. Correlation
between
IL-17 and atTG.
N: number of patients, il-17: interleukin-17, Sig: significance, tG: triglycerides
Correlation is significant at the 0.05 level (2-tailed). N: number of patients, IL-17:
interleukin-17, Sig:Table
significance,
TG: triglycerides
2. Correlation
between Il-17 and CHol
IL-17
CHOL
Pearson Correlation
1
.338(*)
Sig. (2-tailed)
0.033
N
40
40
IL-17
CHOL
CHOL
Pearson Correlation
.338(*)
1
IL-17
Pearson Correlation
1
.338(*)
Sig. (2-tailed)
.033
Sig. (2-tailed)
0.033
N
40
40
N
40
40
level (2-tailed).
CHOL
Pearson correlation
Correlation is significant at the 0.05
.338(*)
1
N: number of patients, il-17: interleukin-17, Sig: significance, cHol: cholesterol
Sig. (2-tailed) Table 2. Correlation between
.033 IL-17 and CHOL.
N
40
40patients, IL-17:
Correlation is significant at the 0.05 level (2-tailed).
N: number of
IL-17
126
interleukin-17, Sig: significance, CHOL: cholesterol

Table 2. Correlation between IL-17 and CHOL.
Correlation is significant at the 0.05 level (2-tailed). N: number of patients, IL-17:
Il-17 a proatherogenic biomarker?
It was showed that IL-17 plays an important role

in the development of diet-induced atherosclerotic
lesions [5]. Other data demonstrate that IL-17A plays
a pro-atherogenic inflammatory role during atherogenesis, by promoting monocyte/macrophage recruitment into the aortic wall [6].
Long-term sleep restriction has been shown to
determine persistent changes in the immune system:
lymphocyte activation and production of proinflammatory cytokines, such as: IL-1b, IL-6, and IL-17,
increased production of IL-17 together with CRP (Creactive protein), thus increasing the risk of developing
cardiovascular diseases (atherosclerosis and stroke)
[12]. It has recently been reported that IL-17 stimulates the expression of CRP in hepatocytes [12] and in
coronary artery smooth muscle cells [13], and that
CRP facilitates the formation of atherosclerotic
plaques, leading to an increased risk for cardiovascular diseases [12].
Novel therapeutic strategies include targeting
the inflammatory mediators. There are higher levels
of IL-17 expression in the target tissues of eAe (experimental autoimmune encephalomyelitis) animal
models. Therapeutic neutralization of IL-17 with the
IL-17-receptor-Fc (fusion construction)-hybrid protein
in eAe, has lead to an improvement of clinical symptoms. Neutralization of IL-17 with a monoclonal antibody also improved the disease course [14].
Th17 cells play a crucial role in mounting an
immune response against both intracellular and
extracellular pathogens. IL-1b in combination with
IL-23 induces Th17 differentiation [15, 16]. The Il-23IL-17 axis is required for the immune response
against gram-negative bacteria including Klebsiella
pneumoniae. IL-23 has proved important for IL-17
production and could function to expand antigenprimed IL-17-expressing T cells [17-19].
such inhibitors can act on pathways upstream of
IL-17 or on other cytokines in addition to IL-17. since
IL-23 is involved in IL-17 production, the inhibition
of IL-23 is a way to control the Th-17 pathway by acting upstream. Treatment with a monoclonal antibody
against p40, which is common to IL-12 and IL-23, has
shown efficacy in psoriasis and Crohns disease. specific inhibitors of IL-23 remain to be compared with
the p40 inhibitors [20-22].
Other cytokines have anti IL-17 properties, too:
IL-4, TNF, IL-1, IL-6 [23]. IL-25/IL-17e is a member of
the IL-17 family [24], with anti-inflammatory properties on Th-17 cells [25].
Recent evidence indicates that simvastatin, a
clinically utilized cholesterol-lowering agent, does
attenuate the expression of IL-6 and IL-23 and of the
gene encoding IL-17 in activated CD4 cells, resulting

in inhibition of IL-17 secretion [26]. Another cholesterol-lowering agent, widely used to treat hyperlipidaemia, is pravastatin. Its immunosuppressive effect
has been confirmed in various immune-mediated
models [27].
CoNClUSIoNS
similarities of atherosclerosis and other chronic
inflammation diseases (cirrhosis, rheumatoid arthritis,
glomerulosclerosis, pulmonary fibrosis and chronic
pancreatitis) were observed [28], therefore inflammatory responses have almost the same pattern in arteries and other tissues. Generally, the value of inflammatory markers as independent predictors is controversial; therefore we must interpret their variation in
the context of clinical data.
Beside other molecules/biomarkers, IL-17 may
contribute to atherogenesis by promoting monocyte/
macrophage recruitment into the aortic wall, by stimulating the expression of CRP in hepatocytes and in
coronary artery smooth muscle cells, which facilitate
the formation of atherosclerotic plaques leading to an
increased risk for cardiovascular diseases. A large
number of novel therapies have been developed or
are in experimental phases, but not all are benefic for
humans: anti-cytokine (TNF-a) therapies - treatment
with Infliximab results in worsening of cardiac function; etanercept (anti-TNF-a) therapy leads to a paradoxical, rapid elevation of TNF-a levels and worsening heart failure; so, in general, antagonizing of
cytokines in heart failure is a wrong strategy. Treatment with cyclooxygenase-2 inhibitors (Rofecoxib Vioxx) revealed an increased incidence of cardio-vascular events [29, 30]. There are a lot of questions to
ask, such as: is IL-17 specific for atherosclerosis? Does
this biomarker independently predict risk beyond
conventional tools? Is atherosclerosis a local or a systemic disease? Do therapies that decrease plasma levels of inflammatory and lipid markers also reduce cardiovascular risk? Can we place an equal sign between
findings in animal and human atherosclerosis?
However, understanding of the mechanisms of
IL-17 effects in atherogenesis is still in its infancy, and
therefore the specific role of this molecule as biomarker for the development of cardiovascular diseases
needs to be studied in more detail.
127
CTAN et al.
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HePATITIs B, C AND D COINFeCTION IN HIV-INFeCTeD PATIeNTs:

