pneumonia , sepsis , meningitis due to infection with gbs are common causes of morbidity , mortality , and long - term neurological sequelae in neonates .
vaginal smears of up to 40% of pregnant women show colonization with s. agalactiae , 50 to 70% of their children will be colonized postpartum [ 2 , 3 ] ; up to 2 newborns per 1000 live births will develop gbs infection [ 4 , 5 ] , which is characterized by unspecific clinical signs such as temperature instability , respiratory distress , palor , and abdominal symptoms .
the majority of neonates develop early onset sepsis within the first 24 hours of life , which rapidly progresses in septic shock and hemorrhage .
depending on the gestational age , case fatality rate can increase up to 30 percent in preterm infants < 32 weeks of gestation [ 6 , 7 ] .
more than 50% of affected newborns suffer from neurological deficencies like sight or hearing loss and mental retardation .
in addition 19,000 cases of gbs disease occur annually in the united states , and up to 35% of the symptomatic infections in elderly and immunosuppressed patients suffer from chronic diseases .  
gbs has characteristic virulence factors , including the ability to bind to extracellular matrix components , such as fibronectin , fibrinogen , and laminin , allowing to pass cellular barriers and to become invasive .  
following bacteremia , immune response is partially insufficient , presumably due to immature innate immunity of the neonate .
lipopolysaccharides of gram negative bacteria are able to modify the barrier function of endothelial cells .
could previously demonstrate that gbs are able to penetrate the blood - brain barrier by targeting human brain microvascular endothelial cells .
however it is unclear if gbs is also able to invade human coronary artery endothelial cells .
endothelial dysfunction is a major component in the pathophysiology of septicemic gbs infection [ 13 , 14 ] .
therefore , analysis of the interaction between gbs and endothelial cells is of major interest , especially the septic and haemorrhagic complications in newborns .
genome wide expression profiling allows detailed insights in the pathogenesis of gbs sepsis and helps to better understand the underlying pathophysiological mechanisms .  
we studied the interaction between human coronary artery endothelial cells ( hcaecs ) and s. agalactiae by genome wide high density microarrays .
the results of this study indicate that gbs are able to regulate transcription of a wide range of genes , involved in infection , inflammation , and apoptosis .

 streptococcus agalactiae lancefield 's group b ( atcc13813 strain lehmann and neumann , serotype v , septic , nonhemolytic ) was obtained from atcc ( lgc promochem , wesel , germany ) .
bacteria were grown overnight on tryptic - soy agar plates supplemented with 5% sheep blood ( institut fr hygiene und medizinische mikrobiologie , university of wuerzburg , germany ) and cultured in 20  ml lysogeny broth ( lb )   medium , supplemented with 1% trypton , 0,5% yeast extract , and 1% nacl at 37c for 90 minutes . a final bacteria concentration of 2 to 4  10/ml was achieved after adjustment of the optical density at 600  nm .
primary human coronary artery endothelial cells ( hcaecs , cryopreserved , third passage ) were obtained from lonza ( basel , switzerland ) and cultured in ebm2 medium ( lonza ) , and supplemented with egm-2-mv singlequots ( lonza ) with 5% fbs .
all cells were subcultured at a 1  :  6 ratio and were used for experiments from passages 5 to 16 . after reaching 80% confluence , endothelial cells were washed three times with phosphate buffered saline ( pbs ) and detached from culture flasks using trypsin - edta solution .
the reaction was stopped with tns solution ( cambrex ) , and cells   were resuspended in complete medium .
gbs , diluted in lb medium , were added in multiplicity of infection ( moi ) of 20 to hcaec .
, cells were trypsinised , resuspended in trypsin neutralization solution ( tns , cambrex ) , and centrifuged at 800  rpm for 5 minutes after 6 hours of infection , because preliminary data have shown that the maximum of gene regulation occurs between 4 and 8 hours . for additional conformation experiments ,
confirmation of gene array data was selectively carried out by real - time rt - pcr , western blot analysis , and elisa at different time points . for real - time rt - pcr hcaecs
were detached from cell culture dishes by a cell scraper , snap - frozen and stored at 80c until rna isolation .   for western
blotting , protein extracts were prepared by detaching cells , followed by centrifugation at 800  rpm for 5 minutes .
cell pellets were carefully washed once with 4  ml phosphate buffered saline solution ( pbs ) and centrifuged again .
