Streptococcus pneumoniae were observed during infection in the host by quantitative real time RT-PCR; one was characteristic of bacteria in blood and one of bacteria in tissue, such as brain and lung. Gene expression in blood was characterized by increased expression of pneumolysin, pspA and hrcA, while pneumococci in tissue infection showed increased expression of neuraminidases, metalloproteinases, oxidative stress and competence genes. In vitro situations with similar expression patterns were detected in liquid culture and in a newly developed pneumococcal model of biofilm respectively. The biofilm model was dependent on addition of synthetic competence stimulating peptide (CSP) and no biofilm was formed by CSP receptor mutants. As one of the differentially expressed gene sets in vivo were the competence genes, we exploited competence-specific tools to intervene on pneumococcal virulence during infection. Induction of the competence system by the quorum-sensing peptide, CSP, not only induced biofilm formation in vitro, but also increased virulence in pneumonia in vivo. In contrast, a mutant for the ComD receptor, which did not form biofilm, also showed reduced virulence in pneumonia. These results were opposite to those found in a bacteraemic sepsis model of infection, where the competence system was downregulated. When pneumococci in the different physiological states were used directly for challenge, sessile cells grown in a biofilm were more effective in inducing meningitis and pneumonia, while planktonic cells from liquid culture were more effective in inducing sepsis. Our data enable us, using in vivo gene expression and in vivo modulation of virulence, to postulate the distinction – from the pneumococcal point of view – between two main types of disease. During bacteraemic sepsis pneumococci resemble planktonic growth, while during tissue infection, such as pneumonia or meningitis, pneumococci are in a biofilm-like state.Two main patterns of gene expression of  Streptococcus pneumoniae is the main cause of community-acquired pneumonia and meningitis in children and the elderly and of septicaemia in HIV-infected individuals. It is also one of the principal causes of otitis media. Bacteraemia has been reported to accompany more than 25% of cases of pneumococcal pneumonia , comD (CSP receptor histidine kinase) or comE (regulator of the to ComD kinase) are biofilm defective , comE (regulator of CSP specific two-component system) and comX . Other regulators assayed were the two-component systems TCS02 (micAB), TCS04 (pnpSR), TCS05 (ciaRH) and TCS13 (blpRH or spiRH), the putative quorum-sensing gene luxS, the heat shock regulators hrcA and ctsR, the serine/threonine protein kinase stkP, the virulence gene regulator mgrA, the putative iron-regulated regulator SP1638 (dtxR or smrB) and the putative sugar regulators SP1899 (msmR or araC) and regR  challenge, pneumonia after intranasal (IN) challenge and sepsis after intravenous (IV) challenge. Brain samples from mice with meningitis, lung washes of mice with pneumonia and blood of mice with sepsis were collected at the onset of severe symptoms and analysed individually. Pneumococcal gene expression during infection in mice showed two principal patterns; one found in the blood and one during infection of lungs and brain  and indicate a reduced variation in this condition, with respect to the reference condition of growth liquid medium. While different from blood, the relative gene expression values of pneumococci in tissue of infected mice, either lung or brain after direct inoculation, are similar. When comparing gene expression in blood against gene expression of pneumococci isolated from lungs and brain it is obvious that some genes show marked variations. A striking feature is that in the tissues each of the three competence genes  had much elevated expression compared with sepsis. The expression of other regulators and virulence genes varied according to site. The virulence genes igA, nanA, nanB, nox, sodA, pbp2x, zmpB and zmpC were upregulated in tissues, whereas ply and pspA showed increased expression in blood. Also regulator genes were differentially expressed, for example comE, comX, ctsR, dtxR, msmR and regR were more expressed in tissues, while the major heat shock regulator gene, hrcA, was upregulated in blood. Some of the monitored genes, namely blpR, ddlA, luxS, mgrA, pnpR and stkP showed increased expression in all in vivo conditions over liquid culture in TSB .in vitro reference condition of exponentially growing bacteria in liquid medium, we also assayed for gene expression of pneumococci on blood agar plates. On agar, the cps4A gene, unchanged in all other conditions, showed a significant upregulation, while most other genes showed no changes or diminished levels of expression  was evaluated in flat bottomed polystyrene wells after 18 h of incubation. Direct microscopic examination of the bottoms of the wells after repeated washes showed the presence of pneumococci attached to the plastic support when incubated in CSP-supplemented medium . Samples were observed both as wet preparations by phase contrast microscopy and after fixation with methanol and staining in bright field. Bacterial cells covered the whole bottom of the wells and were unevenly distributed forming various types of aggregates and clusters  did not produce any biofilm structure .The optimal concentration of CSP that induced attachment to plastic was assayed by incubation of pneumococcal isolates of different serotype and CSP-pherotype with threefold serial dilutions of CSP, ranging from 3 to 300 ng ml−1 . All str1 allele . Indepenin vitro and in vivo environments was reflected in virulence, we did challenge experiments in the presence and absence of CSP. We chose IV and IN challenge as representatives of the two different patterns of competence gene expression . Using transformation of pneumococci in vitro as detection tool to determine the stability of CSP in vivo we found that the half-life of CSP in blood was less than 1 min (data not shown).In order to assay if the divergent competence gene expression seen in different pression . The dat5 and with 2 × 106 TIGR4 and FP184 drawn from the identical frozen stocks as for the previous experiment (P < 0.05) and challenged with FP184 (**P < 0.002) is significantly higher than survival of the group of mice challenged with TIGR4 and treated with CSP. When comparing the data of the pneumonia experiment with the data of the bacteraemia experiment it is obvious that induction of competence by CSP or inhibition of the competence cascade by mutation of comD has opposite effects in the two models. These virulence data show that in bacteraemic sepsis, where competence genes are found to be less expressed, further reduction of competence (i.e. in the comD mutant) results in more severe disease, while upregulation of competence (i.e. by using CSP) decreases the severity of disease. In contrast, when competence genes are more expressed, as in pneumonia and meningitis, reduction of competence results in decreased virulence, while upregulation of competence increases disease severity.Intranasal challenge was done with 2 × 10periment . Groups periment , and trein vitro, either planktonic or sessile, conferred characteristics with a direct impact on virulence we used bacteria from biofilm and form liquid culture for challenge. Pneumococci detached from microtiter wells and from mid log exponential growth were used in parallel for challenge in our IV sepsis model, the IN pneumonia model and the IC meningitis model. As shown in P = 0.021) and for the IC challenge (P = 0.045). In the IN experiment biofilm bacteria are more virulent than liquid culture bacteria, as in the IC model, but data are not statistically significant at this sample size (P = 0.067). It should be noted that none of the two challenge strains in this set of infections was mouse passaged, indicating that mouse passage is not essential for pneumococcal virulence.In order to evaluate if the physical state in vivo conditions of sepsis without primary focus of disease and tissue infection were correlated to the in vitro models of growth in liquid medium and to a newly developed biofilm model respectively. Recent data have suggested that the competence regulon could be involved in the regulation of such in vivo and in vitro systems  are similar in their signal to sensor ratio when compared with the concentrations inducing transformation (2–8 ng ml−1 of CSP for cell densities below 106 cfu ml−1)  with in vivo models , we chose to use quantitative real time RT-PCR. This approach was chosen as this is the only technology able to quantify a range of targets ranging from 104  to over 108 (severe sepsis), which is the range of concentrations bacteria reach in pneumococcal infection models  out of the 29 genes monitored showed increased expression in this environment, when compared with pneumococci in tissue. Gene expression patterns in lung  or brain  were identical to each other but differed from that in blood  renders the bacteria even more virulent in a disease were the planktonic state is the hallmark. The main challenge in this work was to evaluate if this in vivo situation, in which we could induce or reduce disease severity, could be repeated in a disease model with an opposite pattern of competence expression. We chose for this our IN infection model, as pneumococci in pneumonia were shown to upregulate competence genes. As predicted, the co-administration of CSP with bacteria significantly increased disease severity and morbidity in pneumonia. This is due to the fact that in a disease in which pneumococci behave as a biofilm, the forcing of this shift towards a biofilm state, by addition of synthetic CSP, exacerbates disease. In contrast, the comD mutant was less virulent in this model. These data are in accordance with data of others  (agr system) (com system). The quorum-sensing regulatory mechanisms of these models have been proposed as a novel drug target using derivatives of the quorum-sensing molecules as lead compounds  the application of validated tools to modulate virulence in vivo, we have demonstrated that pneumococci during infection of the host are in different physical states depending on the disease. Pneumococci during infection of tissues (meningitis and pneumonia) resemble a biofilm-like mode of growth, while bacteria in the blood of septic mice resemble the in vitro model of growth as in liquid medium. The recognition of two diverse physiological conditions of pneumococci during infection and the description of in vitro correlated models may open new approaches for understanding the pathogenesis of pneumococcal infection.In a three-step process consisting in (i) the description of gene expression patterns in models of infection, (ii) the identification of 2-enriched atmosphere. Bacterial stocks were prepared from mid log cultures and stored frozen at −80°C in 