PReVALeNCe AND PROGRess
Bogdan Ionescu* and Grigore Mihescu
University of bucharest, Faculty of biology, Microbiology and immunology Department, bucharest, romania
ABsTRACT
The HAART therapy has improved life expectancy enabling long latency conditions caused by the
hepatitis viruses that became the leading cause of death in HIV infected patients.
In this study a group of 300 patients aged from 18 to 63 years were selected in order to assess the
prevalence and consequences of HIV and the hepatitis B (HBV), C (HCV) and D (HDV) viruses
coinfections. study groups were designed for each coinfection. These groups were in turn divided
in case groups formed of coinfected participants and control groups consisting of mono-infected
participants.
This classification was obtained by testing the participants for the presence of specific infection
markers using the eLIsA technique. As a result, in regard to the HIV/HBV coinfection the study
group consisted of 16 coinfected participants and 114 HBV-infected participants resulting in a
prevalence of the coinfection of 14%. In the case of the HIV/HDV coinfection the study group consisted of 5 coinfected participants and 45 HDV-infected participants. The prevalence of the
HIV/HCV coinfection was 25% out of the 170 HCV-infected participants.
The effect of the coinfections on the expression and levels of the infection markers was analyzed
in constrast to those encountered in the case of the mono-infection. The observed changes in the
expression of the specific hepatitis markers indicate the impact of the coinfection with HIV on the
progression of the hepatitis infections. In addition, the inadequate immune response towards the
hepatitis viruses in the case of the coinfected participants leads to the development of cirrhosis and
end stage liver disease.
Keywords: HAART, HIV, HBV, HCV, HDV, coinfection, eLIsA
INTRoDUCTIoN
A total of 370 million people around the world
are estimated to be infected with HBV, 170 milion
with HCV, 15 milion with HDV and 40 million with
HIV. Many of the countries affected by hepatitis
viruses are also characterized by a high HIV burden
because of the similar routes of transmission that
have lead to an estimate of 4-5 million coinfected
individuals around the world [1, 2, 3, 4]. Furthermore, studies were conducted on the risk factors
associated with the acquisition of hepatitis viruses
and HIV in order to better understand the clinical picture of the respective coinfections [5].
The introduction of the highly active antiretroviral treatment (HAART) has enabled conditions such
as chronic viral hepatitis and end-stage liver disease
to emerge as major health concerns. studies conducted on the effects of HIV on hepatitis infections
have shown that the rate of progression and complications from viral hepatitis are accelerated in patients
with HIV coinfection [6, 7].
For the purpose of finding a suitable therapy for
the emerging health concern represented by these
coinfections, more and more studies are focusing on
developing treatments and analyzing the side-effects
exhibited by the existing ones [8-12].
According to the statistics provided by the
National Program of Healthcare, Romania is considered the country with the highest prevalence of
chronic hepatitis C in the european Union (4.9%)
and with one of the highest prevalence of hepatitis B
infection (6%) [13].
In Romania aproximatively 1.5-2 milion people
are infected with at least one type of hepatitis virus
and only 10% are diagnosed.
*corresponding author: Bogdan Ionescu, e-mail: bogdanionescu87@yahoo.com, tel.: 0728122746
129
IoNESCU and MIHESCU
The purpose of this study was to determine the

prevalence and progress of hepatitis viruses coinfections in HIV-infected patients.
Study group and samples
The study was performed over a duration of 4
months (January- April 2011) on 300 participants, out
of which 130 were HBV positive, 50 HDV positive
and 170 HCV positive. Three study groups consisting
of participants positive for each hepatitis viral infection were formed. In order to observe the HIV and
hepatitis mono-infections and the effect of the coinfections on their progress, comparisons between the
hepatitis infections and the HIV infection have been
made [Tables 1, 2]. The study groups were later divided
into case groups and control groups for each coinfection here discussed. The case groups consisted of HIVcoinfected participants and the control groups consisted of participants suffering only from viral hepatitis.
The mono-infections were diagnosed by determining the presence of the viral hepatitis infection

markers.
In case of the hepatitis B virus the specific

diagnosis
markers were HBsAg, HbeAg, HbsAb and
HBeAb. A classification of hepatitis B infections was
made based on the presence of specific markers for