cells were resuspended in 100  l pbs and 100  l sds stop solution ( 200  mm trizma - base ph 6.7 , 15% glycerin , 0,03% bromphenole blue , 6% sodium dodecyl sulfate ( sds ) ,  - mercaptoethanol , 9  :  1 , 95c ) for 5 minutes at 95c . until further analysis , protein extracts were stored at 20c .   in parallel ,
cell culture supernatants were collected and frozen at 20c until further analysis by elisa ( upa ) . for il-8 and cxcl-6 elisa ,
cells were detached in 150  l pbs , snap - frozen , and stored at 80c . for rna extraction , cell pellets
were resuspended in  - mercaptoethanol and rlt lysis buffer ( qiagen , hilden , germany ) . for disruption and homogenization lysates
were loaded on qiashredder spin columns ( qiagen ) and centrifuged at 13.000  g for 2  min .
rna purification was carried out using rneasy spin columns ( qiagen ) according to the protocol of the manufacturer .
rna was eluted in 35  l rnase - free water , and rna concentration was determined photometrically ( nanodrop nd-1000 , nanodrop technologies , wilmington , usa ) .   for gene array analysis , quality of rna was determined using a bioanalyzer ( agilent technologies , waldbronn , germany ) , and rna integrity numbers ( rins ) were between 9.6 and 9.7 .
two g of total rna were reverse transcribed , performing second strand synthesis ( one - cycle cdna synthesis kit , affymetrix , santa clara , usa ) .
synthesis of biotinylated cdna was carried out using the ivt labelling kit ( affymetrix ) .
fifteen g of the fragmented , labelled crna were hybridized to affymetrix hg - u133 plus 2.0 arrays .
fluorescence intensity was measured by a genechip scanner 3000 ( affymetrix ) . for normalization and data analysis ,
after normalization by variance stabilization , data quality was verified by density plots , degradation plots , and box plots .
resulting genes were functionally clustered according to gene ontology using gene ontology tree machine ( gotm ) ( http://dbmi.mc.vanderbilt.edu/ ) .
for real - time rt - pcr , cdna synthesis was performed using the quantitect reverse transcription kit ( qiagen ) for 25 minutes at 42c and 3 minutes at 95c .
quantitative real - time rt - pcr for cxcl6 , tlr2 , and tlr4 quantification was carried out using the lightcycler instrument . to normalize real - time pcr data , serially diluted cdna copies of the housekeeping gene -aminolevulinic acid synthase ( h - alas ) were coamplified . analysis was performed comparing the number of cdna copies of the samples with the h - alas copies in each sample , respectively .
45 cycles of repeated denaturation ( 95c 9  sec ) , annealing ( 54c 15  sec ) , and elongation ( 72c 25  sec ) were performed .
the pcr mastermix ( lightcycler fast start master hybridization , roche , mannheim , germany ) contained 0,125  m of each primer ( cxcl6 : 5-ttgcacttgtttacgcgtt , 5-tcagtttttcttgtttccactgt ; tlr2 : 5-tgtcttgtgaccgcaatggta , 5-gcttgaaccaggaagacgat ; tlr4 : 5-ggagccctgcgtggaga , 5-tatgccccatcttcaattgtc ) and 0,15  m of the hybridization probes ( cxcl6 : 5-gcaagtttgtctggacccgga - fl , 5-lc640-gccccttttctaaagaaagtcatccagaa - p ; tlr2 : 5-ctacagaggtgtgtgaacctccaggc - fl , 5-lc640-ctggtgctgacatccaatggaattaac - p ;
tlr4 : 5-cccttcaccccgattccattgct - fl , 5-lc640-cttgctaaatgctgccgttttatcacg - p ) , labeled with fluorescein and lc red 640 , respectively .
cellular proteins were loaded onto sds - polyacrylamid stacking gels ( 3% ) , separated in running gels ( 12% ) , and run for 1 hour at 160  v in tris - sds - glycine electrophoresis buffer according to laemmli .
( immobilion - p 45  m , millipore , bedford , usa ) at 1 a for 60 minutes at 4c . after being blocked for one hour in tris
buffered saline / tween ( 25  mm tris ph7.6 , 150  mm nacl , 0,05% tween20 ) containing 6% dry milk , membranes were incubated with primary antibodies ( hmox-1 : 1  :  250 , milk , mouse , bd bioscience pharmingen , san jose , usa ; bim : 1  :  100 , milk , rabbit , bd bioscience pharmingen ) overnight at 4c .
membranes were washed briefly and incubated with horse - raddish - peroxidase - conjugated antirabbit or antimouse igg ( goat anti - mouse / rabbit hrp , 1  :  3000 , milk , goat , bio - rad , munich , germany ) for one hour at room temperature .
immunoreactivity was detected using an ecl or ecl - plus detection kit ( amersham pharmacia biotech , freiburg , germany ) .
the protein bands on x - ray films ( fuji photo film gmbh , dsseldorf , germany ) were scanned , and the intensity of bands was analyzed using nih image software ( version 1.61 ) .