10% glycerol. Antibiotics where appropriate were used at the following concentrations: kanamycin 500 μg ml−1, spectinomycin 100 μg ml−1, chloramphenicol 3 μg ml−1 and novobiocin 10 μg ml−1.Pneumococcal strains used in this work are reported in comC was obtained by transforming TIGR4 with a genetic cassette amplified from FP5  were diluted 1:100 in 200 μl of TSB with or without the addition of CSP  at various concentrations (0–300 ng ml−1). Plates were incubated for 18–24 h at 37°C in a CO2-enriched atmosphere. Turbidity of bacterial cultures (OD590) was measured by using the VERSAmax Microplate Reader ELISA apparatus . To remove planktonic bacteria, wells were washed four times with ice-cold TSB, and added with 100 μl of TSB containing 10% glycerol. To detach biofilm bacteria, plates were subjected to a 2 s sonication in a water bath . Detached cells (100 μl) were recovered from plates for cfu counts and real time RT-PCR or directly frozen. Sonication for 5 or 10 s did not increase cell recovery, while sonication for 30 s killed bacteria.Bacteria were grown in flat-bottom polystyrene tissue culture plates . Frozen pneumococcal cultures  in 2 ml of CSP-supplemented TSB for 18 h in anaerobiosis. After washing the plates with TSB, wells were air-dried, fixed with methanol for 1 min, stained with 2% alcian blue 8GX (Sigma) for 10 min, rinsed, stained with 4% crystal violet (Sigma) for 5 s, rinsed again, and finally dried. Phase contrast microscopy was carried out on washed samples without fixation and staining. Examination of biofilm morphology was done by using a Leica DM1000 Microscope  and a Leica DFC digital camera .in vivo , (ii) time-course pneumonia model for gene expression analysis in vivo , (iii) pneumonia model to study the effect(s) of CSP in vivo  and (iv) an assay for infectivity of pneumococci harvested from either biofilm or from liquid culture in models of pneumonia, meningitis and sepsis . Mouse-passaged pneumococci, prepared as previously described    (n = 12) were lightly anaesthetized with 2.5% (v/v) fluo-thane  over oxygen (1.5–2 l min−1) prior to IN infection with 50 μl of PBS containing 1 × 106 cfu of TIGR4. At set time-points, groups of mice were deeply anaesthetized with 5% (v/v) fluothane. Blood, bronchoalveolar lavages and lung samples were collected. In experiment (iii), CD1 mice were infected IN as described (n = 16/group) were challenged with either TIGR4, or FP184, or with TIGR4 and CSP2 (1.3 μg mouse−1). Experiments were performed with two different bacterial doses (2 × 105 and 2 × 106 cfu). In experiment (iv), the inoculum preparation differed from the previous three ones. Bacteria were harvested either from mid-log phase cultures by centrifugation or from the bottom of microtiter plates by sonication. Both types of inocula were resuspended in TSB glycerol, stored frozen as aliquots, and diluted in TSB for challenge. Pneumococci were not mouse-passaged prior to infection. MF1 mice were infected either IC with 102 cfu (n = 5), or IN with 2 × 105 cfu (n = 10), or IV with 103 cfu (n = 6). All mice that became severely symptomatic were humanely sacrificed. Statistical analysis of survival was done by using the chi-square test.Meningitis, pneumonia and sepsis models of infection were used. MF1 and CD1 mice were purchased by Harlan Nossan  and Charles River Italia  respectively. Experiments were carried out either at the University of Siena or at the University of Leicester according to the respective national and institutional guidelines. Four different types of experiments were performed: (i) end-stage disease by using meningitis, pneumonia and sepsis models to analyse gene expression 590 = 0.2) were chilled on ice, harvested by centrifugation, and the bacterial pellet was stored frozen at −80°C until use. Bacteria cultured on TSA plates were collected after 18 h of incubation at 37°C. Plates were incubated in a candle jar with a burning candle to avoid excessive oxidative stress or pH drop resulting from adding CO2 for anaerobic incubation. Bacteria were recovered from agar in 1 ml of ice-cold TSB, put on ice, collected by centrifugation and frozen at −80°C. Pneumococci were detached from biofilms as described above. Mouse samples were obtained from individual animals immediately after the sacrifice. Brains homogenates, lung lavages and blood were collected from mice with meningitis, pneumonia and sepsis respectively. As the amount of CSF obtainable from mice does not exceed few microlitres, we chose to use whole brain homogenates as representative samples for evaluation of gene expression in meningeal infections. Blood was obtained by retrorbital bleeding into tubes containing 1/10 volume of 0.1 M sodium citrate; brains were homogenated in 1 ml of ice-cold PBS, and lung washes were done with 1 ml of PBS. All samples were put on ice and subjected immediately to a low speed centrifugation . The supernatant, containing the bacteria, was transferred into a new tube and centrifuged . The pellet of all samples was resuspended in 100 μl of TE buffer  before storage at −80°C.Bacteria were sampled from liquid broth, solid medium and infected mice. Pneumococcal cells grown in TSB until mid log phase (OD2O. Samples were stored frozen at −80°C. Retrotranscription was carried out by using the ‘ImProm-II Reverse Transcriptase Kit’ (Promega) according to the manufacturer's instructions as described. Briefly, annealing was performed at 25°C for 10 min and extension at 37°C for 1 h. Samples were inactivated at 70°C for 15 min and immediately subjected to real time PCR. Quantitative real time PCR was performed as previously described (2 to 106 target copies per reaction (104−108 target copies per sample), and only oligonucleotides with comparable efficiency were chosen. Primers spanning 100–150 bp segments were designed by standard procedures and are reported as Supplementary material (gyrB (SP0806), comA (SP0042), lytA (SP1937), comX1 (SP0014) and dprA (SP1266) were as previously described  towards expression in TSB. For each analysis, three to five distinct biological replicas were done, and quantitative data were expressed as mean ± SD. Values of fold change in gene expression above 2 or below 0.5 as determined by the 2–ΔΔCT method were considered significant.RNA was extracted by using ‘SV Total RNA Isolation System Kit’ (Promega) according to the manufacturer's instructions with few modifications: treatment with DNAse was shortened to 10 min and elution of RNA was in 50 μl of nuclease-free Hmaterial . PrimersT method . The ref


Despite bacteraemia is present in the majority of patients with pneumococcal, little is known about the influence of the systemic infection on the meningeal inflammatory response.6 CFU Streptococcus pneumoniae, type 3, and the 26 rabbits were either provided with ~1 × 106 CFU S. pneumoniae intravenously at 0 hour , immunized with paraformaldehyde-killed S. pneumoniae for 5 weeks prior to the experiment , or not treated further . WBC and bacterial concentrations were determined in CSF and blood every second hour during a 16 hours study period together with CSF IL-8 and protein levels. We also studied CSF and blood WBC levels in 153 pneumococcal meningitis patients with and without presence of bacteraemia.To explore the role of systemic infection on the meningeal inflammation, experimental meningitis was induced by intracisternal injection of ~1 × 10P < 0.05), whereas no differences in CSF bacterial levels were observed (P > 0.05). Blood WBC decreased in bacteraemic rabbits between ~10–16 hours after the bacterial inoculation in contrast to an increase for both the immunized rabbits and controls (P < 0.05). The CSF pleocytosis was attenuated in bacteraemic rabbits as compared to the two other groups between 12–16 hours from time of infection (P < 0.017), despite accelerated CSF IL-8 levels in bacteraemic rabbits.As designed, blood bacterial concentrations were significantly different among three experimental groups during the 16 hours study period , but there was a significant correlation between CSF and blood WBC .In patients with pneumococcal meningitis, no significant difference in CSF WBC was observed between patients with or without bacteraemia at admission  vs. n = 50, 1961 cells/μL (673–5182), respectively, Our results suggest that a decrease in peripheral WBC induced by enhanced bacteraemia in pneumococcal meningitis results in an attenuated CSF pleocytosis. Streptococcus pneumoniae meningitis is characterized by an accumulation of leukocytes within the subarachnoidal space. It has been suggested that host inflammatory reactions to the invading pathogen are largely responsible for the poor outcome of pneumococcal meningitis, because pathophysiological alterations leading to death continue to evolve after the bacterial eradication by antibiotic therapy , whereas only sparse information has been generated about the influence of pneumococcal bacteraemia on the recruitment of leukocytes to local infections sites. In contrast to our results, Sato et al. [Several studies have previously documented an altered leukocyte function during endotoxemia  declare that they have no competing interests.C∅ provided the scientific idea of the study, designed the study, and drafted the manuscript. TOR, CB, NFM, and JDL took part in preparation of the manuscript. All authors approved the final manuscript.The pre-publication history for this paper can be accessed here:


Subacute sclerosing panencephalitis (SSPE) is a rare, slowly progressive neurological disorder caused by the persistent infection with measles virus (MV). Despite much research into SSPE, its pathology remains obscure. We examined autopsy tissues of eight SSPE patients by real time quantitative PCR, immunohistochemistry and immunoblotting to determine viral load. MV N, M and H gene RNA could be detected in the central nervous system (CNS) of all patients and in two non-CNS tissues of one patient. The viral burden between patients differed up to four-fold by quantitative PCR and corresponded with detection of MV protein. The level of both viral RNA and antigen in the brain may correlate with disease progression. Subacute sclerosing panencephalitis (SSPE) is a rare, slowly progressive neurological disorder of children and young adults, caused by the persistent infection with measles virus (MV). After acute infection with MV, typically at a very young age, the patients appear to recover normally. Following a period of asymptomatic persistence, usually 3 to 10 years, neural dysfunctions develop and progress over months to years, resulting eventually in coma and death. Although much effort has gone into researching SSPE, its pathology remains largely obscure. The progression of SSPE greatly differs in individual patients [Viral load has been correlated to disease development, progression and outcome in several infections, including HIV, SIV, cytomegalovirus ,3 and EpAs MV can usually be detected in brain tissue from biopsy or autopsy by RT-PCR and/or immunohistochemistry, it is thought to persist and spread within the CNS. Although the brain appears to be the main location of persistent MV in SSPE, it is not entirely clear if this is also the site of persistence and the only target during disease. Viral antigen or RNA have been detected outside the CNS in SSPE cases -10. To aThis study was carried out to determine whether differences in viral load caused diversity of disease progression. Autopsy tissues of brain and non-brain origin of eight confirmed cases of SSPE were analysed by quantitative PCR to determine amounts of the MV RNAs encoding the nucleoprotein (N), matrix protein (M) and haemagglutinin (H) and by immunological techniques to detect the N protein.Autopsy or biopsy tissues of eight SSPE patients were analysed, details are shown in Table 6 copies of N RNA per ng total RNA in occipital brain patient UK111 to 4 × 102 copies/ng RNA in temporal brain of case UK585, respectively. In addition to relating copy numbers to the amount of total RNA, they were standardised as a function of GAPDH mRNA. GAPDH  is a ubiquitously expressed message, which is frequently used to standardise across tissues. Although expression of GAPDH may not be uniform across all tissue types, this method was found acceptable in a comparison of nine CNS and non-CNS tissues from three SSPE patients. The maximum fold change between CNS tissues was <8-fold, between CNS and non-CNS tissues <11-fold  in all available CNS tissues from eight SSPE patients and in two non-CNS tissues of one patient. The concentration of MV N gene RNA in CNS tissues ranged from almost 2 × 106 copies of H RNA per ng total RNA. Again the weakest positive CNS sample was temporal brain of UK585 with just under 2 × 103 copies/ng RNA. This is approximately 9 × 103 %GAPDH and 3% GAPDH, respectively. The M gene signal was approximately ten-fold weaker, but the different cases were similar in relation to each other as with the N and H genes. Occipital brain of UK111 contained 4 × 105 copies M gene/ng RNA or 3 × 102 %GAPDH. This gene could not be detected in temporal brain tissue of patient UK585. In PCRs with MV N, M and H gene targets, occipital brain of UK111 was the strongest positive when looking at copies/ng RNA, while the brain of UK85 contained most MV RNA when expressed as a function of GAPDH. The efficiency of all three PCR reactions was found to be similar when titrations of p(+)MV, a MV genome containing plasmid, were used as template (data not shown), which indicates that the numbers obtained with the different PCRs can be compared. The M and H gene results confirm the relative findings with the N gene. All quantities are illustrated in Figure MV N RNA positive samples were also used for detection of MV M and H gene RNA. The H gene PCR lead to very similar results as the N gene PCR, both in copy numbers and %GAPDH. Occipital brain tissue of UK111 contained just under 2 × 10To determine whether the detected viral RNA is actively replicating, immunological methods were used to visualise MV protein in tissues from patients UK111, UK98 and UK585. In occipital brain tissue of UK111, a large number of MV N protein positive cells could be detected by immunohistochemistry, as seen in Figure To detect MV protein with higher sensitivity, tissue lysates were analysed by immunoblotting Figure . In agreA weak inverse correlation between viral loads measured by qPCR and SSPE disease duration was detected. Quantities of viral RNA were compared to durations of SSPE and of infection from primary MV acquisition to sample date. Results are shown in Figure 3 and 1 × 105 %GAPDH, respectively, was detected. The M gene of virus in case UK585, on the other hand, was strongly mutated and low virus levels of <1% GAPDH were measured. In case UK83, however, viral load was high (1 × 103 %GAPDH), despite presence of virus with a highly mutated M protein.To investigate a possible link between virus integrity and viral spread, MV loads detected here were compared with the extent of MV M gene and protein mutation reported previously . In caseBy Spearman's ranked correlation, no statistically significant links were found between viral load, duration of disease or infection and number of total or expressed mutations in the M gene of the isolated viruses.6 copies of MV N RNA could be detected per ng total RNA, approximately 100 times more than in the brain samples of UK98. The brain of UK111 was the only analysed tissue positive by immunohistology and was a much stronger positive by immunoblotting than specimens from UK98. In the brain of UK585, which was negative by immunoblotting, only 4 × 102 to 2 × 103 copies could be detected. Thymus and appendix of UK98 were the only non-CNS samples positive for the N gene by real time PCR. GAPDH normalisation of these samples may be less reliable due to greater heterogeneity of tissue origin. However, taking into account exclusively the copy numbers per total RNA, they were weak positives, contained fewer than 102 copies per ng RNA.Great variation in levels of MV RNA and protein were detected in different SSPE patients, while findings with quantitative PCR and immunological methods are in agreement. In the occipital brain of UK111, 2 × 10A weak correlation between the amount of MV RNA and the duration of SSPE was observed. As some CNS samples contained relatively little MV RNA, high viral load alone did not seem to be responsible for death, but may have accelerated destruction of tissue. MV specific interferon-gamma release by host immune cells may determine disease progression . It is fAs the MV genome is encoded in RNA, PCR detection does not distinguish between genomic and mRNA. It is therefore not known if the identified virus is actively transcribed, but presence of MV protein indicates that the virus is transcribed. However, it is not clear whether a larger number of MV RNA copies indicate a greater number of infected cells or greater number of MV RNA copies per infected cell. Increased MV RNA levels per cell may indicate a more actively replicating virus, which may causes faster progressing disease. Alternatively, infection of a greater number of cells may lead to destruction of more tissue, which may also lead to accelerated disease.Of patient UK98, tissues from five brain areas, frontal, parietal, occipital, temporal lobes and cerebellum, were available for analysis. Virus levels in the cerebrum – frontal, parietal, occipital and temporal lobes – were within a factor of 3.5, which may indicate that the virus did not favour any one particular area. Alternatively this may be an indication of very slow disease progression, as is the case in this patient, during which the virus had sufficient time to spread into all regions. In contrast, infection of the cerebellum was 30 to 100 times lower. The cerebellum is a very extensively connected part of the brain, which integrates information from the cerebrum as well as the periphery. Both efferent and afferent pathways link it to the cerebrum. Lack of axonal connection does therefore not cause this lower level of infection. It may rather be the different cell types present within this organ. The cerebellar neurons, the granule cells, may be less susceptible to MV infection.The detection of MV RNA in non-CNS tissues was very low, which indicated that these tissues were not reservoirs of infection. The detected RNA may have derived from infiltrating lymphocytes that were infected within the CNS. Peripheral lymphocytes of individuals with SSPE have been shown to contain MV . AlternaIn summary, the amount of MV protein and RNA differs greatly in different cases of SSPE, which may influence progression of the disease. Further studies of greater numbers of cases are required to confirm these findings.® SYBR® Green qPCR SuperMix UDG , 0.2 μM of each primer and 6.5 μl nuclease free water, using a Rotor-Gene 3000  with the following cycling conditions. Hold at 50°C for 2 min, hold at 95°C for 3 min, followed by 45 cycles of 95°C for 15 sec, 55°C for 30 sec, 72°C for 30 sec. Data was acquired at 72°C during each cycle with excitation wavelength of 470 nm and detection at 585 nm. Amplification was followed by melt analysis from 50–90°C. All samples were run as duplicates and analysed using Rotor-Gene 6.0.7 software. Integrity of all samples was demonstrated by amplification of GAPDH mRNA. DNA and RNA standards were included in each run and each reaction was performed in duplicate. For verification of correct amplification, PCR products were run on agarose gel and sequenced.Tissues were homogenised and total RNA extracted with RNeasy Mini kit . RNA was treated with DNase to avoid amplification of genomic DNA. The concentration of extracted RNA was determined by spectrophotometry and 100 ng RNA reverse transcribed using random primers. PCR primers were designed using Primer3 software  to yieldDNA and RNA standards were used for qPCR and RT-qPCR, respectively. The concentration of a plasmid containing the entire MV genome, p(+)MV , was determined by spectrophotometry. A ten-fold dilution series was made and used as DNA standard. From this plasmid, the whole N, M and H genes were amplified by PCR and cloned into pGEM . The pGEM plasmid was linearised with SpeI and the MV fragments transcribed in vitro using the T7 Riboprobe System  according to the manufacturer's instructions. The resulting RNA was purified and the concentration determined by spectrophotometry. Ten-fold dilution series were made and included in the RT reactions as RNA standards.In order to compare results from different tissues and correct for use of different numbers of cells in each sample, two normalisation methods were employed. First, the same concentration of total isolated RNA was used for each RT-PCR. This lead to results expressed as copies of MV RNA per ng total RNA input. Alternatively, a housekeeping gene was employed to compare RNA levels between different samples. To find an appropriate gene to normalise sample input, 5 ng RNA from nine samples were used for qPCR with primers against all three housekeeping genes, beta-actin, GAPDH and 18S rRNA. Results are shown in Table 2O2 in methanol. Tissues were washed in water and blocked in blocking solution ) for 10 min. Sections were incubated with a monoclonal mouse antibodies against the N protein  at a diTissues were homogenised in lysis buffer  and protein concentrations of whole lysates, without removal of cell debris, determined by spectrophotometry. Equal amounts of total protein of each sample were analysed by SDS-PAGE and immunoblotting. The 7C11 MV N specific antibody was used to probe the blots, followed by detection with an anti-mouse-HRP antibody  and ECL plus reagents .MKS conceived the study, carried out the laboratory work and drafted the manuscript. JL and DWGB participated in the design of the study and the revision of this manuscript. All authors read and approved the final manuscript.