each type of infection [Table 3].
The screening of the hepatitis C infection was focused on the HCVAb and in case of hepatitis D virus,
on HDVAg and the HDVAb detection. Concerning
the HDV, the existence of one of the two investigated
markers ensured the presence of the infection.
The groups were also tested for the presence of
the HIV infection confirmed by the existence of
HIVAb.
Biomarkers assessment by ElISA technique
The results of this study were obtained by using
the General Biological, Diasorin and Biorad eLIsA
kits. eLIsA method is used to detect the presence of
antigens or antibodies in the serum or plasma. The
principle of the eLIsA technique is based on the interaction between antibodies and antigens in vitro. The
Ag-Ab complexes formed as a result of these interactions are detected using a colorimetric reaction catalized by an enzyme which transforms the coluorless
substrate into an colored compound, whose optical
density will be equivalent to the concentration of the
Ag or Ab in the serum or plasma. The participants in
the study were screened to determine the presence of
the specific infection markers that allowed the design
Table 1. Comparison between specific characteristics for HIV and HBV mono-infections
Table
1. Comparison between specific characteristics for HIV and HBV mono-infections
Table 1. Comparison between specific characteristics for HIV and HBV mono-infections
HIV
HIV
Discovery year of the virus
1983
Discovery
year of affected
the virus
1983
Number
of people
40
million
Number of people affected
40 million
GenomHRNA
GenomHRNA
Cellular
target
CD4 cells
Cellular
target
CD4 cells
Main sources of transmission
Sexual
contact
Mainofsources
of transmission
Sexual contact
Rate
chronicization
100%
Rate of chronicization
100%
Asymptomatic period
10 years
Asymptomatic
period
10 years
Indicators of outcome
CD4
cell count and viral load
Indicators
of
outcome
CD4
Treatment
Antivirals
Treatment
Antivirals
HBV
HBV
1942
1942
350
million
350 million
'NA
'NA
Hepatocytes
Hepatocytes
Parenteral
Parenteral
17.5
%
17.5 %
180 days
180 days
Hepatic
fibrosis
Hepatic
fibrosis
Immunomodulators
Immunomodulators
Table 2. Comparison between specific characteristics for HIV and HCV mono-infections
Table
Comparison between
between specific
for for
HIVHIV
and HCV
mono-infections
Table
2. 2.
Comparison
specificcharacteristics
characteristics
and HCV
mono-infections
Discovery year of the virus
Discovery
year of the
virus
Number
of infected
people
Number of infected people
Genetic material
Genetictarget
material
Cellular
Cellular
targetof transmission
Main
sources
Main
sources
of transmission
Asymptomatic period
Asymptomatic
Predictors
of theperiod
outcome
Predictors of the outcome
Treatment
Treatment
130
Type of hepatitis B
Typeinfection
of hepatitis B
infection
Chronic hepatitis B
HIV
HIV
1983
1983
40
million
40 million
RNA
RNAcells and T lymphocytes
CD4
CD4 cells
and T lymphocytes
Sexual
contact
Sexual
contact
100%
100%
10 years
10 years
CD4
CD4 cell count and viral load
Antivirals
Antivirals
HCV
HCV
1969
1969
170
million
170 million
RNA
RNA
Hepatocytes
Hepatocytes
Parenteral
Parenteral
76%
76%
30 years
30
yearsfibrosis
Hepatic
Hepatic fibrosis
Immunomodulators
Immunomodulators
Table 3. Classification of hepatitis B infection

Table 3. Classification of hepatitis B infection
HBs Ag
HBs Ag
HBs Ab
HBs Ab
HBe Ag
HBe Ag
HBe Ab
HBe Ab
HBc Ab
HBc Ab
Main sources of transmission

Sexual contact
Parenteral
100%
76%
Asymptomatic period
10 years
30 years
Predictors of the outcome
CD4 cell count and viral load
Hepatic fibrosis
Hepatitis B, C and D coinfection in HIV-infected patients: prevalence and progress
Treatment
Antivirals
Immunomodulators
Table
3. Classificationof
of hepatitis
hepatitis BBinfection
Table
3. Classification
infection
Type of hepatitis B
infection
HBs Ag
HBs Ab
HBe Ag
HBe Ab
HBc Ab
Chronic hepatitis B
infection
with HBe Ag present
Chronic hepatitis B
infection
without HBe Ag
Hidden hepatitis B
infection
Carriers of the hepatitis B

virus
1
the study groups for each coinfection consisting of
of
mono-infected individuals and respectively HIV coinfected individuals.
The results obtained by eLIsA method were correlated with those offered in other studies in order to
describe in detail the features of the coinfections.
RESUlTS
HIV/HBV coinfection
The study group in the case of the HIV / HBV
coinfection consisted of 16 coinfected participants and
114 HBV-infected participants. The mean age of the
coinfected participants proved to be around 33 years.
As a result the prevalence of the HIV infection amongst
the HBV study group was assessed to be 14%.
The case group exhibited a higher rate of HBV
multiplication, accelerated loss of anti-HBs, higher levels of HBV DNA and lower ALT levels in comparison to the control group.
HIV/HDV coinfection
The study of the HIV/HDV coinfection was conducted on 5 coinfected participants and 45 HDV infected participants with a mean age of 25 years. The
prevalence of the HDV-HIV coinfection was determined to be 10%.
The 5 case group participants showed a decrease
in the HBV viral load and a higher rate of HbsAg
clearance. A decrease in the expression of the surface
antigen (HBsAg) was also noticed.
HIV/HCV coinfection
The prevalence of the HIV / HCV coinfection
amongst the 170 HCV-infected participants was 24.7%.
The mean age of the participants registered in this
study group was 27 years.
The 42 case group participants registered higher
levels of HCV RNA compared to those observed in
the control group.
DISCUSIoN
Hepatitis infections are seemingly with HIV opportunistic infections, encountered with a high prevalence, and leading to a rapid progress to end stage
liver disease and cirrhosis, which determined them to
become the major cause of death in HIV infected
patients.
In this study we observed the progress and
effects of the HIV-hepatitis viruses coinfections. study
groups were designed for each coinfection consisting
of a case group including coinfected participants and
a control group formed out of participants suffering
from mono-infections with hepatitis viruses.
In the case of the HIV- HBV it was observed that
HIV decreases the clearance rate of the HBsAg and
HBeAg. The degree of immunosuppression associated
with the HIV infection [14] is considered to be responsible for the dysregulation in viral hepatitis markers expression. Other studies have shown that the
rate of surface antigen-negative occult HBV infection seems to be higher in the case of HIV coinfected
patients [15]
131
IoNESCU and MIHESCU
Furthermore, the increased rate of HBV multiplication, accelerated loss of anti-HBs, higher levels of
HBV DNA and lower ALT levels seen in the case of
the coinfected patients indicate a more rapid progress
of the hepatitis B infection towards end stage liver
disease.
The lower ALT levels suggest less hepatocyte destruction caused by the depressed immune response
[16, 17].
studies conducted on the effect of HBV on the
HIV progress have revealed an increased rate of highly
active antiretroviral therapy (HAART)-related hepatotoxicity, a higher level of HIV multiplication and a
lower CD4 cell count caused by the hepatitis B infection [18, 19].
The hepatitis D virus is a defective virus requiring the helper function provided by the hepatitis B
virus in order to multiply. As a result the hepatitis D
infection is encountered only in hepatitis B infected
participants. studies have shown that the HDVHBV
coinfection is estimated to occur in 5% of the patients
positive for HBV [5].
The case group exhibited a lower HBV viral load
and a higher HBsAg clearance. Noteworthy, the
inhibitory effect exhibited by HDV on the DNA polymerase responsible of the HBV multiplication influences the expression of the HBV markers.
Concerning the HIV-HCV coinfection the
incresed levels of HCV RNA were observed in the
case group. In the case of the HCV mono-infection
the multiplication takes place in the hepatocytes but
the number of multiplication sites increases when the
coinfection is present. As a result HCV can multiply
in the lymphoid cells as well, such as macrophages,
CD4 and CD8 lymphocytes. The higher rate of multiplication leads to a massive increase of HCV viral
load following HIV seroconversion [18].
Thus, the progress of the hepatitis infections
seems to be positively influenced by the HIV seroconversion and the high number of multiplication
sites for the hepatitis C virus represented by hepatocytes and lymphoid cells such as macrophages, CD4
and CD8 lymphocytes.
studies focused on the effect of the HAART therapy on hepatitis C progress have revealed an increase
in the level of liver enzymes in approximately 30% of
HIV-HCV coinfected individuals after beginning the
treatment. some anti-HIV drugs such as nevirapine,
ritonavir and stavudine are more likely to cause elevations in liver enzymes [19, 20, 21]. The majority of
studies conducted on this coinfection concluded that
HIV accelerates the progression of HCV-related liver
disease [21, 22, 23].
132
With reference to all the case groups the low CD