loading control was performed with  - actin ( 1  :  20 , milk , rabbit , santa cruz biotechnology inc . ,
, michigan , usa ) , il-8 ( quantikine kit , r&d systems , wiesbaden , germany ) , and upa ( imubind , american diagnostica , stamford , usa ) levels were quantified by elisa , according to the suppliers protocol .
mean fluorescence was quantified by measurement of optical density , using the softmax system ( molecular devices gmbh , ismaning , germany ) .
in total , 124 genes were differentially expressed ( 89 upregulated , 35 downregulated ) based on a more than 3-fold difference to control hcaec   ( see supplementary material available online at doi : 10.1155/2011/971502 ) .   signal log ratios ( base e ) varied between 2.7 and 1.5 comparing control cells versus hcaec after 6 hours of coincubation with s. agalactiae .
the highest level of induction was observed for genes encoding transcriptional regulation ( 14.3-fold upregulated ) ; the highest level of inhibition was achieved in genes involved in dna - specific binding ( 4.5-fold downregulated ) .
differencial expression of selected genes ( table 1 ) was validated by one additional method , including western blots , real - time rt pcr , and elisa assays .
we selected regulated genes , which are involved in apoptosis , hemostasis , oxidative stress response , infection , and inflammation for further analysis .
previously , we could demonstrate cleavage of caspases 3 and 8 after infection of hcaec with s. agalactiae ( data unpublished ) .
bim protein levels were increased by 1.3-fold after 4 hours , 1.5-fold after 8 hours , and 2.6-fold after 24 hours of infection , compared to uninfected cells ( figure 1 ) .
heme oxygenase 1 plays an important role in the regulation of apoptosis and cell protection during inflammatory processes .
expression of hmox-1 is species- and cell - specific , induced by oxidative stress , due to lps , ischemia , or cytokines .  
infection of hcaec with the septicemic s. agalactiae strain atcc 13813 led to hmox-1 upregulation , compared to unstimulated cells ( 2.3-fold after 4 hours , 4.7-fold after 8 hours , and 6.6-fold after 24 hours of infection , figure 2 ) .
cxcl6 is a strong mediator of inflammation , attracting mainly neutrophils , but also eosinophils , lymphocytes , and monocytes .
cxcl6 production can be induced by il-1- , tnf - alpha , hypoxemia , and lps .
downregulation of cxcl6 after gbs stimulation was observed ( 2-fold after 4 hours and 12.9-fold after 8 hours of infection ) .
tlr 2 and tlr4 are major receptors of the human innate immune response against various pathogens , interacting with lipoteichoic acid and bacterial lps , respectively .
tlr4 expression was reduced after gbs stimulation ( 3.4-fold after 4 hours , 2.9-fold after 8 hours , and 11-fold after 24 hours of infection ) .
we also observed downregulation of tlr2 ( 4.3-fold after 4 hours , 3.8-fold after 8 hours , and 1.2-fold after 24 hours of infection , figure 4 ) .
interleukin 8 , a member of the cxc chemokine superfamily , attracts neutrophils and monocytes to sites of inflammation .
production of il-8 can be induced by different cytokines , viral infections , or gram negative bacteria .
il-8 secretion was reduced by factor of 2.5 ( 4  h ) , 1.7 ( 8  h ) , and 1.5 ( 24  h ) .
therefore , hemorrhagic dysregulation , observed in gbs infected neonates might be enhanced from the observed upregulation of urokinase .
we found upregulation in urokinase levels in hcaec cell culture supernatants ( 1.6-fold after 4 hours , 2.6-fold after 8 hours , and 3.7-fold after 24 hours of infection , figure 5 ) .
cox2 is induced during inflammation among others in macrophages , monocytes as well as in endothelial cells .
we observed a weak upregulation of cox2 expression after 8 hours of infection ( 1.5-fold ) , whereas cox2 levels remained unchanged after 4 and 24 hours of infection compared to uninfected hcaec .
the present study demonstrates the use of cdna microarray technology to characterize transcriptional responses in hcaec coincubated with gbs .
( ii ) gbs leads to upregulation of the proapoptotic bcl2-like - interacting protein ( bim ) and of heme - oxygenase-1 ( hmox-1 ) .
( iv ) urokinase ( upa ) and cyclooxygenase-2 ( cox2 ) were activated after gbs infection .
doran et al .   performed a study analyzing the interaction between s. agalactiae and human brain microvascular endothelial cells .
in contrast to our study , this group used a haemolytic strain and revealed  - hemolysin / cytolysin as the most important pathophysiological player in a murine model of hematogenous meningitis .  
our data show that the proapoptotic protein bim is upregulated during the course of gbs infection .
one reason for bim upregulation during infection with s. agalactiae could be elevated levels of cytokines and chemokines in hcaec .
bim might cause the release of mitochondrial cytochrome c , activating the intrinsic way of apoptosis induction .
our own previous results ( data unpublished ) indicate that gbs can cause a breakdown of the mitochondrial membrane potential in hcaec , reflecting an initiation of programmed cell death .  
activation of toll - like receptors leads to an augmented phosphorylation and inactivation of bim , which in consequence avoids apoptosis .
our group was able to demonstrate a downregulation of tlr2 and tlr4 after infection with the septicemic gbs strain atcc 13813 after 4  h , 8  h , and 24  h , respectively , compared to unstimulated cells .
gbs - induced downregulation of tlr4 might be responsible for bim - induced apoptosis , which might support tissue invasion of gbs .   according to smith et al .