Restoration of p21 expression was accomplished using adenoviral infection. As anticipated from the loss of a cell cycle inhibitor, p21(-/-) FLS grow more rapidly than WT FLS. In examining migration towards biologically relevant RA synovial fluid, p21(-/-) FLS display a marked increase  in migration compared to WT cells. Moreover, this effect is independent of the cell cycle since chemical inhibitors that block the cell cycle have no effect on migration. In contrast, p21 is required to repress migration as restoration of p21 expression in p21(-/-) FLS reverses this effect. Taken together, these data suggest that p21 plays a novel role in normal FLS, namely to repress migration. Loss of p21 expression that occurs in RA FLS may contribute to excessive invasion and subsequent joint destruction.Rheumatoid arthritis (RA) is characterized by synovial hyperplasia and destruction of cartilage and bone. The fibroblast-like synoviocyte (FLS) population is central to the development of pannus by migrating into cartilage and bone. We demonstrated previously that expression of the cell cycle inhibitor p21 is significantly reduced in RA synovial lining, particularly in the FLS. The aim of this study was to determine whether reduced expression of p21 in FLS could alter the migratory behavior of these cells. FLS were isolated from mice deficient in p21 (p21 Proper regulation of the mammalian cell cycle is vital for cellular homeostasis. Alterations in the cell cycle components have been associated with several disease states. Progression through the different phases of the cell cycle is dependent on the activities of cyclin dependent kinases (cdks) bound to their cognate cyclins ,2. AnothRheumatoid arthritis (RA) is a chronic inflammatory and destructive disease . The fib(-/-)  and wild-type  mice were purchased from the Jackson Laboratory . Mouse knees were excised from WT or p21(-/-) mice and pooled. Isolated mouse synovial tissues were digested with collagenase, dispase, and DNAse I, and single cell suspensions were obtained [p21obtained ,25,26. ASynovial fluid (SF) specimens were collected during arthrocentesis from patients who met the American College of Rheumatology criteria for a diagnosis of RA. All specimens were obtained with approval of Midwestern University's Institutional Review Board.(-/-) fibroblasts were plated at 1.8 × 104 cells/well in 1 ml DMEM + 10% FBS in a standard 24-well tissue culture plate and incubated at 37°C. At different time points cells were washed with PBS, trypsinized and their number quantified by hemocytometer counts with trypan blue. Analysis was performed in triplicate wells and results are expressed as the mean ± standard error (SE).WT and p21(-/-) FLS were cultured for 18 h in DMEM containing 1% FBS. One hour prior to the assay, this media was removed, cells were rinsed twice with PBS, and media was replaced with DMEM containing 0.1% FBS. FLS (2.6 × 104 cells/well in DMEM + 0.1% FBS) with or without mitomycin C  were added to the bottom wells of a 48-well microchemotaxis chamber . The chambers were inverted and incubated for 2 h (3 h when MMC was present) in a 5% CO2 atmosphere at 37°C, allowing for cell attachment to the membrane. Upon righting the chambers, dilutions of synovial fluids collected from patients with RA (or control agents) were added to the top wells and the chambers were incubated overnight at 37°C. PBS served as a negative control, whereas recombinant human basic fibroblast growth factor  served as a positive control. The next morning, non-migrated cells were detached with a cotton swab, membranes were removed, fixed in methanol, and stained with Diff-Quik . Checkerboard analysis was performed in a similar manner, except that the concentrations of RA SF were varied in the upper and lower chambers. Dilutions of RA SFs  were added to the cell suspension in the bottom wells as well as on the opposite side of the membrane, when appropriate. Each condition was analyzed in quadruplicate and migrated cells from membranes mounted on glass slides were quantified in three representative high power fields. Quantification of high powered fields was accomplished by analyzing photographs of chemotaxis spots taken with a Nikon Coolpix E5000 (5.0 megapixel) camera mounted on a Nikon Eclipse TS100 inverted microscope. Chemotaxis data appeared normally distributed based on examination of histogram plots, and statistical analysis was performed using a Student's t-test.Chemotaxis was performed with minor modifications to a protocol previously optimized for RA synoviocyte chemotaxis . WT and (-/-) FLS were treated with identical conditions as used for chemotaxis assays described above. Cells were trypsinized, counted using trypan blue, plated at 2.6 × 104 cells/well into two separate chemotaxis chambers, and inverted to allow cell adherence to the membrane. FLS from one chamber were fixed with MeOH exactly 2 h after plating, allowing sufficient time for cell attachment but not enough time for proliferation. Counts from this chamber allowed an exact determination of the number of cells plated for each group. WT and p21(-/-) FLS in the second chamber were plated with or without 10 μg/ml MMC. After 2 h, the second chamber was righted and PBS was added to the top side of all wells. After an 18 h time period, the time allowed for migration in all chemotaxis experiments, the membrane was removed and the WT and p21(-/-) FLS were fixed with MeOH and counted.To assure that local proliferation on the membrane was not occurring, WT and p21(-/-) FLS were infected with an adenovirus overexpressing p21 (Adp21) and compared to mock infected WT FLS, mock infected p21(-/-) FLS or p21(-/-) FLS infected with an adenovirus that produces an irrelevant bacterial protein . Initially, FLS infected with Adp21 and AdlacZ were subjected to β-galactosidase staining to estimate a concentration that resulted in infection of nearly 100% of cells . All infections were completed by incubation with virus for 4 h in DMEM + 5% FBS. Mock infected WT and p21(-/-) fibroblasts were incubated with the same media in the absence of virus. After incubation, cells were washed three times with PBS and full growth media was replaced for 4 h. Cells were incubated overnight in a 1:10 dilution of the full growth media. Chemotaxis assays were performed as described above the following day.To restore p21 expression, p21(-/-) mice. We first assessed whether FLS from p21(-/-) mice possessed proliferative characteristics, as was expected from the loss of a cell cycle inhibitor. Figure (-/-) FLS were plated into 24-well plates. At various time points, cell number was determined and by 44 h after plating the cells, more p21(-/-) FLS were consistently present relative to WT FLS  (data not shown) FLS migrate to RA SF in a dose-dependent manner. Even RA SF dilutions of 1:1000 from some patients could significantly increase FLS migration when compared to background migration represented by PBS (p < 0.05).Little is known about whether the reduced p21 expression exhibited in RA FLS may be related to alterations in FLS migration. To address this issue, we isolated FLS from knee synovium of WT and p21S Figure  and p21((-/-) FLS in response to RA SF, we employed 48-well microchemotaxis chambers and determined cell motility towards dilute RA SFs. Cell counts were normalized to their background migration by representing the data as fold-increase over PBS. Figure (-/-) FLS when compared with WT FLS (p < 0.05). A representative chemotaxis experiment is shown in Figure (-/-) FLS. The data shown are representative of three independent experiments, which consistently suggest that p21(-/-) FLS exhibit significantly enhanced migration towards RA SFs (p < 0.05).To directly compare migration of WT and p21(-/-) FLS may simply be the result of a non-specific enhancement of chemokinesis, we performed several checkerboard assays. A representative assay (Table To examine whether the increased migration of p21(-/-) FLS in the presence and absence of MMC at the end of the assay and compared it with the number of cells plated at the start of the assay. Figure (-/-) FLS treated with MMC was significantly below the number of p21(-/-) FLS that were not MMC treated (p < 0.05). In contrast, there were no significant differences between WT and p21(-/-) FLS that were or were not MMC treated. This suggests that MMC completely halted proliferation and that any demonstrable changes in chemotaxis in the presence of MMC are not due to proliferation on the membrane.To examine whether the enhanced migration could be explained by differences in proliferation, we performed chemotaxis assays using cells that were pretreated with MMC to stop cell division. To assure that local proliferation on the membrane was not occurring in the presence of MMC, we determined the number of WT and p21(-/-) and WT FLS chemotaxis assays using seven separate RA SFs in nine independent experiments, there is significantly more migration by the p21(-/-) FLS compared with WT FLS (p < 0.05). Figure (-/-) FLS. It should be noted that the counts in chemotaxis assays using mouse FLS isolated on different dates occasionally varied greatly between experiments (background migration as well as induced migration), although the trend was always identical, such that the p21(-/-) FLS migrated significantly more than the WT FLS. Therefore, in the presence of MMC, a cell division inhibitor, we still demonstrate that loss of p21 confers an increased migratory behavior on FLS when compared with WT cells (p < 0.05). Of note in Figure (-/-) FLS.Figure (-/-) and WT fibroblasts. Therefore, the enhanced migration may be specific for another chemoattractant, or a combination of chemoattractants found in the RA SF.To begin to assess whether the enhanced migration may exhibit specificity with regards to the chemoattractant used, we performed several chemotaxis assays using bFGF as the chemoattractant. Figure (-/-) FLS with p21 would reduce their excessive migration. First, to demonstrate that our adenoviral construct produced functional p21, we infected p21(-/-) FLS and compared the growth rate with that of p21(-/-) FLS infected with an adenovirus producing green fluorescent protein. Growth of FLS expressing p21 was significantly inhibited compared with FLS expressing green fluorescent protein . Next, to establish whether p21 is solely responsible for the enhanced migratory characteristics, we reconstituted p21(-/-) FLS with p21 and compared chemotaxis to cells infected with AdlacZ. Figure (-/-) FLS infected with Adp21 was comparable to WT FLS that were mock-infected. This migration was significantly below mock-infected p21(-/-) FLS or those infected with an adenovirus producing an irrelevant bacterial protein, β-galactosidase.We next aimed to use an adenovirus to increase p21 and establish whether reconstituting p21(-/-) mice. We first assessed whether FLS from p21(-/-) mice possessed proliferative characteristics, as was expected from the loss of a cell cycle inhibitor. Figure Expression of p21 in RA synovial tissue is significantly decreased when compared with the same tissue from osteoarthritis patients. Expression of p21 inversely correlates with thickness of the RA synovial lining . Similar(-/-) FLS migration to RA SF suggests that p21(-/-) FLS exhibit significantly enhanced migration (p < 0.05). Our checkerboard assays (Table (-/-) FLS proliferate quicker than WT FLS, the non-MMC treated FLS in Figure (-/-) mice migrated more than those from WT mice. Overall, chemotaxis assays performed in the presence of a cell cycle inhibitor suggest that the changes noted are not the result of enhanced proliferation by migrating cells. Moreover, reconstitution of p21 into p21(-/-) FLS is sufficient to significantly reduce migration of these cells, suggesting that the loss of p21 in FLS may enhance their ability to migrate towards the proinflammatory constituents of the RA joint.A direct comparison of WT and p21(-/-) embryonic fibroblasts were also examined in this study, but were determined to not exhibit differences in migration when compared with WT cells [(-/-) fibroblasts that were isolated from synovium obtained from 5–8 week old mice, whereas the previous study employed fibroblasts derived from an embryo. The previous study examined cell migration by wounding of a confluent monolayer of cells, whereas our study examined specific directed chemotaxis and chemokinesis. An additional key difference is that our study used RA SF as the chemoattractant to assess whether the multiple biological constituents of these disease-related fluids may display differences in chemoattraction between p21(-/-) and WT fibroblasts. In fact, when we utilized bFGF as a sole chemoattractant in seven independent chemotaxis assays, we do not see consistent differences between migration of p21(-/-) and WT fibroblasts, which is in-line with the previously published findings where p21(-/-) assays were performed in the presence of growth serum [Recently, Besson and coworkers  demonstrWT cells . There ath serum .(-/-) VSMCs [(-/-) cells with exogenous p21, transduction of Gax cDNA once again inhibits VSMC migration. Thus, increasing Gax upregulates p21 and inhibits VSMC migration. This appears consistent with our studies, in which a loss of p21 in FLS results in excessive migration and reconstituting p21 is capable of reducing the exuberant migration to RA SF. Overexpressing p21 alone in WT VSMCs did not influence cell migration. In our hands, similarly, infecting WT FLS with a high titer of Adp21 did not inhibit cell migration to RA SF (data not shown).Previous studies in vascular smooth muscle cells (VSMCs) involving the homeobox transcription factor growth-arrest specific homeobox (Gax) have also established a tie between p21 and cell migration . Overexp-) VSMCs . Upon rein vitro has recently been associated with the rate of joint destruction in vivo [FLS locomotion can be regarded as an important pathogenic mechanism contributing to the invasion of cartilage and bone in RA. Grafting of RA FLS alone to SCID mice, in the absence of a functional immune system, results in chronic arthritis, underscoring the potential key role of these cells in driving disease ,33. This in vivo . Invasiv in vivo , and the in vivo . We demoWe previously demonstrated that RA FLS exhibit decreased expression of p21, and now observe that a lack of p21 may contribute to excessive migration of FLS. These data suggest that p21 plays a novel role in normal FLS in repression of migration. Further, a lack of p21 expression, as occurs in RA FLS, may contribute to excessive invasion towards the biological chemoattractants found in RA SF.AdlacZ = adenovirus expressing β-galactosidase; Adp21 = adenovirus expressing p21; bFGF = basic fibroblast growth factor; cdk = cyclin-dependent kinase; DMEM = Dulbecco's modified Eagle's medium; FBS = fetal bovine serum; FLS = fibroblast-like synoviocytes; Gax = growth-arrest specific homeobox; MMC = mitomycin C; PBS = phosphate-buffered saline; RA = rheumatoid arthritis; SF = synovial fluid; VSMC = vascular smooth muscle cell; WT = wild-type.The authors declare that they have no competing interests.JMW designed and developed all aspects of the study, performed chemotaxis experiments, drafted the manuscript, and addressed reviewers concerns. KK, DJS, NGS, and MSR each contributed a significant number of chemotaxis assays, worked with adenoviruses, and participated in growth and maintenance of FLS. DJP performed the majority of chemotaxis counting, while MVV participated in chemotaxis counting as well as in the design of experiments and interpretation of data. JCS performed all animal work and isolation of FLS cultures from mice. HP conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.


Cardiopulmonary bypass (CPB) induces hemodilutional anemia, which frequently requires the transfusion of blood products. The objective of this study was to evaluate oxygen delivery and consumption and clinical outcome in low risk patients who were allocated to an hematocrit (Hct) of 20% versus 25% during normothermic CPB for elective coronary artery bypass graft (CABG) surgery.This study was a prospective, randomized and controlled trial. Patients were subjected to normothermic CPB (35 to 36°C) and were observed until discharge from the intensive care unit (ICU). Outcome measures were calculated whole body oxygen delivery, oxygen consumption and clinical outcome. A nonparametric multivariate analysis of variance for repeated measurements and small sample sizes was performed.n = 28; 20% Hct, n = 26), calculated oxygen delivery (p = 0.11), oxygen consumption (p = 0.06) and blood lactate (p = 0.60) were not significantly different between groups. Clinical outcomes were not different between groups.In a total of 54 patients (25% Hct, These data indicate that an Hct of 20% during normothermic CPB maintained calculated whole body oxygen delivery above a critical level after elective CABG surgery in low risk patients. The question of whether a transfusion trigger in excess of 20% Hct during normothermic CPB is still supported requires a larger prospective and randomized trial. Hemodilution occurs during cardiac surgery when cardiopulmonary bypass (CPB) is instituted. Hemodilution reduces blood viscosity and vascular resistance, and may increase large vessel blood flow to maintain whole body oxygen delivery . It does2) during CPB. Hct level is used as a measure for triggering transfusion, but transfusion carries a wide range of complications and appears to worsen outcome after coronary artery bypass graft (CABG) surgery [Debate exists on the minimum safe level Hct necessary to maintain oxygen delivery (DO surgery . Moreove surgery  and tran surgery . Results surgery  found a  surgery  reported surgery  describe2) was kept stable at Hct levels of 0.14, 0.11 and 0.10 in the respective temperature groups. The authors of this animal study concluded that the compensatory increase in cerebral blood flow at an Hct of 0.14 met the increased rate of cerebral oxygen demand even at a body temperature of 38°C. Although results from controlled clinical studies reported similar or beneficial outcome after normothermic CPB [Most data on the relationship between hemodilutional anemia and outcome have been measured during hypothermic (28 to 32°C), moderate hypothermic (32 to 34°C) or normothermic CPB. Temperature regulation favoring hypothermia during CPB was found to reduce cerebral oxygen consumption , preservrmic CPB ,18, cardResults from controlled trials investigating the effect of hemodilution on oxygen delivery and clinical outcome in patients undergoing normothermic CPB have not yet been published. Therefore, the objective of this prospective, randomized and controlled study was to investigate oxygen delivery and consumption and the clinical outcome of patients who were randomly allocated to one of two Hcts (20% or 25%) during normothermic CPB.After institutional approval by the local ethics committee and preoperative written informed consent, 57 patients were considered eligible for this randomized, controlled clinical trial from February 2004 until November 2004. Patients were allocated to the trial groups according to a computer-generated random list. One patient had to be excluded from analysis as the donated autologous blood showed multiple clots and could not be retransfused. Informed consent was withdrawn by one patient and one patient had to be excluded due to the preoperative decision for a combined surgical procedure. In total, 54 patients (28 in the 25% Hct group and 26 in the 20% Hct group) remained for statistical analysis according to the Full Analysis Set (Intention To Treat).Inclusion criteria were age >18 and <75 years, elective coronary artery bypass graft surgery, weight >70 kg and preoperative Hct >36% (hemoglobin >12 g/dl).1 <70% or FEV1/VC max <70% or partial pressure of oxygen in arterial blood (paO2) <60 mmHg, known acute or chronic hepatitis or hepatic disease with impaired synthesis of coagulation factors or bilirubin >2.0 mg/dl, known inflammatory bowel disease, known renal insufficiency or anuric renal failure or creatinine >1.5 mg/dl, ingestion of aspirin or clopidogrel until 3 days prior to surgery, and treatment with glycoprotein-receptor antagonists within 2 days before surgery. Furthermore, patients were excluded from the study if the Hct could not maintained within the targeted range during CPB, if an emergency situation  occurred before initiation of CPB and if the autologous blood could not be retransfused.Exclusion criteria were withdrawal of consent, Jehova's Witnesses, stroke in patient's history or with persistent neurological residue, unilateral occlusion of carotid artery >70% or bilateral occlusion of carotid artery >50%, combined cardiac procedure, left ventricular ejection fraction <40%, unstable angina, a left main stem stenosis >70%, ventricular arrhythmia >LOWN IVa, symptomatic chronic pulmonary disease requiring long-term medication or FEVThe standard anesthetic practice was an opioid-based anesthetic supplemented with midazolam and isoflurane as required. In all patients, a femoral artery was cannulated with a 4-Fr.-cannula  prior to induction of anesthesia. A central venous catheter and a pulmonary artery catheter  were inserted via the right internal jugular vein.®, Fresenius-Kabi, Bad Homburg, Germany) was performed to reduce the Hct to a level of 5 ± 1% above the target Hct level of 20 ± 1% or 25 ± 1%. For all measurements of Hct and blood lactate, a blood gas analyzer  was used.The standardized CPB priming consisted of 600 ml of crystalloid fluid, 500 ml of 10% hydroxyethylstarch solution and a total dose of 50,000 KIU aprotinin per kg bodyweight prior to and during CPB. Pump flow was adjusted to maintain a mean arterial pressure (MAP) of 55 to 60 mmHg and an oxygen saturation >75% during CPB. Norepinephrine was used during CPB when MAP could not be maintained within the targeted range by adjusting the pump flow. During bypass an arterial partial pressure of oxygen of 150 to 250 mmHg was maintained. Body temperature was kept between 35.5 and 36°C during CPB. CPB technique was normothermic using intermittent antegrade warm blood cardioplegia as described by Calafiore and colleagues . Before Measurements of Hct were done 2 and 5 minutes after initiation of CPB and every 15 minutes when the target hematocrit was stable. In cases where the initial Hct exceeded the targeted Hct range, crystalloid fluid was substituted, and if the hematocrit was below the target range, autologous blood was transfused.2 >60 mmHg with an inspired oxygen fraction (FiO2) of 40%, adequate neurological reaction and sufficient muscle strength. Packed red blood cells were substituted according to the following protocol: Hct <23% or lactic acidosis or ST-segment elevation or secondary organ failure attributed to hemodilutional anemia. In the ICU, the patients were not infused with crystalloids according to a standard protocol. Hydroxyethylstarch solution (6% HES 130/0.4) was given when patients showed clinical symptoms of hypovolemia.In the intensive care unit (ICU), patients were extubated as soon as possible after an observation period of 6 hours if they fulfilled the following criteria: paO2 >60 mmHg with an oxygen insufflation of 4 l per minute and a normal partial pressure of carbon dioxide in arterial blood (paCO2) and no need for continuous loop diuretics to maintain urinary output or renal replacement therapy.Discharge from ICU to the intermediate care unit was feasible when patients were in a stable clinical condition, that is, awake without neurological deficit or agitation, = 5 μg/kg/minute dopamine or no inotrope support, paOPrimary outcome measures of this trial were calculated whole body oxygen delivery, oxygen consumption and mixed venous blood lactate during CPB, at the end of surgery and in the ICU . Secondary outcome measures were: drainage loss, transfusion utilization and Hct in the ICU, incidence of secondary organ failure, hemodynamic parameters and stay in ICU.Oxygen delivery and oxygen consumption were calculated using standard formulae . Hemodynamic parameters such as MAP, central venous pressure, mean pulmonary artery pressure and pulmonary artery occlusion pressure were measured before hemodilution, at the end of surgery after retransfusion of autologous blood, one and six hours after admission to the ICU and before discharge from the ICU. Cardiac index, systemic vascular resistance and pulmonary vascular resistance were calculated using standard formulas whereas extravascular lung water and intrathoracic blood volume index were calculated at the same time points using the PiCCO plus-monitor . During CPB, pump-driven cardiac index, body temperature and blood lactate were recorded 15 minutes after institution and at the end of CPB whereas the cumulative amount of norepinephrine and urine volume were taken at the end of CPB. The incidence of acute cardiac failure, defined as need for epinephrine and/or enoximone, the need for intraaortic balloon counterpulsation for separation from CPB, and the dosage of dopamine for weaning from CPB were recorded.In the ICU, the following indicators for secondary organ failure were assessed: neurological complications defined as transitory ischemic attack, agitated arousal reaction or palsy of extremities or hemiplegia. Myocardial infarction was determined by electrocardiogram  and a ratio of creatine kinase and myocardial subtype of creatine kinase >10%. Acute cardiac failure was defined as the need for inotrope support , respiratory failure as the need for reintubation due to respiratory failure, prolonged respiratory support (>24 hours) or the need for continuous positive airway pressure breathing. Renal insufficiency was assumed when patients required renal replacement therapy, continuous intravenous loop diuretics or increase of creatinine >2.0 mg/dl. Additionally, chest drainage loss, transfusion requirements and Hct, cumulative urine volume, creatinine, ICU stay in hours and mortality were recorded.2 or Fisher's exact test for categorical and dichotomous variables, respectively. In the case of continuous variables, we applied the Mann-Whitney U test for inter-group analysis. A non-parametric multivariate analysis of variance (nonparametric MANOVA) for repeated measurements and small sample sizes in two and three-factorial designs [p < 0.05 was considered statistically significant. The statistical analysis was performed using SPSS for Windows, 11.0 .Because of the limited sample sizes and/or non-symmetrically distributed observations we applied only nonparametric statistics. Results were expressed as median and interquartile range in the case of continuous variables. Absolute and relative frequencies were used for categorical and dichotomous variables. The effect of hemodilution regarding primary and secondary outcomes was analyzed using χ designs , respectn = 52) with two women who were randomized in the higher hematocrit group.Basic patient characteristics (Table 1) did not differ between the groups. The majority of patients were men . Oxygen consumption was significantly lower in the 20% Hct group 6 hours after admission to the ICU . In the non-parametric MANOVA the difference in oxygen delivery did not remain significant (p = 0.11) between groups over the study period whereas oxygen consumption tended to be lower (p = 0.06) in the 20% Hct group. The blood lactate levels were not different between groups throughout the study period. For blood lactate, the non-parametric MANOVA did not find a significant difference between groups throughout the study period (p = 0.6).Figure 1 gives the results for oxygen delivery and consumption throughout the study period. Calculated oxygen delivery differences between groups almost reached statistical significance at the end of surgery (767 versus 647 ml/mp < 0.01). The non-parametric MANOVA confirmed the significantly lower Hct levels in the 20% Hct group (p < 0.01).The Hct was significantly lower in the 20% Hct group from the end of surgery until the end of ICU stay. Notably, an Hct of 30% was reached in the 20% Hct group at 6 hours after admission to the ICU  that were measured prior to, during and after CPB. All intra-operative measures were not significantly different between groups.p = 0.28), the incidence of neurological complications (p = 0.99), cardiac (p = 0.99), respiratory (p = 0.99) and renal failure (p = 0.99) and the combined endpoint of organ failure (p = 0.57) were not different between the groups. Five patients in the 20% Hct group and one patient in the 25% Hct group (p = 0.10) were transfused in the ICU. In particular, no acute myocardial infarction was recorded. ICU stay in hours was not significantly different between the groups.The clinical outcome in the ICU with regard to chest drainage loss, transfusion use and secondary organ failure are given in Table 3. Blood loss from chest drainage .2 in anesthetized humans, as reported by Shibutani and colleagues [2 from being significantly reduced. It may also be that the microcirculation compensated for the differences in Hct levels used in our study [2. Patients in the 20% Hct group used significantly less oxygen in the ICU . In both groups, oxygen consumption continuously increased from the lowest values at the end of surgery to the highest values before discharge from the ICU while oxygen delivery was maintained well above critical levels. Forces upon oxygen consumption during the ICU period and after extubation, which include, for example, lower level of sedation, increased muscular activity, fever or pain, may have caused the increase in oxygen consumption. Prior work in awake humans has shown that oxygen consumption is stable until an Hct of 15% [This study demonstrated that an Hct of 20% during normothermic CPB for elective CABG surgery did not 'critically' reduce calculated whole body oxygen delivery. Over the study period, calculated oxygen delivery was maintained above a critical threshold of 330 ml/minute/mlleagues . An incrur study  reportedt of 15% , indicatt of 15%  and cardt of 15%  and seemt of 15% .2 above a critical level and may be considered safe in regard to adequate whole body oxygen supply in normothermic CPB for elective CABG surgery in low risk patients.Both groups had normal blood lactate levels throughout the study, indicating that a gross mismatch of whole body oxygen delivery and consumption during CPB did not occur. This further supports our conclusion that hemodilution to 20% Hct maintained DOThe clinical outcome in regard to drainage loss, transfusion requirements and secondary organ failure were not different between the study groups. In particular, no myocardial infarction was detected in our group of patients, which may support the hypothesis that myocardial oxygen delivery during and after CPB was maintained in the 20% Hct group. Our results are in accordance with previous results that an Hct of <34% on admission to the ICU  patients, 1 patient in each group), which confirms previous results . It has p = 0.24) and confirms previous results from elective CABG surgery [The median stay in the ICU in our patients was 22 hours in the 25% Hct group versus 23 hours in the 20% Hct group ( surgery . The com2)' would result in a sufficient power of more than 80%. A sufficiently powered clinical trial investigating 'mortality' as a clinical endpoint would require a far larger number of patients to detect a clinically relevant difference between the groups.Our study does have some limitations. First, the sample size of this pilot study was generally not large enough to detect the differences observed with sufficient power. Therefore, our results may serve as a database for larger studies investigating the effect of hemodilution during CPB. A power calculation for the outcome 'ICU stay' with the given sample sizes would correspond to a power of not more than 20% for 'ICU stay' whereas for 'oxygen delivery (DOp = 0.1) but may have become so in a larger study. Transfusions in the postoperative period may have non-beneficial effects on safety and outcome in cardiac surgery [Second, patients studied were low risk patients. This precludes that our results can be generalized to cardiac patients with certain comorbidities in whom an individual risk-benefit analysis prior to transfusion is required. A univariate analysis of all risk factors that may have influenced the length of ICU stay was performed but did not find the lowest Hct during CPB to be predictive for ICU stay. Therefore, and due to the limited sample size, a multivariate analysis was not considered appropriate. Third, there were only two women in our study population, which does not reflect the gender distribution of CABG patients. In particular, women with a lower body weight are at risk of being exposed to a low Hct and allogeneic blood products during CPB. Therefore, our results need to be confirmed in a larger study including a sufficient number of women. Fourth, five times as many patients in the 20% Hct group were transfused after surgery. This was not statistically significant ( surgery ,4 and ne surgery , who traOur results from a small and low risk group of CABG patients suggest that hemodilution to an Hct of 20% during normothermic CPB did not induce a 'critical' imbalance between oxygen delivery and consumption that would have resulted in a worse clinical outcome. Although these results require confirmation by larger studies, patients without severe comorbidities who are at a high risk of receiving allogeneic blood products may safely tolerate an Hct of 20% during normothermic CPB. If further investigations determine an Hct of 20% during normothermic CPB to be safe, a reduction in exposure to allogeneic blood products seems to be possible.• 	An Hct of 20% versus 25% during normothermic CPB for CABG surgery did not impair oxygen delivery.• 	No significant difference in oxygen consumption between the study groups was detected, indicating that a critical 'oxygen balance' was not induced by lowering the hematocrit to 20% during normothermic CPB.• 	The clinical outcome of the patients was not significantly different between the groups, although the study was not sufficiently powered for this outcome.2 = oxygen delivery; Hct = hematocrit; ICU = intensive care unit; MAP = mean arterial pressure; paO2 = partial pressure of oxygen in arterial blood.CABG = coronary artery bypass graft; CPB = cardiopulmonary bypass; DOThe authors declare that they have no competing interests.CvH: conception and design of the work, sampling of data, analysis of the data and drafting the article for content. MS: conception and design of the work, sampling of data, analysis and interpretation of the data and revising the article for content. AF: conception and design of the work, interpretation of the data and revising the article for content. BDS: interpretation of the data, revising the article for content and approval of the version to be published. AH: conception and design of the work and sampling of data. JB: conception and design of the work, interpretation of the data and revising the article for content. MK: conception and design of the work, sampling of data, revising the article for content. JG: conception and design of the work, sampling of data, interpretation of the data and revising the article for content. PD: conception and design of the work, interpretation of the data and revising the article for content. SD: conception and design of the work, interpretation of the data and revising the article for content. JH: conception and design of the work, sampling of data and revising the article for content. WFK: revising the article for content and approval of the version to be published. K-DW: analysis and interpretation of the data, revising the article for content, approval of the version to be published. CS: conception of the work, interpretation of the data, revising the article for content and approval of the version to be published.Standard formulae used for calculating outcome measures.Click here for file


Otitis media is one of the most common infections in young children. Although exposure to environmental tobacco smoke is a known risk factor associated with otitis media, little information is available regarding the potential association with air pollution.We set out to study the relationship between exposure to traffic-related air pollution and otitis media in two birth cohorts.2.5)], and elemental carbon—were calculated for home addresses of approximately 3,700 and 650 infants from birth cohort studies in the Netherlands and Germany, respectively. Air pollution exposure was analyzed in relation to physician diagnosis of otitis media in the first 2 years of life.Individual estimates of outdoor concentrations of traffic-related air pollutants—nitrogen dioxide, fine particles [particulate matter with aerodynamic diameters ≤ 2.5 μm , 1.10 (1.00–1.22), and 1.14 (1.03–1.27) in the Netherlands and 1.24 (0.84–1.83), 1.10 (0.86–1.41), and 1.14 (0.87–1.49) in Germany, respectively.Odds ratios (adjusted for known major risk factors) for otitis media indicated positive associations with traffic-related air pollutants. An increase in 3 μg/mThese findings indicate an association between exposure to traffic-related air pollutants and the incidence of otitis media. Given the ubiquitous nature of air pollution exposure and the importance of otitis media to children’s health, these findings have significant public health implications. Otitis media is one of the most common childhood infections in young children. Three of four children experience otitis media by 3 years of age, with most infections occurring before age 2 . Otitis Of particular relevance to a possible association between air pollution exposure and otitis media is the strength of environmental tobacco smoke (ETS) exposure as a risk factor . A quantPrevious analysis of this Dutch birth cohort indicated a significant association between a combined measure of severe upper respiratory tract and ear/nose/throat infections for the 12-month period before the child’s second birthday and exposure to traffic-related air pollutants , but didThe Prevention and Incidence of Asthma and Mite Allergy (PIAMA) study is a prospective birth cohort study with an initial enrollment of 4,146 children . The cohThe participants of the LISA-Munich (Influence of Lifestyle factors on the development of the Immune system and Allergies in East and West Germany) birth cohort study were recruited during pregnancy. From December 1997 to January 1999, newborns from six obstetrical clinics in Munich whose parents were born in Germany and had German nationality were defined as the target population for the study. A detailed description of selection, exclusion criteria, and characteristics of study population has been published previously . For thi2.5)] were collected with Harvard impactors , nitrogen dioxide was collected by Palmes tubes, and light-absorbing carbon was measured as the reflectance of the PM2.5 filters, a method shown to be highly correlated with thermal elemental carbon measurement  within a 300-m radius, and an indicator for the region of the country were used in the model to predict light-absorbing carbon concentrations. In Munich, the light-absorbing carbon model used traffic intensity within a 50-m distance, traffic intensity in a circular area between 50 and 250 m, and the population density within a 300-m radius and in a circular area between 300 and 5,000 m to predict concentrations. Models with similar variables describing traffic intensity were developed for PMherlands  and 56,  Germany . We then Germany .We assessed information on otitis media using a parent-completed questionnaire. Specifically, in the Netherlands the question “Did a doctor diagnose infection of the middle ear in your child in the last 12 months?” was asked when the child was 12 months and 24 months of age. In Munich, the question “Did a doctor diagnose otitis media in your child during the last 6 months?” was asked every 6 months. A series of potential confounding variables  with 95% confidence intervals (CIs). We adjusted for potential confounding factors such as sex, parental atopy, maternal education, siblings, maternal smoking during pregnancy, ETS exposure at home, use of gas for cooking, indoor moulds and dampness, number of siblings, breast-feeding, and presence of pets in the home . ORs aresampling ], and 10In both cohorts, the prevalence of otitis media increased in the second year and was nearly identical at 2 years of age . By that2 were similar between the Netherlands and Germany whereas PM2.5 concentrations were higher in the Netherlands, probably due to higher regional background concentrations. Interquartile ranges were nearly twice as large in the Netherlands  than in Munich , reflecting the fact that the Dutch cohort included suburban and semirural areas that were not heavily affected by traffic-related air pollutants, whereas the German cohort was restricted to the Munich metropolitan area in which traffic emissions contribute to the urban background as well as to variability within the area.Exposures to traffic-related air pollution are summarized in Crude ORs indicated elevated risks for otitis media in association with all air pollutants, with associations reaching statistical significance in the larger Dutch cohort . These O2 OR = 1.13  at 1 year and OR = 1.10  after 2 years of age] but still elevated. Additional stratified analyses indicate that the effect of air pollution on otitis media occurrence was not restricted to those children who attended child care.Because attendance at a child care facility has been independently associated with respiratory tract infections in the Dutch cohort  and is aThis analysis represents the first examination of the relationship between air pollution exposure and otitis media in a large cohort study. In two birth cohorts with a common exposure assessment approach, we have identified associations between individual estimates of traffic-related air pollution exposure and the incidence of otitis media. Given the widespread nature of air pollution exposure and the high prevalence of otitis media, these findings indicate an important and previously unrecognized societal impact of air pollution. Further, given the high direct and indirect costs associated with otitis media episodes, our results suggest a potentially important preventable risk factor for this common childhood disease.2 and TSP in all three areas, prevalence rates for otitis decreased from 31% in 1992–1993 to 26% in 1995–1996 and 27% in 1998–1999  and 1.42  for a 100-μg/m3 change in SO2 concentration  for current and frequent ear infection. Social indicators such as parental education and literacy also differed between the three districts. Further, temporal changes in ambient particulate matter concentrations were also associated with changes in frequent ear infection prevalence. Although this study has several limitations , the results are consistent with an association between prevalence of otitis media and ambient air pollution.In a somewhat similar design, ear infections were examined in two cross-sectional surveys of approximately 400 children 11–13 years of age from three districts of São Paulo, Brazil, conducted in 1986 and in 1998 . The thrOther studies that have evaluated air pollution and otitis media typically lack exposure measurements or were conducted on small sample sizes  but alsoAlthough our study has several major advantages over previous investigations , there are also inherent limitations to our approach. First, as is common with air pollution epidemiologic analyses, we estimated exposures instead of directly measuring them using personal monitoring. Additionally, for those children who moved or attended child care, estimating exposures at these locations may reduce exposure misclassification. Second, we asssessed otitis media by questionnaire-based self-reporting of physician diagnosis and not by any objective measure. Third, we did not assess severity or address issues such as recurrent otitis media and interactions between air pollution and different treatment regimes. Such analyses, although important to understanding the public health significance of our findings, are probably best addressed in a longitudinal study design in which detailed information regarding otitis media occurrence and treatment is evaluated in relation to short-term changes in air pollution exposure. Finally, although we adjusted analyses for a large number of potential risk factors, the possibility for residual confounding remains, especially given that exposure estimates were based on spatial contrasts in air pollution that may also lead to spatial contrasts for other unmeasured otitis media risk factors. These limitations are, however, largely unavoidable for cohort studies of large populations.2 and coarse particles may be especially active in this regard (2 exposure before infection has been demonstrated to lead to increased severity of asthma exacerbations (These findings indicate an association between exposure to traffic-related air pollutants and the incidence of otitis media. This association is supported by a wealth of evidence linking exposure to high levels of air pollution indoors in developing countries with acute lower respiratory infections , more lis regard . Additios regard —making prbations . Alterna


To better understand maize genome organization and to build a framework for genome sequencing, we constructed a sequence-ready fingerprinted contig-based physical map that covers 93.5% of the genome, of which 86.1% is aligned to the genetic map. The fingerprinted contig map contains 25,908 genic markers that enabled us to align nearly 73% of the anchored maize genome to the rice genome. The distribution pattern of expressed sequence tags correlates to that of recombination. In collinear regions, 1 kb in rice corresponds to an average of 3.2 kb in maize, yet maize has a 6-fold genome size expansion. This can be explained by the fact that most rice regions correspond to two regions in maize as a result of its recent polyploid origin. Inversions account for the majority of chromosome structural variations during subsequent maize diploidization. We also find clear evidence of ancient genome duplication predating the divergence of the progenitors of maize and rice. Reconstructing the paleoethnobotany of the maize genome indicates that the progenitors of modern maize contained ten chromosomes.Maize  As a cash crop and a model biological system, maize is of great public interest. To facilitate maize molecular breeding and its basic biology research, we built a high-resolution physical map with two different fingerprinting methods on the same set of bacterial artificial chromosome clones. The physical map was integrated to a high-density genetic map and further serves as a framework for the maize genome-sequencing project. Comparative genomics showed that the euchromatic regions between rice and maize are very conserved. Physically we delimited these conserved regions and thus detected many genome rearrangements. We defined extensively the duplication blocks within the maize genome. These blocks allowed us to reconstruct the chromosomes of the maize progenitor. We detected that maize genome has experienced two rounds of genome duplications, an ancient one before maize–rice divergence and a recent one after tetraploidization. Cereal crops, such as rice, maize, and wheat, are the major caloric source for humans and farm animals. The cereals shared a common ancestor some 50 million years ago (MYA) and even though their genome sizes vary considerably, their genetic map organization is highly conserved ,2. Rice Based on recent alignments of orthologous regions of rice and sorghum chromosomes with two homoeologous regions in maize, the progenitor of sorghum and the two progenitors of maize diverged at nearly the same time about 11.9 MYA. Furthermore, the two progenitors of maize may have hybridized as recently as 4.8 MYA, explaining the divergence of the duplicated regions in maize . AlignmeSequence analysis of random sheared fragments , BAC  deep-coverage large insert BAC libraries  name ZMMBBc). In the c library, the first half (288 384-well plates) was from the EcoR1 digest, and the second half was from the MboI digest. Clones ending in “sd1” are sequenced BACs downloaded from GenBank  was employed as an internal size standard, which was run in each capillary. Reaction products were resolved on ABI3700 automated DNA sequencers. ABI GeneScan version 3.7.1 software (Applied Biosystems) was used for band detection and extraction. Only fragments ranging in size from 75 to 500 bp were used for HICF assembly. Because FPC can only input one set of band values for each clone, we used the technique of Ding et al. [For the HICF method ,30,31, BN markers and have a Sulston score of less than M, using the following  pairs: , , , or . False-positive overlaps generally result in a stack of clones within the contig whose bands do not align well with the underlying consensus band map; these clones are called Q (questionable) clones [FPC software  was used) clones . The FPCTo automatically build the HICF map, possible contaminated clones were screened for same-plate overlaps at 1e−45, followed by removal of all remaining clones over 175 bands. The initial contigs were built at 1e−70 at the tolerance of 4, then the DQer reassembled all contigs that had >15% Q clones. The end-merging function was run at 1e−61, 1e−52, 1e−45, 1e−40, and 1e−21, where the end-merging function checks the clones at the ends of contigs, and if at least two pairs of clones overlap based on the given cutoff, the contigs are merged. Singletons were placed at 1e−43 to their best location, before the final end-merge.N and at least M shared markers, where the valid  pairs were , , , or . We also extensively used the HICF map, marker information, and maize–rice synteny for contig merging. A new marker type called HICF was created, which was used to link each pair of clones that are bridged by an HICF clone that did not occur in the agarose map. We know that the pair of clones lie close to each other (within 150 kb) and probably overlap, but it cannot be confirmed. The agarose method produced an average of 30 bands for each maize clone (band size range from 1 to 23 kb), while the HICF method generated an average of 107 bands for each clone .N CB units, then its approximate length in base pairs is N × 4,900. The 4,900 was calculated as the average size restriction fragment as follows: A total of 20 completely sequenced maize BACs that had corresponding fingerprints were downloaded from GenBank. The average size of all sequenced BACs was divided by the average number of fingerprinted bands, which resulted in an average band size of 4,900 bp. Since the number of bands was taken from the real fingerprint and not from the simulated fingerprint, this average size took into account missing bands. This estimation is similar to that in the rice physical map [The metric of a FPC contig is the consensus band (CB) unit. If the length of a contig is ical map .As an essential component to establish an integrated map for the maize genome, a saturated and evenly distributed genetic map was developed to anchor and fully orient physical contigs along the chromosome. We built a dense genetic framework using the IBM mapping population . The inbThe number of genetically mapped markers was increased by intercalating locations of additional markers into the IBM, constructing “neighbors'” maps , i.e., bWe used probe hybridization on BAC filters, primer amplification methods on 6-D BAC pools, and overgo probe hybridizations to integrate genetically mapped markers with the related BAC clones in the maize physical map. Since genetic markers can hybridize to multiple contigs because of paralogous sequences, it was necessary to distinguish between marked contigs that could be genetically placed and all other copies. To this end, anchoring markers retained their original name, and any unanchored copies were assigned a suffix (.A). Markers included RFLPs, SSRs, ESTs, SNPs, and InDels. Filter hybridization has low throughput, so we used only 90 RFLP probes on filters  and carrHybridization of overgos was conducted on four filters of a 6 × 6 grid with a total of 165,888 BACs, half from each of the HindIII and EcoRI libraries. The 40-bp overgo probes were designed from unigenes of the DuPont/MMP/Incyte Genomics partnership and were screened with a maize repeat database to ensure that each probe was low copy. The overgos were classified into five marker types where the prefix identifies the marker type: (1) the prefix CL identifies clusters assembled from public EST sequences; (2) PCO represents public sequences combined with DuPont sequences (Unigene Consensus deposited in GenBank); (3) dd signifies anonymous clusters assembled by DuPont (sequences are not available); (4) si represents public singletons that did not cluster with DuPont sequences; and (5) SOG markers are overgos from the Paterson lab, derived from probes that have been mapped in sorghum and other grasses. Except for the SOG markers, which were designed for sequence conservation, all other overgos were designed for their diversity. We employed a 2-D 24 × 24 pool strategy for overgo hybridization and scored dual grid spots as positive clones .PCR methods were used to screen BACs with SSR or genomic sequence markers. A 6-D pool strategy  was usedhttp://www.agcol.arizona.edu/symap.We used the SyMAP  to compuFigure S1The distribution of EST-derived overgo markers and genetic markers was plotted against maize chr1. The overgos, which hybridized to at least two BAC clones in each contig and hit fewer than ten contigs, were counted for the plot. The overgos and genetic markers were counted as a total in each Mb region.(44 KB PPT)Click here for additional data file.Figure S2(A) General view of maize–rice synteny alignment is presented. This is the block view generated from dotplot analysis in http://www.agcol.arizona.edu/symap), an individual block view with rice alignment or a comparative block view like (B) Physical synteny map of rice chr2 with maize chromosomal regions is presented. Each maize physical block is indicated by chromosome number and FPC contig numbers. When clicked in our interactive website ((72 KB PPT)Click here for additional data file.Table S1(16 KB XLS)Click here for additional data file.Table S2(26 KB XLS)Click here for additional data file.Table S3(16 KB XLS)Click here for additional data file.Table S4(30 KB XLS)Click here for additional data file.Table S5(31 KB XLS)Click here for additional data file.