4 cell count and alteration of cytokines with specificity towards hepatitis viruses are considered to be
the major causes of the inadequate immune response
to the hepatitis infections.
The observed changes in the expression of the
specific hepatitis markers indicate the impact of the
coinfection with HIV on the progression of the hepatitis infections. In addition, the inadequate immune
response towards the hepatitis viruses in the case of
the coinfected participants leads to the development
of cirrhosis and end stage liver disease.
It is therefore of paramount importance that a
suitable therapy is used in combating this emerging
health threat to HIV positive people. Furthermore,
more in depth research is required to establish the
prevalence of these coinfections at a national scale
in order to prevent them from reaching even higher
levels.
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133
THe GUT MICROBIOTA IN THe MeTAGeNOMICs eRA:

sOMeTIMes A FRIeND, sOMeTIMes A FOe
Daniela Elena erban*
iuliu Haieganu University of Medicine and pharmacy cluj-Napoca, romania
ABsTRACT
The normal intestinal microflora (microbiota) represents a complex, dynamic, and diverse collection of
microorganisms, which usually inhabit the gastrointestinal tract. Normally, between this flora and the human
host a mutually beneficial long-term symbiotic relationship is established, where the host contributes essential nutrients necessary for the survival of the microbiota and the latter fulfils multiple roles in host nutrition
and development. several achievements have recently converged to renew interest in studying the normal
gut microbiota: the development of molecular methods of studying the microbial communities, the improved
understanding of host-microbe interactions in health and disease, and the potential for therapeutic manipulation of the microbiota. We present recent data concerning the molecular technologies of studying the
microbiota and new findings regarding the composition of the normal flora. We underline the beneficial
activities of the gut flora on the human host. We emphasize the recent findings in the alterations of the microbiota in various medical conditions (celiac disease, irritable bowel syndrome, obesity, colorectal cancer,
allergic disorders, and especially inflammatory bowel diseases). The results of these new studies suggest that
changes of the microbiota could be linked to the etiopathogenesis of these diseases. These outstanding
findings could be used for further diagnostic tools and/or therapy.
Keywords: gut microbiota, metagenomics, metabolomics, inflammatory bowel disease, celiac disease, irritable
bowel syndrome, colorectal cancer, allergic disorders
INTRoDUCTIoN
The normal intestinal microflora (microbiota)
represents a complex, dynamic, and diverse collection of microorganisms, which usually inhabit the
gastrointestinal tract [1]. Normally, between this flora
and the human host a mutually beneficial long-term
symbiotic relationship is established [2], where the
host contributes essential nutrients necessary for the
survival of the microbiota and the latter fulfils multiple roles in host nutrition and development.
several achievements have recently converged
to renew interest in studying the normal gut microbiota: the development of molecular methods of
studying the microbial communities, the improved
understanding of host-microbe interactions in health
and disease, and the potential for therapeutic manipulation of the microbiota [3].
The role of intestinal microflora in health and
disease is becoming increasingly recognized. The
composition and activities of the GI microflora can
have beneficial, but also harmful outcomes on the
host.
MolECUlAR TECHNoloGIES oF STUDYING

THE MICRoBIoTA
since most bacterial species cannot be cultured,
over the past 10 years sophisticated culture independent methods have been developed [4]. Most of
them have so far targeted the small subunit (ssU) 16s
ribosomal RNA (rRNA) gene sequences (from amplified bacterial nucleic acid extracted from faeces or
biopsies), containing regions of both highly conserved and hypervariable nucleotide sequences [5]. 16s
rRNA analyses can only appraise the phylogenetic
composition of a sample and provide no direct information about its functional capabilities [6]. The availability of bacterial sequence data has facilitated the
development of molecular probes for various sophisticated methods (Table 1) [7-9].
Metagenomic analyses reconstruct the entire genome of the microbial community, in order to determine its component microbial lineages and genes (the
microbiome). According to this approach, humans
are actually superorganisms, whose genome is the
sum of genes of their own genome and of the micro-
*corresponding author: Daniela elena erban, Iuliu Haieganu University of Medicine and Pharmacy Cluj-Napoca, Romania, second
Pediatric Clinic, str. Crian nr. 5, Cluj-Napoca, Romania; Tel: 40-264-532216, e-mail: danitiserban@yahoo.com
134
The gut microbiota in the metagenomics era: sometimes a friend, sometimes a foe
biome [10]. Functional omic methods study the

communitys expressed RNA (meta-transcriptomics),
protein products (meta-proteomics), and its metabolic
network (meta-metabolomics) [10]. This approach
allows the detection of the structures and functions of
microbial communities, as well as their interactions
with the habitats they occupy. Metagenomic and metabolomic profiling of the microbiota, although at an
early stage, have demonstrated the range and complexity of the gut ecosystem and cast insights into
several diseases [11].
Reports on the assessment of the gut microbiota
have to be carefully evaluated, based on the study
design and population size, as well as in the frame of
advantages and limitations of the technologies and the
site concerned - luminal or surface-adherent flora [8].
endocrine system) [15]. Microbiota mediates many

key functions, some of them not being able to be
achieved by the gut tissue alone [3]. The metabolic
activity of the microflora is considered to be equal to
that of the liver [16].
studies on germ-free animals have shown the
presence of less mucosal cell turnover, vascularity,
digestive enzyme activity, muscle wall thickness, local cytokine production, motility as well as smaller
Peyers patches, fewer intraepithelial lymphocytes,
lower serum immunoglobulin levels. Conversely,
these germ-free animals have more enterochromaffin
cells, infection and stress-susceptibility, as well as
requirement of caloric intake [4].
Human gut contains 10 times more bacteria

than eukaryotic cells in the entire human body
[12]. This gut flora has around 100 times as many
genes in aggregate as there are in the human genome
[13]. A very complex metagenomic study showed in
March 2010 that gut microflora comprises 1000-1500
species and each individual has at least 160 such species, which are also largely shared. Over 99% of the
genes are bacterial, the remainder being mostly
archaeal, with only 0.1% of eukaryotic and viral origins. They also have shown the existence of a common core between humans, 75 species being common to >50% of individuals and 57 species common to >90% [13]. Another very recent 16s rRNAbased study has detected, as well, a common bacterial species core, shared among at least 50% of individuals [14].
The most frequent phyla are represented by: Firmicutes, 30.6-83% (Clostridium, Ruminococcus, eubacterium, Dorea, Peptostreptococcus, Peptococcus, Lactobacillus); Bacteroidetes, 8-48% (Bacteroides); Actinobacteria, 0.7-16.7% (Bifidobacterium) and Proteobacteria, 0.1-26.6% (enterobacteriacee) [8, 14].
The most common genera of fungi include Candida
and saccharomyces. The content of bacteria increases
from mouth (less than 200 species) to the colon (bacteria reaching 1010-1012/g of luminal content).
Beneficial Activities of The Gut Flora on

Human Host
Protection against pathogenic species: pathogen
displacement, nutrient competition, receptor competition, production of anti-microbial factors [4]
Structural effects (gut maturation): growth and
development of the epithelial barrier [1], apical
tightening of the tight junctions [4], stimulation of intestinal angiogenesis [17], control of the intestinal
epithelial cell differentiation and proliferation
Development/modulation of gut-associated lymphoid tissue: education of nave neonatal innate
immunity [1]; induction of Ig A; development of oral
tolerance [4]
Antialgic effects: Lactobacillus acidophilus
Metabolic functions: fermentation of nondigestible carbohydrates to produce short-chain fatty
acids - sCFAs [4]; regulation of host fat storage [7];
protein fermentation with production of amino acids
indispensable to humans [18]; vitamin synthesis biotin, folate and vitamin k [4]; ion absorption (Mg,
Ca, Fe) [1, 4]; biliary acids metabolism; xenobiotics
and dietary carcinogens degradation [4].
Most of functions of gut flora are achieved
through sCFAs [4]: pH lowering (reducing the formation of secondary biliary acids - pro-carcinogens and
reducing the counts of damaging bacteria); increasing
gut absorption of water; nutrient for growth of commensal bacteria; increasing growth of human gut cells
(epithelial and lymphoid cells); ion absorbtion [15].
IMPoRTANCE oF THE GUT FloRA oN THE

HUMAN HoST
FACToRS INFlUENCING THE CoMPoSITIoN oF

GUT MICRoFloRA
These microorganisms are metabolically active

and interact continuously with their environment (including other bacteria, the gut epithelium, mucosal
immune system, the central nervous system, and the
In neonates, gestational age, mode of delivery,

faecal and vaginal bacteria of maternal origin, sanitary conditions, geographical distribution of bacterial
species, maternal dietary intake, and babys diet are
CoMPoSITIoN oF THE GUT MICRoFloRA

(1014)
135
DANIElA ElENA ERBAN
important factors. Numerous studies have found a

predominance of Lactobacilli (L) and Bifidobacterium
(BF), respectively a more diverse flora, including Bacteroides, BF, staphylococci, e coli, and Clostridia, in
the stool of breast-fed infants relative to formula-fed
infants [6, 19]. However, on the one hand, these reports are controversial, on the other hand, with the
advent of probiotic and/or prebiotic-enriched formulas, the differences in the stool flora may not vary that
much.
Later on, other factors can influence the composition of microbial gut communities: age, sex, diet,
various conditions of the host (digestive, immune and
endocrine functions, and genetic background), bacterial mucosal receptors, emotional stress, and drugs
[6, 19].
DElETERIoUS EFFECTS oF THE GUT MICRoBIoTA
In some instances, the interest-based friendship
between flora and host can be broken: harmful properties of some components of the microflora [12],
disruption of the ecological balance between prohealth and anti-health organisms (dysbiosis), and predisposing conditions of the host (modified genetics,
immunology, gut barrier) [4]. Besides, over the millennia, the socio-economic development has led to
modifications in the composition of the flora and
some of them have detrimental effects on the host
health (the hygiene hypothesis) [9]. Various diseases,
from infections, to degenerative, inflammatory,
immune, and allergic disorders may appear [1]. Very
interesting papers have shown alterations in the microbiota in various diseases. However, it is very difficult sometimes to asses if the changes in the microbiota are the cause or the effect of these diseases [11].
Alteration of The Normal Flora (especially by
antibiotics, immunosuppressors, radiation or chemotherapy) could lead to dysbiosis, bacterial overgrowth,
antibiotic-associated diarrhoea (Clostridium difficile,
klebsiella oxytoca, salmonella spp, Candida albicans),
and sepsis, by microbes translocation [5]. Oral Ampicillin or other Penicillins suppress the normal aerobe
and anaerobe flora (including L, BF, streptococcus),
with increase of klebsiella, Proteus and Candida.
After using Amoxicillin, the stool flora decreases at
most on the 4th day and returns at normal levels on
the 30th day, in most of cases [20].
Irritable Bowel Syndrome (IBS)
There is growing evidence that the gut microbiota plays a role in the pathogenesis of IBs: the causal
136
relationship between the gut microbiota and the postinfectious IBs, the improvement of IBs symptoms by
treatments targeting the microbiota (antibiotics, probiotics, prebiotics) and the alterations in the gut microflora in IBs [21]. A significant difference between
IBs-patients and healthy controls (HC) has been found
in both mucosal and stool flora [22]. A very recent
study demonstrated that, in faecal samples, the microbiota of patients, compared with HC, had a 2-fold
increased ratio of the Firmicutes to Bacteroidetes (an
approximately 1.5-fold increase in numbers of Dorea,
Ruminococcus, and Clostridium spp.; a 2-fold
decrease in the number of Bacteroidetes; a 1.5-fold
decrease in numbers of BF and Faecalibacterium spp)
[23]. Moreover, significant differences have been
identified in the composition of the flora between the
IBs-subtypes and with the HC [21]. IBs-D patients
were depleted in several Firmicutes and Bacteroides
spp., while IBs-C patients had elevated amounts of
many of Firmicutes. Actinobacteria were depleted in
all IBs subtypes compared with the HC. High faecal
amounts of L and/or streptococci have been reported
in IBs-D patients [24]. A recent paper showed that L
and Veillonella spp. correlate positively with digestive symptoms and impaired quality of life [25]. A
very recent research has shown that in IBs-children
there is a significantly greater percentage of the class
Gammaproteobacteria (0.07% vs 0.89% of total bacteria). Also, a novel Ruminococcus-like microbe was
associated with IBs and a greater frequency of pain
was correlated with an increased abundance of several bacterial taxa from the genus Alistipes [26].
The use of metabolomics in IBs has started and
with promising results. Recent findings have shown
elevated levels of amino acids (alanine and pyroglutamic acid) and phenol compounds (hydroxyphenyl
acetate and hydroxyphenyl propionate) in faeces of
IBs-patients. These results were highly correlated with
the abundant Lactobacillus and Clostridium [27]. All
these findings could lead to a better therapy of these
patients.
Celiac Disease
Although its pathogenesis is related to gluten
sensitivity in genetically predisposed subjects, alterations in the gut flora have been recently described in
celiac disease (CeD). A recent study involved 3 groups
of children: untreated with CeD, CeD on gluten-free
diet and HC. The Ig A-coated faecal bacterial levels
were significantly reduced in both untreated and
treated CeD patients vs HC, as were the IgG and IgMcoated bacterial levels in treated CeD patients vs
untreated CeD patients and HC. Also, BF, Clostri-
rationale for therapeutic manipulation of enteric bacteria using pharmabiotics.

Table 1. Molecular technologies of studying the gut microbiota a)

Table 1. Molecular technologies of studying the gut microbiotaa)
Method
Comments
Quantitative, but low sensitivity [7]

Dot-blot hybridization
Fluorescent in situ hybridization (FISH)
Quantitative, but rather insensitive and laborious
FISH combined with epifluorescent light
Highly dependent on the 16S rRNA gene
microscopy or flow cytometry
sequences deposited in the databases
Polymerase-chain reaction (PCR) combined with
Very laborious, rather insensitive and not
denaturing - or temperature-gradient gel
quantitative
electrophoreses (PCR-DGGE/PCR-TGGE)
Quantitative real-time PCR
Quantitative, but time consuming
454-pyrosequencing 16s RNA
High fidelity taxonomic information
DNA microarray [6]
Quantitative information
a) - reviewed in Bibiloni et al [8] and Kalliomaki et al [9]
dium histolyticum, Clostridium lituseburense and
Faecalibacterium prausnitzii group proportions were
less abundant in untreated CeD patients than in HC.
On the contrary, Bacteroides-Prevotella group proportions were increased in untreated CeD patients vs
HC. Levels of IgA coating the Bacteroides-Prevotella
group were significantly reduced in both CeD patients vs HC [28]. Another study has detected in untreated children increased levels of Gram-negative
(escherichia coli and Bacteroides groups) and potentially proinflammatory bacteria in the duodenal biopsies vs treated children [29]. Very interesting recent
findings in children at diagnosis vs on a gluten free
diet vs HC have been recently published. Bacteroides
and Clostridium leptum groups were more abundant
in faeces and biopsies of CeD children vs HC, regardless of the stage of CeD. Also, the levels of escherichia coli and staphylococcus counts in faeces and
biopsies of non-treated CeD patients were higher
than in HC, but their levels normalized after glutenfree diet. BF levels were reduced in faeces of both
groups of CeD patients and in biopsies of untreated
CeD patients vs HC [30].
Colorectal Cancer (CRC)
early studies have shown that Bacteroides and
Clostridium increase the tumour rate, whereas some
strains of L and BF may inhibit colon tumour development [15]. In certain situations, commensal bacteria could fuel the progression towards colorectal malignancy by generating reactive metabolites, converting pro-carcinogens to carcinogens, altering host carbohydrate expression, generation of toxic products of
the protein metabolism [2, 3, 15].
In a very recent series, the authors compared stool
bacterial DNA in patients with CRC and HC. Among all
dominant and subdominant species, those of Bacte-
roides/Prevotella were higher in CRC than in HC.

IL17 immunoreactive cells were significantly expressed more in the normal mucosa of CRC patients than
in HC [31]. Another interesting paper revealed that
microflora dysbiosis occurs at the mucosal surface in
colonic adenomas a 20-fold relative reduction of
mucosa adherent bacteria compared with normal tissue and may represent a potential factor for dysplastic cell proliferation. Concomitantly, a-defensin expression and production are significantly increased in
adenomas, while adenoma mucosa showed increased antibacterial activity in vitro compared with normal mucosa [32].
obesity And Hyperglycemia
Recent researches have shown that the gut
microbiota is involved in obesity and metabolic disorders, as it influences nutrient absorption and energy storage [33]. Obese mice and human subjects
have alterations in the composition of the gut microbiota compared to their lean counterparts - high levels of Firmicutes and low levels of Bacteroides. These
modifications are reversible with weight loss [33, 34].
Moreover, germ-free mice are leaner than the conventional ones and transplantation of the microbiota
of normal mice results in 60% increase in total body
fat and insulin resistance within 2 weeks, despite a
reduction in the food intake. This could appear as a
result of the suppression of Fiaf in the gut epithelium
(fasting-induced adipocyte factor, which inhibits the
lipoprotein-lypase) by the microbes [7]. Moreover,
bacteria stimulate the breakdown of polysaccharides,
with more absorption of mono-saccharides, increased
lypogenesis in the liver and hyperglycemia [33].
Differences in faecal microbiota of infants have been
associated with the risk of being overweight/obese at
7 years of age. Children of normal weight had higher
137
DANIElA ElENA ERBAN
BF and lower staphylococcus aureus concentrations at

ages 6 and 12 months than children who became overweight/obese [35]. These results suggest that differences in the microbiota precede overweight/obesity.
Allergy (Dermatitis, Rhino-Conjunctivitis)
Many studies have shown that alterations in gut
microbiota may precede the development of allergy
[reviewed in 9], like the decrease in the amount of BF
and an increase in Clostridia and escherichia coli.
However, results of the studies are difficult to interpret, as there were: different study designs (prospective vs cross-sectional), different methods of bacterial
analysis, timing and number of sampling, different
duration and type of follow-up and many differences
in the study populations [9]. Increase in the amounts
of BF adolescentis, with a decrease of BF bifidum,
were described in children with atopic eczema vs HC
[36]. Others have found that BF catenulatum/psseudocatenulatum was associated with eczema [37].
Inflammatory Bowel Disease (IBD)
There is mounting evidence, from both animal
models and human studies, that alterations in enteric
microbiota/dysregulation of the mucosal immune response to antigens present in the normal bacterial
flora, in genetically predisposed individuals, are involved in the pathogenesis of IBD (especially Crohns
disease - CD) [38]. Arguments that the intestinal microbiota may drive intestinal inflammation include
the following: absence of IBD in germ-free animals
[39]; presence of the highest concentrations of bacteria in most common diseases locations (distal ileum
and colon) [40]; association between ileal CD and
the NOD2/CARD15 gene polymorphisms, related to
bacterial peptidoglycan recognition [41]; healing of
intestinal lesions in CD after diversion of faecal stream,
with early recurrence after restoring the digestive
continuity [42]; benefits of probiotics (in ulcerative
colitis and pouchitis) [43]; improvement of some intestinal lesions with antibiotics in CD [40]. Also, bacteria with possible implications have been discovered: increased levels of mucosal adherent escherichia coli in ileal lesions (30% in CD vs 6% in HC)
[44], at the disease onset [45] and of Campylobacter
concisus in intestinal biopsies of children with CD
(51% vs 2% in HC) [46]. On the other hand, low levels of ileal Fecalibacterium prausnitzii, a protective
bacterium, were associated with higher risk of postoperative recurrence of ileal CD [47].
Recent findings revealed also the growing role
attributed to the loss of tolerance to Candida albicans
(ALCA, AMCA, ACCA antibodies) in the pathogene138
sis of CD [48]. AsCA were positive in 72% of patients

with CD and in 34% of their healthy relatives vs 4%
of HC, as C. albicans had high amounts in stool from
CD patients (44%) and their healthy relatives (38%)
vs 22% of controls [49]. Moreover, an increasing
number of positive antibodies (ALCA, anti-L Ig A,
AMCA, ACCA, anti-C Ig A, gAsCA, pANCA) was
associated with early age of CD diagnosis and longer
disease duration and more aggressive disease (penetrating phenotype, perianal disease location and need
for abdominal surgery) [50]. Also, according to many
studies, the more numerous the antibodies against
infectious antigens (OmpC, I2, cBir1 and/or those of
Candida albicans), the more complicated the course
of the CD [50, 51]. A recent study has shown the predominance of cBir1 antibodies in children less than 8
years 66% (suggesting the role of Clostridia) and of
AsCA and ompC after this age (suggesting a possible
intervention of escherichia coli and fungi) [52].
Recently, it has been shown that patients with
CD have a different faecal microbiota composition
from that of healthy relatives of CD patients and of
HC. The faecal microbiota of CD patients was characterized by a decrease in Dialister invisus, an uncharacterized species of Clostridium cluster XIVa,
Faecalibacterium prausnitzii, and BF adolescentis,
and an increase in Ruminococcus gnavus characterized compared with that of HC. The unaffected relatives had faecal microbiota characterized by lower
levels of Collinsella aerofaciens and a member of the
escherichia coli-shigella group, and a higher level of
Ruminococcus torques than HC [53]. Another study
has revealed that Fusobacterium nucleatum is present
in more patients with IBD than in HC [54].
Metagenomic and metabolomic profiling of the
microbiota have also shown important differences
between CD, ulcerative colitis and HC. Faecal extracts
of both CD and ulcerative colitis patients have shown
decreased levels of butyrate, acetate, methylamine,
and trimethylamine and high levels of amino acids.
Metabolic differences in faecal profiles were more
marked in the CD group [11].
DIRECTIoNS FoR THE FUTURE
We are only just beginning to understand and
appreciate the many roles that these microbes play in
human health and development. That is why in 2007,
the National Institutes of Health, aiming to identify
and characterize the microorganisms found in association with healthy and diseased status, initiated The
Human Microbiome 5-year Project [55]. The beneficial effects of some strains and deleterious contribu-
tion of others certainly sustain the rationale for therapeutic manipulation of enteric bacteria using pharmabiotics.
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ROMANIAN ARCHIVES

ROMANIAN ARCHIVes OF MICROBIOLOGY AND IMMUNOLOGY

Editor-in-Chief: Gabriel Ionescu

2011 CANTACUZINO INsTITUTe Bucharest

ROMANIAN ARCHIVes OF MICROBIOLOGY AND IMMUNOLOGY

IMMUNOLOGICAL AsPeCTs IN VIRAL HePATITIs B AND C INFeCTION

CORReLATION OF ANTI-Helicobacter pylori CagA IgG ANTIBODIes WITH ResIsTANCe

INFLUeNCe OF ORTHODONTIC TReATMeNT ON ORAL sTRePTOCOCCI

DYNAMICs OF eNDOTHeLIAL PROGeNITOR CeLLs

A HIGHLY PURIFIeD VeGeTAL FRACTION ABLe

Is INTeRLeUkIN -17 A PROATHeROGeNIC BIOMARkeR?

HePATITIs B, C AND D COINFeCTION IN HIV-INFeCTeD PATIeNTs:

JULY - sePTeMBeR 2011

ROMANIAN ARCHIVes OF MICROBIOLOGY AND IMMUNOLOGY

Manuscripts should be divided into the following sections and order:

Department, regional institute of Gastroenterology and Hepatology

Viruses or products of the viral replication are

IFN-g, which negatively regulates the viral replication

rate and destruction of infected cells [19,20]. All

Immunological aspects in viral hepatitis B and C infection

disease [26]. Immunohistochemical studies showed

CORReLATION OF ANTI-Helicobacter pylori CagA IgG

Hospital, Gastroenterology, bucharest, romania;

2University of bucharest, Faculty of biology, Microbiology immunology Department, bucharest, romania;

undergo morphological changes, vacuolization and

gastric adenocarcinoma. The estimated risk has ranged

Fig 1. Distribution of cases resistant to first line

For this purpose 2-5 ml of serum were taken

RESUlTS AND DISCUSSIoN

Fig. 2. Distribution of bleeding gastroduodenal ulcers in

CagA IgG antibodies can persist up to four years,

Fig. 3. Predominance of anti-cagA IgG positive in

Fig. 5. Images of diffuse infiltrative tumor (type IV in

The presence of the cagA IgG antibodies, with

Fig. 6. Relation between selected cases (resistance

INFLUeNCe OF ORTHODONTIC TReATMeNT ON ORAL sTRePTOCOCCI

of orthodontics and Dento-Facial orthopedics, Faculty of Dental Medicine,

* corresponding author: Theodor-Cristian Vizitiu, e-mail: theodorvizitiu@yahoo.com

subcultivated on unselective growth media to obtain

Fig. 1. Sex distribution of the patients

Influence of orthodontic treatment on oral streptococci

Fig. 2. Age distribution of the patients

22.5% and 10%), whose frequency increased after 3

Table 1 - Total bacteria concentration

104 < 105

- Isolated oral streptococci strains

Group I (40) (patients)

Group II (34) (patients)

Group III (31) (patients)

decreasing to 9.6% after 6 months of wearing orthodontic appliances.

pared to the beginning of the treatment, for the main

DYNAMICs OF eNDOTHeLIAL PROGeNITOR CeLLs

by markers like CD34 and flk-1 play a major role in

* corresponding author: Leon Zagrean, e-mail: leon.zagrean@gmail.com

RESUlTS AND DISCUSSIoN

Dynamics of endothelial progenitor cells following sevoflurane preconditioning

Fig. 1. Endothelial progenitor cell dynamic in the

us to assume that this could be another way through

could favorize early healing and endothelization, as

Dynamics of endothelial progenitor cells following sevoflurane preconditioning

1. Kamota T, li TS, Morikage N, Murakami M, ohshima

11. Strauer BE, Brehm M, Zeus T, Bartsch T, Schannwell

A HIGHLY PURIFIeD VeGeTAL FRACTION ABLe