, who observed an upregulation of hmox-1 after stimulation with streptococcus pneumoniae , we could demonstrate activation of hmox-1 in hcaec coincubated with streptococcus agalactiae .
after activation , hmox-1 cleaves hemoglobin into carbon monoxide ( co ) , iron ( fe ) , and biliverdin , followed by conversion into bilirubin .
upregulated expression of hmox-1 could reflect a protective mechanism against thrombotic complications after infection with gbs .  
in addition , overexpression of hmox-1 might lead to elevated bilirubin levels , potentially interfering the change from fetal to adult hemoglobin .
hyperbilirubinemia might be responsible for neurological long - term sequelae , observed after gbs newborn infections .
furthermore hmox-1 could indirectly and directly inhibit apoptosis by minimizing reactive oxygen species ( ros )   and by regulation of nfb signaling . in summary , induction of hmox-1 by gbs
could represent a response of hcaec to gbs - induced pathomechanisms such as apoptosis and inflammation . 

cxcl6 , which encodes a strong chemotactic protein mainly for neutrophils , was markedly downregulated in hcaec after infection with s. agalactiae .
impaired cxcl6 levels could be responsible for lower neutrophil recruitment and activation during gbs sepsis .
in addition , cd4 positive t - cells , activated by cxcl6 , secrete augmented levels of igg , leading to opsonisation of microorganisms and thereby to facilitated phagocytosis . in parallel to cxcl6 , a further chemokine , il-8 , was downregulated after gbs infection , potentially leading to impaired chemotaxis of monocytes and neutrophiles .
futhermore , our own data revealed that production of a third chemotactic protein , monocyte chemoattractant protein ( mcp)-1 , was downregulated after incubation with s. agalactiae unpublished .
taken together , these results indicate either a mechanism of the host to avoid excessive inflammation or a bacterial strategy to suppress innate immune response .
the serine protease urokinase plays an important role in fibrinolysis by activating plasminogen to plasmin .
urokinase - type plasminogen activator upregulation might explain the simultaneously observed hemorrhagic and thrombotic complications during gbs sepsis .
cocultivation of hcaec with s. agalactiae revealed marked upregulation of urokinase , as determined by elisa assays .
elevated urokinase levels could lead to resolution of platelet - bacteria - thrombi resulting in facilitated tissue invasion of gbs .   in parallel to urokinase
, we found a second thrombolytic factor , cyclooxygenase 2 , to be upregulated during gbs infection , whereas cox1 is known to be constantly expressed .
imbalance of prostacyclin ( produced by cox2 ) and thromboxane a2 ( produced by cox1 ) might be responsible for hemorrhagical complications .
infection with a highly septicemic bacteria strain leads to marked increase of cyclooxygenase 2 expression .
our data demonstrates that s. agalactiae might be able to escape innate immune mechanisms and to induce hemorrhagical complications in hcaec .
induction of apoptosis , bleeding , and vasodilatation could explain the invasive growth during gbs sepsis .  
knowledge of molecular mechanisms during gbs infection will contribute to a broader understanding of the interaction of streptococci with the endothelium and , in consequence , will support the development of new therapeutical strategies against this severe complication .
 early onset sepsis due to group b streptococcus leads to neonatal morbidity , increased mortality , and long - term neurological deficencies . 
 interaction between septicemic gbs and confluent monolayers of human coronary artery endothelial cells ( hcaecs ) was analyzed by genome wide expression profiling . in total , 
 124 genes were differentially expressed ( 89 upregulated , 35 downregulated ) based on a more than 3-fold difference to control hcaec . 
 regulated genes are involved in apoptosis , hemostasis , oxidative stress response , infection , and inflammation . 
 regulation of selected genes and proteins identified in the gene array analysis was confirmed by real - time rt - pcr assay ( granulocyte chemotactic protein 2 ) , elisa ( urokinase , cyclooxygenase 2 , granulocyte chemotactic protein 1 ) , and western blotting ( heme oxygenase1 , bcl2 interacting protein ) at various time points between 4 and 24 hours . 
 these results indicate that gbs infection might influence signalling pathways leading to impaired function of the innate immune system and hemorrhagic and inflammatory complications during gbs sepsis . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion