Hepatocellular carcinoma (HCC) usually has a dismal prognosis because of its limited response to current pharmacotherapy and high metastatic rate. Sulfated oligosaccharide has been confirmed as having potent antitumor activities against solid tumors. Here, we explored the preclinical effects and molecular mechanisms of isomalto oligosaccharide sulfate (IMOS), another novel sulfated oligosaccharide, in HCC cell lines and a xenograft model.The effects of IMOS on HCC proliferation, apoptosis, adhesion, migration, and invasiveness in vitro were assessed by cell counting, flow cytometry, adhesion, wound healing, and transwell assays, respectively. The roles of IMOS on HCC growth and metastasis in xenograft models were evaluated by tumor volumes and fluorescent signals. Total and phosphorylated protein levels of AKT, ERK, and JNK as well as total levels of c-MET were detected by Western blotting. IMOS-regulated genes were screened by quantitative reverse-transcription PCR (qRT-PCR) array in HCCLM3-red fluorescent protein (RFP) xenograft tissues and then confirmed by qRT-PCR in HepG2 and Hep3B cells.IMOS markedly inhibited cell proliferation and induced cell apoptosis of HCCLM3, HepG2, and Bel-7402 cells and also significantly suppressed cell adhesion, migration, and invasion of HCCLM3 in vitro. At doses of 60 and 90 mg/kg/d, IMOS displayed robust inhibitory effects on HCC growth and metastasis without obvious side effects in vivo. The levels of pERK, tERK, and pJNK as well as c-MET were significantly down-regulated after treatment with 16 mg/mL IMOS. No obvious changes were found in the levels of pAkt, tAkt, and tJNK. Ten differentially expressed genes were screened from HCCLM3-RFP xenograft tissues after treatment with IMOS at a dose of 90 mg/kg/d. Similar gene expression profiles were confirmed in HepG2 and Hep3B cells after treatment with 16 mg/mL IMOS.p53 and worth studying further in patients with HCC, especially at advanced stages.IMOS is a potential anti-HCC candidate through inhibition of ERK and JNK signaling independent of  Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third leading cause of cancer-related death globally . As indiHeparanase inhibitor has recently become an attractive agent for highly malignant tumors, due to its antiangiogenic and antimetastatic activities -10. Two Two distinctive differences in molecular structure exist between isomalto oligosaccharide sulfate (IMOS) and PI-88. IMOS is composed of four sulfated isomaltose molecules with a molecular weight <1500 Da, whereas PI-88 is composed of five sulfated mannose molecules with a molecular weight of 2100 to 2585 Da. Such alterations in structure may affect its toxicity and antitumor effects. In this report, we present our preliminary evidence of the effects of IMOS on experimental HCC growth and metastasis.IMOS, with a patent (patent no. ZL2005 1 0002141.8) granted by the State Food and Drug Administration of China, is designed and successfully synthesized de novo by Herbon Polysaccharide Bio-tech. Figure 2 at 37°C.Four human hepatoma cell lines HepG2, Bel-7402, Hep3B, HCCLM3, and its red fluorescent protein (RFP)-expressing derivative, HCCLM3-RFP, were used in this study . HCCLM3,3 cells/well. Twenty-four hours later, cells were exposed to IMOS at doses ranging from 0 to 64 mg/mL. On days 1, 2, 3, 4, and 5, cells were digested with pancreatic enzymes including ethylenediaminetetraacetic acid (EDTA) and washed with phosphate-buffered saline (PBS). Cell numbers were then counted by the Countess™ automated cell counter .Cell proliferation was assessed by the method described previously . In brie5 cells/well. After treatment with IMOS at 0, 2, 4, 16, 32, or 64 mg/mL for 24 hours, the cells were fixed with ethanol and stained with annexin V for early apoptosis assay by a fluorescence-activated cell sorter (FACS) Calibur cytometer . In the same way, 72 hours after IMOS treatment, the cells were stained with propidium Iodide (PI) for late apoptosis and cell cycle assays.Cell cycle and apoptosis were detected using the Annexin V-FITC Apoptosis Detection Kit™ according to the manufacturer's instructions . Briefly, HCCLM3, HepG2, and Bel-7402 cells were plated into 6-well plates at 4 × 104 cells/well, and incubated for 2 hours at 37°C in 5% CO2. After extensive washing, cells were fixed with 100 μL/well of 4% formaldehyde for 20 minutes and stained with a hematoxylin solution for 10 minutes. The average numbers of adhesion cells in four quadrants were counted by inverted microscope.The 96-well flat-bottom plates were precoated with 50 μL/well of 1:8 PBS-diluted Matrigel at 4°C overnight. After removing all coating solutions, the plates were blocked with 150 μL of 1% bovine serum albumin for 1 hour at 37°C. Then, HCCLM3 cells were treated with 0, 16, 32, or 64 mg/mL IMOS for 4 hours, seeded into Matrigel-coated wells at 5 × 1024 or 48 hours - distance0 hour)/distance0 hour.Cell migration was analyzed by a wound healing assay. When cells grew to 90% of confluency, a scratch wound in the monolayer was made using a pipette tip. After washing away all detached cells with PBS, the remaining cells were treated with 0, 16, 32, or 64 mg/mL IMOS and then the distances of wounds were measured by microscope at 0, 24, and 48 hours after treatment. Cell motility was evaluated the following formula: Cell motility =  for 25 minutes, and their numbers in 10 fields of each triplicate filter were analyzed by inverted microscope.Cell invasion was analyzed by a Transwell™ Permeable Supports system  according to the manufacturer's instructions. HCCLM3 cells were pretreated with 0, 16, 32, or 64 mg/mL IMOS for 48 hours, and then seeded into the Matrigel-coated upper insert at 8 × 10Total and phosphorylated protein levels of AKT, ERK, and JNK as well as total protein of c-MET in HepG2 and Hep3B cells were evaluated by Western blotting. About 20 μg protein was extracted from sham-treated and 16 mg/mL IMOS-treated cells, separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene fluoride membranes, and then reacted with primary rabbit antibodies against total and phosphorylated AKT, ERK, and JNK, c-MET  and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). After being extensively washed with PBS containing 0.1% Triton X-100, the membranes were incubated with alkaline phosphatase-conjugated goat anti-rabbit antibody for 30 minutes at room temperature. The bands were visualized using 1-step™ NBT/BCIP reagents  and detected by the Alpha Imager .A dose-escalation strategy was used in male athymic BALB/c mice  to determine maximum tolerable dose of IMSO. Eighty mice, aged 4 weeks, were divided into groups of 10 mice apiece, with each mouse intraperitoneally injected with IMOS at a dose of 0, 30, 60, 90, 180, 360, 480, or 600 mg/kg/d, respectively. Mouse survival was monitored every day. We planned to halt the dose escalation would be halted if mice died. Serum was collected for assays of hepatorenal function. Plasma was collected for determining thrombocyte counts. Heart, liver, and kidney tissues were subjected to hematoxylin and eosin staining for pathologic examinations. The maximum nonlethal dose was determined for the following in vivo therapeutic study. All procedures were approved by the Animal Care and Use Committee of Shanghai, China.7/0.2 mL of HCCLM3-RFP cells in the right upper flank region to establish subcutaneous xenograft models. Four weeks later, the tumors that had grown to 1 cm in diameter were removed, cut into 1-mm3 pieces, and implanted into livers of another 24 mice to establish orthotopic xenograft models as described previously [3) = width2 (mm2) × length (mm)/2. Metastatic foci in lungs and mesenteries were counted by fluorescent stereomicroscope . Fluorescence area  was quantified by Image-Pro Plus 6.0  as described previously [Antitumor activities of IMOS in vivo were assessed against HCCLM3-RFP xenografts. Three 4-week-old male athymic BALB/c mice were injected subcutaneously with 1 × 10eviously . Then, aeviously .® Green PCR Master Mix (Applied Biosystems). Following each cycle, SYBR green fluorescence was monitored and the melting curve was analyzed to ensure that a single PCR product was obtained. Afterward, the size and specificity of amplicons were confirmed by 2.5% agarose gel electrophoresis. All reactions were repeated in three separate runs and evaluated with the Opticon Monitor software (Version 1.02). GAPDH was used to normalize the samples. RNase-free water (Qiagen) was included as a negative control in RNA extraction and in each run.Tumor tissues from mice treated with IMOS at a dose of 0 or 90 mg/kg/d were enrolled for differentially expressed gene analysis by RT Profiler PCR Arrays  and performed by Kangchen Bio-tech . The mRNA levels of differentially expressed genes in HepG2 and Hep3B cells with 16-mg/mL IMOS treatment were confirmed by qRT-PCR. Total RNA of cells was extracted using RNeasy MinElute Cleanup Kit . Then, 1.5 μg RNA was reversely transcribed into first-strand cDNA using SuperScript™ III Reverse Transcriptase . Primer sequences and amplification conditions are listed in Additional file P < 0.05.Statistical analysis was performed with SPSS 15.0 for windows . Quantitative variables were expressed as means ± SD and analyzed by ANOVA. Results were considered statistically significant at To explore the effects of IMOS on HCC proliferation, HCCLM3, HepG2, and Bel-7402 cells were treated with IMOS at doses ranging from 0 to 64 mg/mL. IMOS dramatically decreased cell numbers of all tested cell lines in a dose-dependent manner, especially when exposed to 16- and 64-mg/mL doses of IMOS Figure . The inh2/M phases were significantly decreased, whereas cell numbers at the G0/G1 phase were markedly increased after IMOS treatment in a dose-dependent manner , similar expression profiles were confirmed in IMOS-treated HepG2 and Hep3B cells as compared with HCCLM3 xenograft (Figure To understand underlying mechanisms, total and phosphorylated protein levels of AKT, ERK, and JNK as well as c-MET were analyzed in sham-treated and 16-mg/mL IMOS-treated HepG2 and Hep3B cells. The levels of pERK, tERK, and pJNK were significantly down-regulated in both cell lines, whereas the level of c-MET was markedly down-regulated only in HepG2 cells. No obvious changes were found in the protein levels of pAkt, tAkt, and tJNK Figure . Furtherd Figure . Except t Figure  and 6D.HCC usually has a limited response to current pharmacotherapy. It has been reported that MHCC97L and HepG2 cells surviving oxaliplatin treatment show enhanced migration and invasion in vitro and increase metastasis to the lung when reinoculated into nude mice . SimilarAccording to previous studies, sulfated oligosaccharides were thought to suppress tumor angiogenesis, growth, and metastasis primarily by their competitive inhibition of the cleavage of heparan sulfate-growth factor complex, thus reducing the release of growth factors, such as vascular endothelial growth factor and basic fibroblast growth factor from the microenvironmental matrix . Howeverp53-dependent or -independent manner [p53, and Hep3B, a cell line with mutant p53, were used for further study to elucidate the mechanism of IMOS. Our preliminary results from Western blots revealed that ERK and JNK but not Akt signaling pathways were significantly inhibited by IMOS in both cell lines, whereas c-MET, a well-known p53 transcriptional target, was only down-regulated in HepG2 [p53 activity in HepG2, but not in Hep3B.Numerous studies have shown that Akt, ERK, and JNK signaling pathways have important roles in cancerous cell proliferation, cell cycle, and apoptosis regulation in a t manner -24. Dysft manner ,26. Therin HepG2 . All thop53-independent manner. Among them, BAI-1 and TP73 were significantly up-regulated, whereas Bcl-2, BIRC5/survivin, PCNA, and CDK1/CDK2 were markedly down-regulated. Our findings are consistent with previous observations that Bcl-2, BIRC5/survivin, and BAI-1 were the mediators of cell cycle arrest and apoptosis regulation [PCNA was able to suppress cell apoptosis [PCNA in HCC after IMOS treatment was probably able to induce cell apoptosis. Furthermore, as an apoptosis-induced gene [P73 in IMOS-treated tissue may also promote HCC apoptosis.Ten differentially expressed genes were screened from HCCLM3-RFP xenografts. Eight of them were confirmed in IMOS-treated HepG2 and Hep3B cells. Because similar profiles of most differentially expressed genes were found in both HepG2 and Hep3B cells, IMOS may inhibit cell proliferation and induce cell apoptosis of HCC in a gulation -30. Ectopoptosis , thus deced gene , enhanceIMOS, a novel sulfated oligosaccharide, at doses of ≤90 mg/kg/d exhibited potent antitumor effects on experimental HCC growth and metastasis. It should be a promising anti-HCC agent and worth further studies in patients with HCC, especially disease at advanced stages.The authors declare that they have no competing interests.CLX, ZHT, and JLW organized the study, analyzed the effects of IMOS on cell proliferation, apoptosis, adhesion, migration, and invasiveness, and helped to write the manuscript. LG contributed to the analysis of the hepatorenal functions and the number of platelets after IMOS treatment. WWL and JLW performed the statistical and cell signal pathway analysis. HCS and LW participated in the design and coordination of the study. ZYT, JF, and WZW contributed to the interpretation of the results and helped write the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/150/prepubPrimer sequences and amplification conditions of qRT-PCR.Click here for fileBody weights of nude mice after IMOS treatment (g, .Click here for fileHepatorenal parameters of nude mice after IMOS treatment (.Click here for filePathologic examinations of heart, liver, and kidney tissues after treatment with IMOS.Click here for file


There is emerging evidence that background exposure to persistent organic pollutants (POPs) are important in the development of conditions predisposing to diabetes as well as of type 2 diabetes itself. We recently reported that low dose POPs predicted incident type 2 diabetes in a nested case-control study. The current study examined if low dose POPs predicted future adiposity, dyslipidemia, and insulin resistance among controls without diabetes in that study.The 90 controls were diabetes-free during 20 years follow-up. They were a stratified random sample, enriched with overweight and obese persons. POPs measured in 1987-88 (year 2) sera included 8 organochlorine (OC) pesticides, 22 polychlorinated biphenyls (PCBs), and 1 polybrominated biphenyl (PBB). Body mass index (BMI), triglycerides, HDL-cholesterol, LDL-cholesterol, and homeostasis model assessment value for insulin resistance (HOMA–IR) were study outcomes at 2005-06 (year 20). The evolution of study outcomes during 18 years by categories of serum concentrations of POPs at year 2 was evaluated by adjusting for the baseline values of outcomes plus potential confounders. Parallel to prediction of type 2 diabetes, many statistically significant associations of POPs with dysmetabolic conditions appeared at low dose, forming inverted U-shaped dose-response relations. Among OC pesticides, p,p'-DDE most consistently predicted higher BMI, triglycerides, and HOMA-IR and lower HDL-cholesterol at year 20 after adjusting for baseline values. Oxychlordane, trans-nonachlor, and hexachlorobenzene also significantly predicted higher triglycerides. Persistent PCBs with ≥7 chlorides predicted higher BMI, triglycerides, and HOMA-IR and lower HDL-cholesterol at year 20 with similar dose-response curves.Simultaneous exposure to various POPs in the general population may contribute to development of obesity, dyslipidemia, and insulin resistance, common precursors of type 2 diabetes and cardiovascular diseases. Although obesity is a primary cause of these metabolic abnormalities, POPs exposure may contribute to excess adiposity and other features of dysmetabolism. There is emerging evidence that background exposure to persistent organic pollutants (POPs) may play an important role in the development of type 2 diabetes. Recently, we reported that serum concentrations of some POPs, in particular organochlorine (OC) pesticides and polychlorinated biphenyls (PCBs), predicted incident type 2 diabetes 1in a nested case-control studyIn the current paradigm for type 2 diabetes, obesity results from energy imbalance, insulin resistance from obesity, and exhaustion of pancreatic beta cells from overproduction of insulin to compensate for insulin resistance, which ultimately progresses to diabetesOne recent animal study strongly supported this concept. Rats exposed to POPs through the consumption of fish oil contaminated with various POPs developed abdominal obesity, hepatosteatosis, and insulin resistanceCARDIA focuses on cardiovascular disease risk development in participants initially aged 18 to 30 years. In brief, 5,115 African American and white participants were recruited at baseline in 1985–1986 (year 0)The study was approved by the University of Minnesota Institutional Review Board, as well as institutional review boards at the Universities of Alabama at Birmingham, Northwestern University, and the Division of Research at Kaiser Permanente Health Care Plan. Written informed consent was obtained at every examination. Participants in this study were 90 controls selected from those who had fasting glucose <100 mg/dL at all 5 examinations  at which glucose was measured. Controls were selected at random from each of several year 0 body mass index (BMI) categories ; these categories were chosen so that controls were frequency matched to cases.2 (kg/m2). Plasma total cholesterol, high-density lipoprotein cholesterol (HDL-C), and triglycerides were determined enzymatically by Northwest Lipids Research Laboratory . Low-density lipoprotein cholesterol (LDL-C) was computed using the Friedewald equation. Fasting plasma glucose was determined using the hexokinase-ultraviolet method and fasting plasma insulin by radioimmunoassay . The homeostasis model assessment value for insulin resistance (HOMA–IR) was calculated as (glucose in mmol/L × insulin in µIU/mL)/22.5.Demographics, health behaviors, anthropometrics, and various clinical variables were measured at baseline and follow-up examinations. Blood samples were collected after an overnight fast. BMI was measured weight/heightTable S1).POPs were measured in stored 2 mL serum samples collected at year 2 . The samples were analyzed by solid phase extraction and final determination using gas chromatography isotope dilution high-resolution mass spectrometry (GC/ID-HRMS) at the Centers for Disease Control and PreventionTable S2. Only measurements with more than 75% of values above the detection limit  were included, placing participants with non-detectable concentrations in the lowest quartile of each POP.Lipid standardized concentrations, the concentration of serum POP value divided by total serum lipid content (mg/dL), have generally been reported in epidemiological studies, since POPs are predominantly carried in the lipid component of the blood. However, according to a simulation studytrend or Pquadratic<0.10.We used general linear models to explore the associations of serum concentrations of POPs at year 2 with BMI, triglycerides, HDL-cholesterol, LDL-cholesterol, and HOMA-IR at the year 20 examination. The covariates included age, sex, race, triglycerides and total cholesterol at year 2. Year 20 HDL-cholesterol and HOMA-IR were additionally adjusted for their baseline values at year 2 and year 7, respectively. We considered P<0.05 as statistically significant, but, considering the small sample size, we also comment on results with P2 at year 2; and 33.0±7.9 kg/m2 at year 20.At baseline . p,p'-DDT also appeared to predict BMI at year 20, but the dose-response curve was close to a linear increase (Ptrend = 0.05). The associations between PCBs and BMI differed depending on PCB congener. For example, PCB170, PCB180, or PCB206 showed the highest increase of BMI in the 2nd quartile, while BMI decreased at higher concentrations of these PCBs, making inverted U-shaped associations. However, PCB156, PCB194, and PCB209 showed decreasing trends of BMI from the 2nd to 4th quartile with little or no increase of BMI from the 1st to 2nd quartile.Among various OC pesticides, the most statistically significant year 2 predictor of BMI at year 20, was p,p'-DDE Table 2.nd quartile with lower triglycerides in either quartile 3 or 4, while hexachlorobenzene predicted triglycerides with a positive linear trend. Many PCBs among those with 7 or more chlorides showed statistically significant low dose effects, having inverted U shaped associations. Opposite to findings concerning highly chlorinated PCBs, PCB74 (with 4 chlorides) showed a U-shaped association.Plasma triglycerides were 77.9±46.4 mg/dL at year 2 and 97.3±61.5 mg/dL at year 20. Among OC pesticides, oxychlordane, trans-nonachlor, hexachlorobenzene, and p'p-DDE predicted future triglycerides at year 20 after adjusting for triglycerides at year 2 Table 3.nd quartile, with no further decrease in the 3rd and 4th quartiles of p,p'-DDE. All PCBs that had statistically significant associations with HDL-cholesterol showed low dose effects. However, unlike the findings with p,p'-DDE, after the initial decrease of HDL-cholesterol from the 1st to 2nd quartile, HDL-cholesterol increased from the 2nd to 4th quartile of these PCBs.Plasma HDL-cholesterol was 53.2±13.7 mg/dL at year 2 and 54.1±14.5 mg/dL at year 20. The only OC pesticide which significantly predicted HDL-cholesterol at year 20 was p,p'-DDE Table 4.rd quartile of p,p-DDE and did not further increase at higher p,p;-DDE concentrations. PCBs with 5 chlorides such as PCB87 and PCB99 linearly predicted HOMA-IR while PCBs with 7 or 8 chlorides showed inverted U-shaped associations.HOMA-IR increased over 20 years from 2.6±1.4 to 3.4±1.9 mmol/L * µIU/mL. p,p'-DDE also predicted HOMA-IR at year 20 Table 5.Table S3).Calculated LDL-cholesterol was associated with only 3 POPs  with slightly different dose-response curves ( and trend or pquadratic<0.1) in , , , Figure S2.For illustrative purposes, we displayed the results on p, p'-DDE and PCB178 in s without diabetes. Similar to observed risk for type 2 diabetesIn this prospective study, some OC pesticides and PCBs predicted excess adiposity, dyslipidemia, and insulin resistance among participantThe past two decades have observed an explosive worldwide increase in the number of type 2 diabetic patients,with obesity regarded as a primary cause. Metabolic changes precede the development of type 2 diabetes by years or even decades. Insulin resistance is regarded as the initial metabolic defect, and dyslipidemia is also commonly observed. At first, the pancreatic beta-cells are able to compensate by increasing insulin secretion, but, as insulin resistance worsens, type 2 diabetes occurs when the pancreas fails to sustain this compensatory activity. Our study showed that POPs are likely involved in all these steps of pathogenesis of type 2 diabetes, even including the development of obesity. Importantly, different kinds of POPs were differentially associated with various aspects of metabolic dysregulation. For example, p,p'-DDE predicted increased BMI, increased triglycerides, decreased HDL-cholesterol, and increased HOMA-IR in this study, while p,p'-DDE did not predict type 2 diabetes in the previous studyAn important finding in this study was that some POPs predicted future BMI. Although imbalance between energy intake and expenditure is the central mechanism leading to obesity, the recent surge of obesity might have other causes besides overeating and physical inactivity. In addition to POPsAmong the 31 POPs we studied, 10 POPs statistically significantly or marginally significantly predicted future higher triglycerides and 14 POPs predicted lower HDL-cholesterol. However, higher LDL-cholesterol was associated with only 3 compounds. Lipid disorders are common in type 2 diabetes and play a crucial role in the development of diabetic cardiovascular complications. In fact, high triglycerides and low HDL-cholesterol, often with normal LDL-cholesterol, are fundamental characteristics of the metabolic syndrome rd quartile. However, in the case of highly chlorinated PCBs, such as PCB178, PCB194, and PCB199, mean HOMA-IR increased from the 1st to 2nd quartile and then clearly decreased through the 4th quartile, forming inverted U-shaped associations.Despite the general occurrence in these findings of inverted U-shaped associations, some dose-response curves followed a different pattern, depending on outcome and/or specific POP. Among OC pesticides, hexachlorobenzene predicted future triglycerides and LDL-cholesterol linearly, while p,p-DDE, oxychlordane, and trans-nonachlor showed low dose effects. Dose-response curves also differed between moderately chlorinated and highly chlorinated PCBs. In the case of moderately chlorinated PCBs such as PCB87, PCB99, and PCB118, the evolution of HOMA-IR occurred above the 3Similar to the associations between low dose POPs and type 2 diabetes, underlying mechanisms for associations between POPs and disturbances in glucose and lipid metabolism have been little studied. As PCB congeners with affinity to Aryl hydrocarbon receptor (AhR), like PCB105, PCB118, and PCB167, were not associated with any outcome in this study, affinity to AhR may not play an important mechanistic role in the metabolic disturbances studied here. Also, estrogenic activity is unlikely to be critical because both estrogenic PCB187 and antiestrogenic PCB170 showed similar associations. In addition, although p,p-DDT has a weak-estrogenic activity, p,p'-DDE, the OC pesticide which was strongly associated with most outcomes in this study and the most persistent metabolite of commercial DDT, is an anti-androgen rather being a weak estrogenThe findings on POPs observed in both current and previous studies may help to explain the current world-wide epidemic of metabolic syndrome and type 2 diabetes. The disturbances of glucose and lipid metabolism due to POPs seem to require at least two conditions; low dose and persistency. As serum concentrations of most chlorinated POPs have decreased during several decades, low doses of POPs among our study subjects which were measured in stored serums in 1987 to 1988 were similar to the highest concentrations currently seen in the general population. A very long exposure to POPs may be another critical condition. Because POPs have been released into the environment since the 1950s, we can assume that our study subjects had lifetime exposure to these chemicals. Therefore, in terms of both exposure dose and duration, the current general population may be at higher risk of developing glucose and lipid dysmetabolism due to POPs than were earlier general populations. In addition, obesity appeared to worsen the problem, because the associations between individual POPs and type 2 diabetes were observed only among obese persons in our previous study. Also, the exposure to xenobiotics such as POPs may contribute to the development of obesity. Finally, findings observed in man may be explained by the effects of individual POPs working together. Combinations of endocrine disrupting chemicals are able to produce significant effects, even when each chemical is present at low doses that individually do not induce observable effectsA limitation of this study is potentially false positive associations among the many associations evaluated. We assessed the possible effect of multiple testing under a binomial model by counting the number of significant findings (p<0.05) among the 62 linear or quadratic tests performed for each outcome, compared to 3.1 tests  expected to achieve p<0.05 under the null hypothesis. As 11 tests with BMI, 7 tests with triglyceride, and 15 tests with HDL-cholesterol achieved statistical significance, many of the associations of POPs with BMI, triglyceride, and HDL-cholesterol observed in the current study were unlikely under the null hypothesis, even considering multiple testing. As only 3 of 62 tests concerning LDL-cholesterol achieved statistical significance, multiple testing might well explain these apparently significant tests. The situation concerning evaluation of HOMA-IR is more complex. Although only 4 of 62 tests achieved statistical significance, the range of HOMA-IR was constrained by excluding anyone with impaired fasting glucose over 20 years. This exclusion restricts glucose explicitly and insulin secondarily. Furthermore, the significant associations that were observed were with p,p'-DDE and PCB178, both consistently showed significant associations with BMI, triglycerides, and HDL-cholesterol. Metabolic syndrome links BMI, triglycerides, and HDL-cholesterol with HOMA-IR, so that it is physiologically reasonable to expect similar associations of POPs with each of these variables. Therefore, the findings concerning p,p'-DDE and PCB178 with HOMA-IR should not simply be dismissed due to multiple testing. Furthermore, great consistency in prediction was observed for highly chlorinated PCBs. Another study limitation was small sample size, which was a compromise related to practicalities of cost and use of precious samples. Volumes of the available blood samples also prevented us from measuring dioxins and furans. The primary strength of the study is the long term followup, with baseline measures of all outcome variables.In conclusion, low dose POPs may be involved in the development of obesity, dyslipidemia, and insulin resistance, although different POPs may relate to different metabolic traits. As the general population is simultaneously exposed to various POPs through food consumption, our findings concerning POPs could help to explain why these metabolic abnormalities tend to occur as a cluster. Together with the findings on type 2 diabetes in our previous reportFigure S1Means of year 20 BMI, triglyceride, HDL-cholesterol, and HOMA-IR according to quartiles of selected organochlorine pesticides which showed Ptrend<0.1 or Pquadratic<0.1 in (PDF)Click here for additional data file.Figure S2Means of year 20 BMI, triglyceride, HDL-cholesterol, and HOMA-IR according to quartiles of selected PCBs which showed Ptrend<0.1 or Pquadratic<0.1 in the (PDF)Click here for additional data file.Table S1Target analytes with detection rate and abbreviations used.(DOCX)Click here for additional data file.Table S2Number of subjects in each quartile of organochlorine (OC) pesticides, polychlorinated biphenyl (PCB) or polybrominated biphenyl (PBB).(DOCX)Click here for additional data file.Table S3Adjusted* means of LDL-cholesterol (mg/dL) at year 20 according to quartiles of organochlorine (OC) pesticides, polychlorinated biphenyl (PCB) or polybrominated biphenyl (PBB).(DOCX)Click here for additional data file.


The interaction between tumor progression and innate immune system has been well established in the last years. Indeed, several lines of clinical evidence indicate that immune cells such as tumor-associated macrophages (TAMs) interact with tumor cells, favoring growth, angiogenesis, and metastasis of a variety of cancers. In most tumors, TAMs show properties of an alternative polarization phenotype (M2) characterized by the expression of a series of chemokines, cytokines, and proteases that promote immunosuppression, tumor proliferation, and spreading of the cancer cells. Tumor suppressor genes have been traditionally linked to the regulation of cancer progression; however, a growing body of evidence indicates that these genes also play essential roles in the regulation of innate immunity pathways through molecular mechanisms that are still poorly understood. In this paper, we provide an overview of the immunobiology of TAMs as well as what is known about tumor suppressors in the context of immune responses. Recent advances regarding the role of the tumor suppressor ARF as a regulator of inflammation and macrophage polarization are also reviewed. This process defined as cancer immunosurveillance was initially described by Ehrlich and subsequently revisited by Thomas and Burnet –3, gaini (STAT1) , natural (STAT1) , 6, γδ T T cells , 8, IL-1 T cells , perfori T cells , and gra T cells  have beeMacrophages are one of the major widely distributed innate immune cells and present essential roles in the primary response to pathogens, maintenance of tissue homeostasis, inflammation, and immunity. Macrophages are derived from bone marrow progenitors as immature monocytes. After circulating in the blood stream, monocytes migrate into tissues where they differentiate into resident macrophages .γ either alone or cooperating with microbial stimuli such as lipopolysaccharide (LPS) or cytokines -α and GM-CSF). These cells secrete high levels of classical proinflammatory cytokines such as TNF-α, interleukin (IL)-1, IL-6, IL-12, or IL-23 and increase their concentrations of nitric oxide (NO), superoxide anions, and oxygen radicals [Macrophages are dynamic cells that might modify their functional profiles in response to a variety of stimuli polarizing to functionally different phenotypes. Two distinct subsets of macrophages have been proposed, including classically activated (M1) and alternatively activated (M2) macrophages  Figure . M1 macrradicals , 15. Furradicals . In contrast, IL-4/IL-13 stimulation induces M2 macrophages that reduce IL-12 and IL-23 expression while upregulate the anti-inflammatory cytokine IL-10 , 18. AddAn alternative metabolic pathway of L-arginine, catalyzed by arginase-1 (Arg-1) provides another feature of distinction among the two macrophage activation states. M1 macrophages upregulate iNOS to catabolize L-arginine to NO and citrulline, but M2 macrophages are characterized by high expression of Arg-1, a cytosolic enzyme which metabolizes L-arginine to ornithine and polyamines, which are precursors for collagen synthesis and cellular proliferation . M1 and M2 macrophages also express a different chemokine repertoire. M1 macrophages produce proinflammatory chemokines such as (C-X-C motif) ligand 9 (Cxcl9), Cxcl10, and Cxcl5 , whereasThis plethora of molecules and genes leads macrophages to display distinct functions in virtue to their polarization state. Thus, classically activated macrophages are vital components in the initiation and maintenance of inflammation, as well as in host defense and priming antitumor immune response . For insγ, IL-2, IL-3, TNF-α and GM-CSF, and also suppresses the antigen-presentation capacity of antigen presenting cells. Furthermore, Ccl17, Ccl22, and Ccl24 production favors the attraction of immune-inhibitory cells such as regulatory T-cells (Treg) [ Opposite, M2 macrophages have poor antigen-presenting capability, produce factors that suppress T-cell proliferation and activity, and mainly participate in parasite clearance, tissue remodeling, immune modulation, and tumor progression . Thus, Is (Treg) . Compelling evidence has emerged in recent years for macrophages playing an important function in tumor development. Although the role of macrophages in tumors is still controversial, in most human cancers such as breast, prostate, ovarian, cervical, lung carcinoma, and cutaneous melanoma, a macrophage-rich microenvironment has been correlated with a poor prognosis , 27. TheAmong the cell surface molecules expressed by TAMs, several members of structurally related C-type lectin receptors such as MR and Macrophage galactose-type C-type lectin 1/2 (Mgl-1/2) are included . The MR Chitinase 3-like 3 (Ym1) is a member of the mammalian chitinase family that also includes Ym2 and BRP-39 in mice, and YKL-40 in humans . Increasα (RELM-α), plays a key role in the regulation of cell growth/proliferation and differentiation. Initially described in lung allergic inflammation [Fizz-1, also known as resistin-like molecule ammation , it is hammation , 42. Fizammation , and itsammation , 39. Stabilin-1, a type-1 transmembrane receptor that mediates clearance of “unwanted self” components, is another marker for M2 macrophages that has been found to be expressed by TAM in several murine tumor models  , 44. StaTAMs derive from circulating monocytic precursors previously recruited to the tumor region in response to the chemokines and cytokines secreted by cancer cells. In the tumor mass, TAMs exert immunosuppressive functions through the release of anti-inflammatory cytokines, modulate the tumor microenvironment producing survival/growth factors , and facilitate the progression of tumors via proangiogenic factors release , 26.β, IL-10 and Arg-1, make a significant contribution to the immunosuppressive condition [A relevant function of TAMs is to diminish the effective antitumor immune response. Several cytokines and proteases derived from TAMs, such as transforming growth factor (TGF)-ondition –48. β inhibits the antitumor response through different mechanisms including (i) inhibition of the cytolytic activity of NK cells [+ T cells into Th2 cells [+ T cells antitumoral activity [TGF-NK cells , 49,  iactivity , and , Ccl22, and to a lesser extent Ccl17 are important chemoattractant for immune-inhibitory cells, such as Treg, which might inhibit antitumor immunity resulting in tumor growth and decreased patient survival . MoreoveAccumulating evidence indicates that TAMs exert a critical function in regulating angiogenesis, the process by which new blood vessels sprout from the existing vasculature. TAMs depletion studies in mice showed reduction of blood vessel density in the tumor tissue  and numeβ, which have been reported to promote angiogenesis in tumors such as gliomas, squamous cell carcinomas of the esophagus, and breast, bladder, and prostate carcinomas [TAMs support tumor cell invasion by secreting a broad repertoire of molecules, including growth factors, cytokines, proteases, and chemokines. For instance, TAMs release potent proangiogenic cytokines such as IL-8 (or Cxcl8) and growth factors as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and TGF-rcinomas –63. Imporcinomas . Moreovercinomas –67. Additionally, TAMs have been found to accumulate in hypoxic regions of human and experimental tumors  . TAMs reMacrophage depletion studies have proven that TAMs are essential for tumor growth  and TAMsAnother process in which TAMs have been involved is in the regulation of metastasis. Indeed, a correspondence between the number of macrophages in metastatic sites and the metastatic potential of the tumor has been observed , and sysTAMs appear to influence the microenvironment to facilitate migration of tumor cells , 75 by tTumor suppressor genes act as sensors of multiple forms of cellular stress, being regulated to induce cell-cycle arrest, senescence, or apoptosis. Nevertheless, in last decades, a growing body of evidence indicates that tumor suppressors play a key role in the modulation of innate immune system, a function more relevant even than their activity as cancer inhibitors. Caenorhabditis elegans has been described to be dependent on p53 function [For instance, innate immune response in the metazoan function . A clearfunction –82. Actifunction , 82–87.In addition, a molecular link between p53, ARF, retinoblastoma protein (Rb), and Toll-like receptors (TLRs) has been shown –89 and rRb was identified as the protein responsible for the congenital tumor retinoblastoma  and playκB pathway in response to virus infection [Furthermore, additional functions of Rb in the control of immune response have been described including a novel role in viral infection surveillance. Thus Rb is required for the activation of the NF-nfection . In addinfection . The mecThe PML gene was originally identified in acute promyelocytic leukemia (APL), being implicated in numerous cellular functions including oncogenesis, DNA damage, senescence, apoptosis, and protein degradation. In addition, accumulating reports have also demonstrated the role of PML in host antiviral defense . PML funThe tumor suppressor p53 also known as “the guardian of genome” is activated in response to several types of cellular stress, including DNA damage and oncogene expression. Under normal conditions, p53 is maintained at very low levels through regulation by murine double minute 2 (Mdm2) protein. Mdm2 inhibits p53 transactivation and prompts p53 for proteasomal degradation by promoting its ubiquitination , 102. Hop53 has been implicated in multiple functions that play key roles in health and disease, including ribosome biogenesis, control of aging, cell cycle arrest, and apoptosis, having a clear importance in tumor suppression . Interes−/− mice and HCT116 p53−/− cells, leading to a dysfunction in both NF-κB and IRF-3 signaling pathways [Pattern recognition receptors such as TLR3 have also been reported to be regulated by p53 . TLR3 plpathways . Upregulpathways .Finally, important proinflammatory chemokines such as monocyte-chemoattractant protein (MCP)-1 have also been reported to be transcriptionally regulated by p53 . MCP-1, Thus, in addition to the well-established function of p53 as a tumor suppressor through regulation of apoptosis or cell cycle, p53 exerts essential roles in the expression of key molecules of the innate immune response. Indeed, the loss of p53 function during carcinogenesis might affect the recognition of tumor cells by the immune system through interfering with inflammatory mediators expression. INK4a/ARF locus (Cdkn2a) that generates two unrelated proteins, the cyclin-dependent kinase inhibitor p16INK4a and ARF, which, respectively, regulate the activity of Rb and the p53 transcription factor [Tumor suppressor ARF  is among the most frequent genes mutated in human cancer . ARF is n factor , 115. ARn factor , 116. Aln factor . Thus, An factor  and trann factor –121. In n factor .β-catenin has been shown after oxidative stress that leads to the induction of apoptosis [Recent studies have shown that the tumor suppressor ARF is more than a simple tumor suppressor and acts a general sensor for different situation of cellular stress. In this context, a regulatory network between Heat shock protein 70 (Hsp70), ARF, and poptosis . ARF defpoptosis . ARF is poptosis .  Furthermore, ARF plays an important role in the regulation of innate immunity and inflammatory processes. Several reports have described an antiviral action of ARF as well as its activation after the expression of viral proteins, viral infection, or type I IFN treatment , 85–87. Interestingly, it has been recently described that ARF is a critical modulator of the inflammatory response and macrophage activation. It has also been reported a molecular link between ARF and TLRs . Additioα, IL-1β), chemokines , and inflammatory mediators (iNOS/PGE2). Mechanisms involved in this inhibitory effect have not been fully explored, although a decrease in NF-κB and Mitogen-activated protein kinases (MAPK) activation have been described in ARF−/− macrophages after stimulation with LPS [κB degradation in ARF-deficient macrophages stimulated with VSV [κB-dependent inflammatory signaling [κB. In contrast, in the absence of ARF, E2F1 is overexpressed, and although NF-κB translocates to the nucleus, excessive E2F1 inhibits its activity by binding to p65 and thereby suppressing NF-κB-dependent genes . Mice lacking the ARF gene are resistant to LPS-endotoxic shock, and a significant reduction of leukocyte recruitment in a model of thioglicollate-induced peritonitis was also reported . Moreovewith LPS , as wellwith VSV . Furtherwith VSV . E2F1 haignaling , 128. ThLoss of ARF gene can abrogate tumor surveillance mechanisms and increase cancer susceptibility. Indeed, mice lacking p19ARF are highly prone to tumor development , 130 andHowever, the link between ARF and immune response in the tumoral context remains an open question. A manuscript by Herranz et al.  goes deeTaken together, it is tempting to speculate that ARF has a profound influence in regulating the polarization of macrophages. Thus, ARF deficiency might modify the immune tumor microenvironment as a result of the induction of multiple activities including (a) immune suppression, through the production of the anti-inflammatory cytokine IL-10 and the secretion of Ccl22 and Ccl2 that attracts Tregs, (b) stimulation of angiogenesis through expression of VEGF and MMP-9, and (c) induction of matrix remodeling through the production of MMP-9, Fizz-1, and Ym1. More and more evidence indicates that tumor suppressors play an essential role in host immunity, placing them as general sensors and modulators of innate immune response. Mechanisms involved in this process remain unclear, although transcriptional regulation of several inflammatory mediators has been reported. In addition, M1/M2 polarization and functional plasticity of macrophages have been shown to be modulated by tumor suppressors. These studies reveal positive effects of tumor suppressors on cancer immunosurveillance that will pave the way for new targeted therapies.Therefore, identification of the role of tumor suppressors in the pathways responsible for the skewing of macrophage function as well as in the regulation of inflammatory mediators will remain an important area of investigation in the years to come.


AbstractAgria mihalyii  is recorded from China for the first time, and both sexes are thoroughly documented using a combination of illustrations, photographs and scanning electron microscopy images. The generic affiliation is corroborated from an expanded definition of genus Agria Robineau-Desvoidy, 1830, and a key to males of the two known species from China is provided. The distribution of coeloconic sensilla on the male pre- and postgonite are shown to possess significant diagnostic and phylogenetic information in this genus. Agria Robineau-Desvoidy is a small genus of the subfamily Paramacronychiinae (Sarcophagidae), occurring in the Holarctic region and comprising eight species worldwide  was known from China . The primary aims of this article are to review the genus Agria from China; to redescribe the newly recorded species; to provide detailed documentation through illustrations, photographs and scanning electron microscopy images of Agria affinis and Agria mihalyii; and to refine the scientific definition of the genus. A key to the known species of Agria from China is also provided.Before the present contribution, only om China . Chao anThe specimens examined were collected by sweeping from brushwood in mountainous regions and are deposited in the Museum of Beijing Forestry University, Beijing, China.Methods for the preparation of terminalia, illustrations, photographs and scanning electron microscopy images follow Terminology of male morphology and terminalia follows http://species-id.net/wiki/Agria_affinisMusca affinis Fallén, 1817: 237. Agria punctata: Robineau-Desvoidy 1830: 377; Pape 1987Agria affinis: Verves 1982: 273; Pape 199639°57'50"N, 115°28'26"E, 1100 m, 6.VII.2009, Coll. R. Bi and F. Li; 1 ♂, Mt. Songshan, 40°30'00"N, 115°49'12"E, 800−1000 m, 30.V.2012, Coll. Y.O. Chen; 1 ♂, Mt. Songshan, 40°30'00"N, 115°49'12"E, 800−1000 m, 30.V.2012, [collector unknown].China: Beijing: 1 ♂, Xiaolongmen, China ; Mongolia; Kazakhstan; Kyrgyzstan; common throughout Europe.PageBreakPageBreakhttp://species-id.net/wiki/Agria_mihalyiiAngiometopa mihalyiiAngiometopa mihalyii: Verves 1981Agria mihalyii: Pape 19924+5-R2+3. Legs dark; fore femur with one strong row of posterior bristles, and with long and dense bristles along anteroventral, ventral and posteroventral margins, fore tibia with four anterodorsal and one posterior bristles; mid femur with two anterior and two posterior bristles, and distal 1/3 with short ventral comb-like posteroventral bristles, mid tibia with two or three anterior and one or two hair-like posterior bristles; hind femur with one row of anterodorsal bristles, and with long and dense bristles along anteroventral, ventral and posteroventral margins, hind tibia with one posterodorsal bristle, one row of anteroventral bristles (7 or 8) and one row of anterodorsal bristles (3 or 4). Abdomen long oval with densely grey pollinosity; tergites each with three distinct black spots; tergite 3 without median marginal bristles, tergite 4 with one pair of median marginal bristles, tergite 5 with strong marginal bristles, tergites 7+8 form a hump-shaped structure, epandrium brownish black, sternites 1−4 with long and dense bristles. Terminalia: Cercus tapering and pointed distally, basal 1/3 with long dense bristles; surstylus long and with oval rounded tip in lateral view  distributed on distal half +3, intra-alars 1(0)+2(3), supra-alars 2, postpronotals 3, postalars 3 or 4, notopleurals 2, scutellum with 1 apical, 1 subapical, 1 basal and 1 discal bristles, with or without prebasal bristle. Pleuron with meropleurals 8−10, katepisternal bristles 2(3)+1, prosternum and metasternum bare, proepisternum bare, proepimeron in lower part with fine bristles, postalar wall bare or with fine bristles. Wing hyaline; subcostal sclerite and basicosta yellow, bare; tegula dark yellow, with black setulae; costal spine not differentiated, several dorsal black bristles at node of Rral view . Ejacularal view  and 8B. ral view , and disral view ; postgontal half ; juxta vral view  and 9A−Cral view ; lateralral view  and 9C.PageBreakPageBreakFirst flagellomere length 1.40 × as long as pedicel. Thorax chaetotaxy: acrostichals 0+2, intra-alars 1+2. Fore femur with one posterior, one posterodorsal and one posteroventral rows of bristles; mid femur with short and sparse ventral bristles, without apical comb-like posteroventral bristles, mid tibia with two posterodorsal and two posterior bristles, one strong ventral bristle; hind tibia with two or three posterodorsal bristles, one anteroventral bristle. Abdomen oval; tergites 5 and 6 entire, tergite 7 membranous like with several bristles on the anterior margin, tergite 8 divided into two plates and each with two strong bristles ; Mongolia; North Korea; Russia ; Ukraine.PageBreakPageBreakParamacronychiinae but his analysis gave very little phylogenetic resolution among the genera. More novel morphological characters should be added; especially the structures of the male paramacronychiine genitalia need a much more detailed comparative study. The scanning electron microscopy was here utilized to achieve more morphological details of the male genitalia of Agria. Two features seem to be particularly valuable in corroborating the monophyly of Agria: (1) the shape of the acrophallus, which from a broad base curves back strongly between the juxta, and (2) the pair of lateral sclerotizations originating just distal to the acrophallus and slanting antero-ventrally partly covering the latter.Agria mihalyii postgonite  costal spine reduced, and (2) male lower calypter at least partly brownish. The material examined for our study has led us to reinterpret Agria mihalyii as having a reduced costal spine, which leaves this character state as phylogenetically uninformative for species-level relationships within Agria. However, two other character states appear to support a basal split between Agria mihalyii and the remaining species of Agria, which all share the following: (a) antero-basal bristle of postgonite reduced; and (b) pregonite with numerous coeloconic sensilla scattered across its surface. The latter character state is documented in Agria affinis and in Agria monachae, Agria hikosana and Agria shinonagai. The distribution of sensilla and other ultrastructural details of male terminalia in Sarcophagidae obviously is a potentially rich source of phylogenetic data, that still remains to be fully explored.The present SEM documentation has revealed the presence of coeloconic sensilla on the distal half of the stgonite  and on bmulation , and is (Diptera , Lepidoppidoptera, Hemipteemiptera , and Hymenoptera . Only ferminalia , 2008, apulation , and the


To investigate the phylogenetic position of Aquificales at the whole-genome level, here we reconstructed the phylogenetic trees of 18 bacteria including six Aquificales species based on the concatenated data of proteins shared by these bacteria. In the phylogenetic tree based on the whole-genome information, Aquificales was more closely related to Thermotogales than to Proteobacteria, suggesting that the Aquificales is a relatively early branching lineage within the Bacteria. Moreover, we classified the phylogenetic tree of each conserved orthologous protein by its topology. As a result, in the most major type of the phylogenetic trees, Aquificales was closely related to the Thermotogales. However, Aquificales was closely related to ε-proteobacteria in 21.0% of all phylogenetic trees, suggesting that many proteins phylogenetically related to the ε-proteobacteria may be encoded in the genomes of the members of the Aquificales. This unique feature may be responsible for the high variability in the branching order of Aquificales in single-gene phylogenetic trees.Species belonging to the order Aquificales are believed to be an early branching lineage within the Bacteria. However, the branching order of this group in single-gene phylogenetic trees is highly variable; for example, it has also been proposed that the Aquificales should be grouped with  Species belonging to the order Aquificales are non-spore-forming, Gram-negative rods that are strictly thermophilic with optimal growth usually occurring above 65°C –3. In teAquifex has been shown to be close to ε-proteobacteria [Presently, the Aquificales species are believed to be the earliest branching lineage within the Bacteria –10. Howebacteria , 12 or tbacteria  in many bacteria . Many ofThe phylogenetic tree based on 16S rRNA sequences provides the presently accepted framework for understanding the evolutionary relationships among bacteria . HoweverAquifex aeolicus was sequenced in 1998 [Hydrogenobacter thermophilus TK-6, Hydrogenobaculum sp. Y04AAS1, Persephonella marina, Sulfurihydrogenibium azorense and Sulfurihydrogenibium sp. YO3AOP1) have recently been sequenced [ε-proteobaceria was analyzed at the whole genome level.The phylogenies inferred from concatenated data of housekeeping proteins amplified the resolving power for delineating the phylogenetic relationships among prokaryotes –23. The equenced , 25. HerAquifex aeolicus, Hydrogenobacter thermophilus TK-6, Hydrogenobaculum sp. Y04AAS1, Persephonella marina, Sulfurihydrogenibium azorense, Sulfurihydrogenibium sp. YO3AOP1, Bacillus subtilis subsp. subtilis str. 168, Burkholderia mallei ATCC 23344, Campylobacter jejuni subsp. jejuni NCTC 11168, Chlamydophila pneumoniae CWL029, Deinococcus radiodurans R1, Thermus thermophilus HB8, Escherichia coli str. K-12 substr. MG1655, Salmonella enterica subsp. enterica serovar Typhimurium LT2, Helicobacter pylori 26695, Pyrococcus horikoshii OT3, Thermotoga maritima MSB8 and Thermotoga petrophila RKU-1. These genome sequences was obtained from GenomeNet (http://www.genome.jp/).In this study, we used 18 genome sequences from Hydrogenobacter thermophilus TK-6 was used as a query) were performed against 18 whole genomes by using stand-alone BLAST program [First, BLASTP searches  were estimated with the bootstrap sampling method (200 replications).Next, we constructed 62 multiple-alignments using MUSCLE . After tIn addition, to reduce the influence of compositional bias, we recoded the alignment without saturated sites in 4 states based on the physicochemical properties of the amino acids  as folloγ-proteobacteria, and two ε-proteobacteria, we used 12 genome sequences from Aquifex aeolicus, Hydrogenobacter thermophilus TK-6, Hydrogenobaculum sp. Y04AAS1, Persephonella marina, Sulfurihydrogenibium azorense, Sulfurihydrogenibium sp. YO3AOP1, Campylobacter jejuni subsp. jejuni NCTC 11168, Deinococcus radiodurans R1, Escherichia coli str. K-12 substr. MG1655, Salmonella enterica subsp. enterica serovar Typhimurium LT2, and Helicobacter pylori 26695.To construct the phylogenetic tree of six Aquificales species, two Thermotogales species, two γ-proteobacteria, and ε-proteobacteria, we used 8 genome sequences from Campylobacter jejuni subsp. jejuni NCTC 11168, Deinococcus radiodurans R1, Thermus thermophilus HB8, Escherichia coli str. K-12 substr. MG1655, Salmonella enterica subsp. enterica serovar Typhimurium LT2, Helicobacter pylori 26695, Thermotoga maritima MSB8, and Thermotoga petrophila RKU-1. The phylogenetic tree based on the concatenate data of the whole conserved orthologous proteins was constructed by the same method as described above. The ML trees of individual proteins were constructed using PhyML [To construct the phylogenetic tree of Thermales-Deinococcales species, Thermotogales species, ng PhyML .ε-proteobacteria with 80% bootstrap support, suggesting that the Aquificales is a late branching group within the Bacteria.First, we constructed ML trees based on the 16S rRNA sequences of 18 bacteria . This phγ-proteobacteria, and ε-proteobacteria) with 100% bootstrap support. Analysis of signature sequences (consisting of conserved inserts or deletions) in highly conserved proteins suggested that the Aquificales diverged after the branching of Thermotogales, Thermales-Deinococcales, Cyanobacteria, Spirochetes, and Chlamydiae, but before the emergence of Proteobacteria [Bacillus subtilis was clustered with Thermales-Deinococcales, the ML tree obtained by the recoded alignment  was very similar to the previous tree . To avoid a potential cause for long branch attraction, we removed the most saturated sites from the whole multiple alignments according to the previously described method by Boussau et al. . As a rebacteria . Howeverbacteria . To reduous tree , implyinε-proteobacteria [ε-proteobacteria, we reconstructed phylogenetic trees of 12 bacteria including six Aquificales species, two Thermotogales species, two γ-proteobacteria, and two ε-proteobacteria based on the concatenated data of proteins shared by these bacteria. First, 271 orthologous gene families that are shared by all 12 bacteria were selected. As a result, 16,532 amino acid sites were considered in the ML analysis. The phylogenetic tree based on this whole-genome information indicated that the 12 bacteria were divided into four major groups  with 100% bootstrap support , which is consistent with the results obtained from the phylogenetic tree based on the 271 conserved proteins . These results suggest that many proteins phylogenetically related to the ε-proteobacteria may be encoded in the genomes of the members of the Aquificales order.Next, to investigate the contribution of each protein to the whole-genome phylogenetic tree, we constructed 271 ML trees from 271 protein sets. We classified these trees into the following three types : A-type,proteins . Interesproteins . For exaγ-proteobacteria, and ε-proteobacteria. As a result, the Thermales-Deinococcales group was clustered with the Thermotogales group with 100% bootstrap support in the phylogenetic tree based on the whole genome conserved proteins , and E-type trees occupied 37.1% (96 trees) of all phylogenetic trees (ε-proteobacteria may be low compared to the Aquificales. To compare this profile with that of other bacteria, we performed the same phylogenetic analysis against 259 conserved proteins among Thermales-Deinococcales, Thermotogales, proteins . Next, wproteins ; D-type,ic trees . In contic trees , suggestε-proteobacteria may be encoded in the genomes of the Aquificales. This unique feature may be responsible for the high variability in the branching order of Aquificales in single-gene phylogenetic trees. Moreover, these results raised the possibility that a large horizontal gene transfer had occurred between the Aquificales and ε-proteobacteria, which was suggested by Boussau et al. [ε-proteobacteria include hydrogen-oxidizing bacteria and sulfur-oxidizing bacteria [These results support the hypothesis that many proteins phylogenetically close to the u et al. . This hybacteria  which ocSeveral house-keeping proteins have often been used for the phylogenetic analyses of bacteria –33. HoweList of 62 proteins to construct the phylogenetic tree based on 18 whole genomesClick here for additional data file.


A20, CYLD, DUBA, ST2, CD180, SIGIRR, TANK, SOCS1, SOCS3, SHIP, IRAK-M, DOK1, DOK2, SHP1, SHP2, TOLLIP, IRF4, SIKE, NLRX1, ERBIN, CENTB1, and Clec4a2 in human and mouse solid organs. Humans and mice displayed significant differences between their respective mRNA expression patterns of these factors. Additionally, we characterized their expression profiles in mononuclear blood cells upon bacterial endotoxin, which showed a consistent induction of A20, SOCS3, IRAK-M, and Clec4a2 in human and murine cells. Furthermore, we studied the expression pattern in transient kidney ischemia-reperfusion injury versus post-ischemic atrophy and fibrosis in mice. A20, CD180, ST2, SOCS1, SOCS3, SHIP, IRAK-M, DOK1, DOK2, IRF4, CENTB1, and Clec4a2 were all induced, albeit at different times of injury and repair. Progressive fibrosis was associated with a persistent induction of these factors. Thus, the organ- and species-specific expression patterns need to be considered in the design and interpretation of studies related to PRR-mediated innate immunity, which seems to be involved in tissue injury, tissue regeneration and in progressive tissue scarring.The cell type-, organ-, and species-specific expression of the pattern-recognition receptors (PRRs) are well described but little is known about the respective expression profiles of their negative regulators. We therefore determined the mRNA expression levels of  Pattern-recognition receptors (PRRs) are germ-line encoded receptors of the innate immune system that translate the recognition of pathogen-associated molecular patterns (PAMPs) and tissue damage-associated molecular patterns (DAMPs) into an immediate and antigen-unspecific inflammatory response . Some ofSocs3-deficient mice [Regulators of the signaling cascade can appear as enzymes, orphan receptors and adaptor proteins. A20 is a deubiquitinase that inhibits TRAF6 downstream of TLR2, -3, -4, and -9 in macrophages and thereby protects mice from endotoxic shock . It alsoent mice . “SH2-coent mice ,23. IL-1ent mice . Downstrent mice . SH2-conent mice . The relent mice . Toll-inent mice . IRF-4 ient mice . Suppresent mice . NLRX1, ent mice ,32. Erbient mice . CENTB1 ent mice . CLEC4A2ent mice ,36.Species-specific differences in inflammation-related gene expression clearly exist . As longA20, CYLD, DUBA, ST2, CD180, SIGIRR, TANK, SOCS1, SOCS3, SHIP, IRAK-M, DOK1, DOK2, SHP1, SHP2, TOLLIP, IRF4, SIKE, NLRX1, ERBIN, CENTB1 and Clec4a2. All of these molecules were constitutively expressed in human spleen but the mRNA expression levels of A20, ST2, DOK2, and ERBIN were low  to quantify the mRNA expression levels of the following PRR signaling regulators in human solid organs: were low . GeneralSOCS3 and IRAK-M were low  disease or whether fibrosis is rather a secondary healing response to stabilize the remaining parenchyma, whenever parenchymal healing is insufficient ,66. In fIRF4 , which wTogether, we identified significant differences in the mRNA expression of PRR signaling regulators in human and mouse solid organs and in their regulation in transient inflammation and progressive tissue fibrosis. These findings can help to generate novel hypotheses on the role of single PRR signaling regulators in specific diseases. Furthermore, the species-specific expression of single PRR co-factors need to be considered in the interpretation of either data and human studies need to verify the functional roles of single factors suggested by studies performed in rodents.GAPDH/G3PDH as the housekeeping gene for the analysis of human solid organ cDNA. The PCR product band was determined by video imaging and computer analysis, and band intensity was determined. If necessary, the concentration of individual cDNA preparations was than adjusted so that the average band intensity for the reference genes used to normalize the panel varied no more than 20%. As only a single pool was available for each organ, no studies on biological replicates allowing statistics could be performed. According to Clontech all human samples were purchased and imported in accordance with all local laws and regulations. Donors were tested to be negative for HIV, hepatitis B virus, and hepatitis C virus. Further exclusion criteria were as follows: manifest infections during the last 4 weeks, fever, symptomatic allergies, abnormal blood cell counts, increased liver enzymes, or medication of any kind except vitamins and oral contraceptives. The study was approved by the Ethics committee of Klinikum der Universität München and does not refer to any other experiments.Human solid organ prenormalized cDNA derived from poly-(A)-selected DNase-treated RNAs, which were purified from tissues of healthy male and female humans of caucasian descent were obtained from Clontech, Mountain View, CA. For RNA isolation, either whole organs or tissue samples representing the entire organ were used. To reduce the risk of selection bias, the purchased cDNA preparations are pools of cDNA from many human individuals for most of the organs as it is stated in the manufacturer’s certificate of analysis.  An equalad libitum. Mice were sacrificed by cervical dislocation, tissues were kept in RNAlater  and high quality, DNA-free, RNA was isolated from same tissue mass (10 mg) with Pure Link RNA Mini Kit  according to manufacture instructions as described [w/v) agarose gels as described. 1 μg of good quality RNA of each individual sample was preceded to cDNA using thermo stable RNAse inhibitor during reverse transcription as described [Twelve week old adult C57BL/6 mice were purchased from Charles River, Sulzfeld, Germany and maintained under standard conditions and 12 h light/dark cycle. Animals were housed in polypropylene cages and allowed free access to food and water escribed . Samplesescribed . Reversen = 5–10) underwent unilateral renal pedicle clamping for 45 min followed by reperfusion as a model of ischemia-reperfusion injury (IRI) as described [Groups of eight week old C57BL/6 mice , arithmetic mean (AM) minimal value, maximal value, standard deviation (SD), variance and coefficient of variance (CV) of the housekeeping genes were calculated  was used for amplification. Quantitative real-time PCR was performed on Light Cycler 480 . Each amplification step included initiation phase at 95 °C, annealing phase at 60 °C and amplification phase at 72 °C and was repeated 45 times. Gene-specific primers  were used as listed in 2O were negative for target and housekeeping genes. Primers were designed to be cDNA specific and to target most CCDS approved transcripts. In silico specificity screen (BLAST) was performed. The lengths of amplicons were between 80 and 148 bp. The kinetics of the PCR amplification (efficiency) was calculated for every set of primers. The efficiency-corrected quantification was performed automatically by the Light Cycler 480 based on extern standard curves describing the PCR efficiencies of the target and the reference gene (ratio = EtargetΔCPtarget (control − sample)/ErefΔCPref (control − sample)). To reduce the risk of false positive Cp the high confidence algorithm was used. All the samples that did not rise above the background fluorescence (crossing point Cp or quantification cycle Cq) of 40 cycles during the amplification reaction were considered not detectable. Crossing points between 5 and 40 cycles were considered detectable. The melting curves profiles were analyzed for every sample to detect unspecific products and primer dimers. Products were visualized on agarose gels, extracted and analyzed for sequence.lculated . PRR sigFor human PBMC isolation whole blood (25 mL) was drawn from healthy volunteers into sterile syringes containing 1.6 mg EDTA/mL blood. Whole blood was diluted 1:1 with PBS, layered on Ficoll -containing density gradient and centrifuged at 2500 RPM for 22 min. Afterwards the PBMC fraction was harvested and washed twice in PBS. 1 million cells per well were plated to 6 well-plates containing 2 mL RPMI with 1% FCS and 1% P/S. For each timepoint, three samples were stimulated with 500 ng/mL LPS  or PBS. After 4, 12, 18 and 24 h cells were harvested and RNA was isolated with Pure Link RNA Mini Kit according to manufacture instructions. cDNA preparation was done with 1 μg per sample as described above.For murine PBMC isolation six week old C57BL/6 mice were sacrificed and whole blood (1 mL per mouse) was drawn into 1.6 mg EDTA/mL containing syringes and pooled. The further steps were done as described for human PBMCs.Kidney tissues were fixed in 4% neutral-buffered formalin, dehydrated in graded alcohols and embedded in paraffin. For periodic acid-Schiff (PAS) staining or immunostaining 4 μm sections were deparaffinized, rehydrated, transferred into citrate buffer, and either autoclaved or microwave treated for antigen retrieval and processed as described . The folp < 0.05 indicated statistical significance).Data were expressed as mean ± standard error of the mean (SEM). Comparison between groups was performed using univariate ANOVA (A value of Together, we identified significant differences in the mRNA expression of PRR signaling regulators in human and mouse solid organs and in their regulation in transient inflammation and progressive tissue fibrosis. These findings can help to generate novel hypotheses on the role of single PRR signaling regulators in specific diseases. Furthermore, the species-specific expression of single PRR co-factors need to be considered in the interpretation of either data and human studies need to verify the functional roles of single factors suggested by studies performed in rodents.


Most cases of the 2009 influenza A (H1N1) infection are self-limited, but occasionally the disease evolves to a severe condition needing hospitalization. Here we describe the evolution of the respiratory compromise, ventilatory management and laboratory variables of patients with diffuse viral pneumonitis caused by pandemic 2009 influenza A (H1N1) admitted to the ICU.This was a multicenter, prospective inception cohort study including adult patients with acute respiratory failure requiring mechanical ventilation (MV) admitted to 20 ICUs in Argentina between June and September of 2009 during the influenza A (H1N1) pandemic. In a standard case-report form, we collected epidemiological characteristics, results of real-time reverse-transcriptase--polymerase-chain-reaction viral diagnostic tests, oxygenation variables, acid-base status, respiratory mechanics, ventilation management and laboratory tests. Variables were recorded on ICU admission and at days 3, 7 and 10.During the study period 178 patients with diffuse viral pneumonitis requiring MV were admitted. They were 44 ± 15 years of age, with Acute Physiology And Chronic Health Evaluation II (APACHE II) scores of 18 ± 7, and most frequent comorbidities were obesity (26%), previous respiratory disease (24%) and immunosuppression (16%). Non-invasive ventilation (NIV) was applied in 49 (28%) patients on admission, but 94% were later intubated.2/FIO2 170 ± 25). Tidal-volumes used were 7.8 to 8.1 ml/kg , plateau pressures always remained < 30 cmH2O, without differences between survivors and non-survivors; and mean positive end-expiratory pressure (PEEP) levels used were between 8 to 12 cm H2O. Rescue therapies, like recruitment maneuvers (8 to 35%), prone positioning (12 to 24%) and tracheal gas insufflation (3%) were frequently applied. At all time points, pH, platelet count, lactate dehydrogenase assay (LDH) and Sequential Organ Failure Assessment (SOFA) differed significantly between survivors and non-survivors. Lack of recovery of platelet count and persistence of leukocytosis were characteristic of non-survivors. Mortality was high (46%); and length of MV was 10 (6 to 17) days.Acute respiratory distress syndrome (ARDS) was present throughout the entire ICU stay in the whole group (mean PaOThese patients had severe, hypoxemic respiratory failure compatible with ARDS that persisted over time, frequently requiring rescue therapies to support oxygenation. NIV use is not warranted, given its high failure rate. Death and evolution to prolonged mechanical ventilation were common outcomes. Persistence of thrombocytopenia, acidosis and leukocytosis, and high LDH levels found in non-survivors during the course of the disease might be novel prognostic findings. On April 2009, a novel influenza A H1N1) virus emerged in Mexico and spread rapidly across the world N1 virus . Unlike The Argentinian population was greatly affected during the pandemic, with a total of 1,390,566 cases of influenza-like illness requiring 14,034 hospitalizations. Of the 11,746 confirmed cases of patients infected with the new strain, 617 died . This reThe severity of disease was rapidly perceived by health authorities and scientific societies. Hence, a committee of experts of the Argentinian Society of Intensive Care Medicine decided to focus on the most acutely ill patients: those presenting with diffuse viral pneumonitis requiring mechanical ventilation. They designed an epidemiological study, recently-published, to determine risk factors and outcomes ; this isThere remains, however, a paucity of data published on physiological evolution during ICU stay . This prThis was a multicenter, inception cohort study that included patients aged > 15 years admitted to the ICU with a previous history of influenza-like illness, evolving to acute respiratory failure that required mechanical ventilation during the 2009 winter in the Southern Hemisphere. These patients had confirmed or probable disease caused by the 2009 influenza A (H1N1) virus and were included in the Registry of Cases of the Argentinian Society of Intensive Care Medicine (SATI), created to characterize local aspects of the pandemic. On 27 June 2009, a form to collect online epidemiological data was posted on the official SATI website. A detailed description and analysis of this information was recently published .There was also an optional, more comprehensive case-report form to complete, developed by experts of the SATI's Respiratory Committee for recording certain pre-specified variables throughout ICU stay, which included mechanical ventilation (MV), respiratory mechanics, oxygenation, blood chemistry and organ failure variables. This information was collected over 10 days and is analyzed in the present study.Patients were characterized as confirmed, probable or possible cases of 2009 influenza A (H1N1) ,24. As aWe collected dates of hospital and ICU admission, and of MV onset; demographics; risk factors for influenza A; actual weight; height; severity of illness , organ failures ; type of MV used, as noninvasive (NIV) and invasive; and date of intubation. Ideal body weight  and body mass index (BMI) were calculated; obesity was defined as a BMI > 30.At MV onset (Day 0) and on Days 3, 7 and 10, until death or discharge, whichever occurred first, we recorded: (1) MV-related variables. (2) MV modes: volume-controlled ventilation (VCV); pressure-controlled ventilation (PCV); bilevel mode; pressure support ventilation (PSV); other. (3) Tidal volume  (4) Pressures: peak, plateau pressures, total positive end-expiratory pressure (PEEP) and driving pressure (plateau pressure - PEEP), in cmH2O. (5) Static compliance (ml/cmH2O). (6) Respiratory rate (RR). (7) Inspired oxygen fraction (FIO2). (8) Use of adjuvants of MV: recruitment maneuvers, prone positioning, or tracheal gas insufflations. (9) Use of NIV: duration (hours); requirement of intubation; types of interfaces and of ventilators used . (10) Date of extubation; use of NIV for extubation failure, need of reintubation.(11) Date of tracheostomy 10. (12) Blood gases and acid-base variables. (13) PaO2/FIO2 relationship. (14) Lung infiltrates in CXR (in quadrants). (15) Use of oseltamivir, corticosteroids and neuromuscular blockers. (16) Blood chemistry. (17) Daily fluid balance. (18) SOFA score. (19) Cause of death.The main outcome measure was hospital mortality; secondary outcomes were length of MV, of ICU (LOSICU) and of hospital (LOSHOSP) stays.In case of missing observations, local study coordinators were contacted to provide the corresponding values. Proportions were calculated as percentages of existing data.No assumptions for missing data were made.P-value of <.05 was considered statistically significant. A Kaplan-Meier curve was constructed to evaluate survival over the follow-up period.Statistical analysis was performed with SPSS 17.0 . Data were analyzed for the entire population; for the subgroups of survivors vs. non-survivors; and for patients receiving NIV on admission vs. those who did not. Descriptive statistics used were: mean ± standard deviations (SD) and median and 25-75% interquartile ranges (IQR) for continuous data of normal and non-normal distribution, respectively; and percentages for categorical data. Differences between subgroups were analyzed with unpaired t test, Mann-Whitney U test, and Chi-square tests, as appropriate. A Over time, normally distributed data were analyzed with two-way repeated measures of ANOVA. At the pre-specified time points, differences within the entire group and subgroups, and between subgroups, were tested using paired and unpaired t tests, respectively.In non-normally distributed data, differences over time within the entire group and the subgroups were analyzed with Friedman's and Wilcoxon tests. Comparisons between subgroups at the pre-specified time points were tested with Mann-Whitney U test. The Bonferroni correction was used to adjustments for multiple comparisons.The local Institutional Review Boards waived the need for informed consent, given the general lack of knowledge on the clinical and outcome characteristics of the ongoing pandemic and to the non-interventional study design.Between 6 June and 28 August 2009, the SATI's online Registry included 337 patients admitted to 35 ICUs with confirmed/probable/possible diffuse viral pneumonitis caused by influenza A (H1N1), with acute respiratory failure requiring MV (14). Of these, 178 consecutive patients admitted to 20 ICUs were followed over time, and are presented in this study. To address any potential concern that unconfirmed cases could belong to a different population of patients, we performed a sensitivity analysis of clinical and outcome characteristics data after exclusion of these patients. The results of this analysis did not differ from those of the primary assessments, so the 178 patients are considered for evaluation.Briefly, patients were middle-aged, with no gender preponderance; they had a history of symptoms of nearly one-week duration and were ventilated at 1 [0 to -2] day after hospital admission. Pre-existent respiratory diseases, obesity, and diseases causing immunosuppression were the most frequent comorbid conditions; and prevalence of pregnancy was higher than in the general population, as expected . Non-sur2O. Non-survivors displayed a trend towards lower Vt and higher plateau pressures, which differed significantly from survivors only at Day 7. Intermediate PEEP levels were used, and decreased in survivors from Day 3 onwards. Driving pressures were similar over time in all patients; only at admission did non-survivors exhibit higher values.During the study period, the entire group had Vt values between 7.8 to 8.1 ml/kg of IBW, with plateau pressures remaining always < 30 cmH2/FIO2 increased significantly over time in all patients and in survivors. It remained, however, < 200 in the whole group throughout the entire ICU stay due to non-survivor values. Non-survivors displayed significantly lower PaO2/FIO2 at all time points.PaOP < 0.01) and then decreased during the study in the entire group, especially at Day 10 , which reflected the improvement in survivors .Lung infiltrates (in quadrants) peaked at day 3 ; PaCO2 44 ± 4 vs. 35 ± 3 mmHg (P = 0.04); pH 7.29 ± 0.03 vs. 7.39 ± 0.05 (P = 0.05), and plateau pressures of 30 ± 2 vs. 25 ± 3 cmH2O (P = 0.03). Recruitment maneuvers became significantly more common in non-survivors at Day 3 , as did prone positioning . After that, only prone positioning remained significantly more used in non-survivors . Six patients received tracheal gas insufflation; only one survived. Neuromuscular blockers were prescribed in 18% of patients on admission; and their use was subsequently more frequent in non-survivors .In Figure The main causes of death were refractory hypoxemia (64%); followed by multiorgan dysfunction syndrome 15%) and shock (10%). Prolonged mechanical ventilation and long ICU and hospital stays were frequent  underwent a trial of NIV on admission; they were significantly less ill and had a lower incidence of immunosuppression. Oxygenation and outcome variables were similar to those of patients not receiving NIV.n = 30) receiving NIV survived; duration of NIV was of 8 (2 to 18) hours. There were no differences between survivors and non-survivors in the duration of the procedure, or in the type of interface or respirator used. Of note, most patients on NIV  had to be intubated and ventilated invasively for hypoxemic failure. Characteristics associated to NIV success/failure are shown in Table Sixty-one percent of patients , with success (reintubation not needed) in 8 cases (66%).P < 0.000 for Days 3, 7 and 10 vs. Day 0), secondary to elevations in survivors. At all time points, platelets differed between survivors and non-survivors. Conversely, white blood cell count showed a progressive increase in the whole group (P < 0.000 for Days 7 and 10 vs. Day 0), due to elevations in non-survivors.The most consistent changes over time were found in platelet count, which increased significantly in the whole cohort (P < 0.000 for Days 7 and 10 vs. Day 0), as a result of the decrease in survivors. SOFA was significantly lower in survivors throughout the study.Creatine-kinase and markers of liver injury  were mildly elevated and displayed no substantial changes. On the contrary, lactate-dehydrogenase levels were significantly higher in non-survivors throughout the study. Creatinine levels were stable over the period, but were significantly higher in non-survivors on Days 0 and 3. Finally, SOFA score diminished over time in all patients  patients that were mechanically ventilated for acute respiratory failure due to diffuse pneumonitis during the pandemic in Argentina. Though most were middle-aged, previously healthy adults, patients with preexistent lung disease, immunosuppression, obesity and pregnancy were also affected. Mortality was high and evolution to chronic critical illness was common, as shown by prolonged mechanical ventilation, high needs of tracheostomy, and lengthened ICU and hospital stays.2/FIO2 differed significantly between survivors and non-survivors, requiring higher FIO2 and PEEP in this last subgroup. Yet the levels of applied PEEP were only in the intermediate range, similar to mean values of 8.7 cmH2O of PEEP in an international study on mechanical ventilation [2 used in our study. Driving pressures were similar in both subgroups most of the time, suggesting an intention to limit alveolar excursion as part of a protective strategy.Patients had characteristically a history of protracted symptoms and displayed severe compromise of oxygenation compatible with ARDS throughout the study period, which only improved in survivors. At all time points, PaOtilation , which m2O [It is striking that, as has been described in similar studies on mechanical ventilation performed during the 2009 influenza A (H1N1) pandemic ,7, tidal2O , indicat2O  since it2O . We, lik2O ,7,32,33,2O ,35 have 2O .Not unexpectedly, VCV was the most common ventilator mode used. PCV use increased throughout the study period, peaking at Day 10. This is in contrast with the recently identified trend towards decreased PCV utilization. Transition to PCV mode was associated with preceding physiological worsening, so clinicians might have perceived PCV utilization as part of a global lung-protective strategy .Refractory hypoxemia was the main cause of death. As in other studies ,7,11, reNIV was the first ventilation approach in 28% of cases, with 94% later requiring invasive ventilation, as has been documented in other studies ,7,11. ThSignificant changes in fluid balance were late and reflected changes in survivors. Negative fluid balances could never be obtained, perhaps suggesting a continuing need for hemodynamic support: 72% of patients presented with shock . On the Another important finding was that arterial pH consistently and significantly differed between survivors and non-survivors, as described elsewhere ,41. DuriWith respect to blood chemistry, the usual findings of thrombocytopenia, leukocytosis and mildly elevated creatine-kinase blood levels were present ,42. RegrThis study has several strengths: first, the clinical characteristics and time course of pandemic 2009 influenza A (H1N1) are thoroughly described and analyzed. Second, data were collected prospectively in consecutive patients and with a standardized case-reporting form, representing a large, nationwide cohort. Third, temporal patterns of mechanical ventilation use, acid-base and blood chemistry variables, as well as fluid balance and organ failures, are carefully analyzed. Prognostic implications are highlighted. Finally, we present the largest experience with NIV use during the pandemic.Study limitations include the focus on mechanically ventilated patients, excluding less severe cases also admitted to the ICU. Many cases could not be confirmed because laboratories were overwhelmed with clinical samples, which is also described elsewhere ,14. DataIn 178 patients with diffuse viral pneumonitis caused by the 2009 influenza A (H1N1) virus admitted to the ICU and followed over time, ARDS was the rule, requiring high ventilation support and frequent use of rescue therapies. Death, organ failures, and evolution to prolonged mechanical ventilation were common. In most cases, noninvasive ventilation failed to prevent endotracheal intubation. Finally, elevated LDH levels, lack of recovery of platelet count and persistent acidosis and leukocytosis in non-survivors behaved as prognostic findings.• In 2009 influenza A (H1N1) patients, hospital admission with prompt indication of mechanical ventilation - a marker of severe disease - was associated with a history of symptoms of nearly one-week duration.• An initial NIV trial was not effective to avoid intubation in most patients; thus, this ventilation approach should likely be discarded in this setting.• Mortality and morbidity were frequent: death was common and was mainly caused by persistent, refractory hypoxemia. Prolonged mechanical ventilation and ICU and hospital stays were typical.• pH, platelet count, LDH and SOFA differed significantly between survivors and non-survivors over time. Lack of recovery of platelet count and persistence of leukocytosis might be markers of poor prognosis.• Every effort should be done to increase adherence to protective ventilation in the real world.2 and inspired oxygen fraction used; PCV: pressure-controlled ventilation; PEEP: positive end-expiratory pressure; PSV: pressure support ventilation; RR: respiratory rate; RT-PCR: real-time reverse-transcriptase--polymerase-chain-reaction; SATI: Argentinian Society of Intensive Care; SOFA: Sequential Organ Failure Assessment; VCV: volume-controlled ventilation; Vt: tidal volume.ALI: acute lung injury; ARDS: acute respiratory distress syndrome; BMI: body mass index; CXR: plain chest X-ray film; IBW: ideal body weight; ICU: Intensive Care Unit; LDH: lactate dehydrogenase assay; LOS: length of stay; MV: mechanical ventilation; NIV: non-invasive ventilation; PaO2/FIO2: relation between patient arterial pOThe authors declare that they have no competing interests.FGR conceived and coordinated the study. FGR and EE drafted the manuscript and performed the statistical analysis. FGR, FV, RV, AG, DV and GC participated in the design of the study. FGR, EE, FV, RV, LA, DP, PV, MB, CO, JA, FS, AG, PEG, MD, AZ, MQ, AC, JO, DV, MEG, GC and the rest of the members of the Registry performed the acquisition of data. All authors read and approved the final manuscript.


Aedes aegypti mosquitoes were identified as the primary vector of the virus.An urban epidemic of dengue in Senegal during 2009 affected 196 persons and included 5 cases of dengue hemorrhagic fever and 1 fatal case of dengue shock syndrome. Dengue virus serotype 3 was identified from all patients, and  Aedes spp. mosquitoes and caused by 4 serotypes of dengue virus (DENV): DENV-1, DENV-2, DENV-3, and DENV-4. DENV belongs to the family Flaviviridae, genus Flavivirus  and Thies (n = 87)  . SamplesIn conjunction with human testing, mosquito sampling was performed during December 2009 in households with confirmed dengue cases, and entomologic risk indexes , and 1 died of dengue shock syndrome. The M:F sex ratio for the case-patients was 0.83 , and the median age was 31 years (range 1–93). The fatal case was in a 71-year-old Lebanese woman hospitalized in a private clinic in Dakar on October 30 with a 4-day history of fever; she had severe thrombocytopenia and elevated transaminase levels. Despite treatment , she died of cardiac arrest. Disseminated intravascular coagulation was probably responsible for the hemorrhagic syndrome. 9/L) and leukopenia (leukocyte count 2.5–3.5 × 109 cells/L); 4 reported headache, and 3 reported myalgia/arthralgia. All 5 patients were hospitalized and received transfusions of fresh frozen plasma, platelet concentrates, and other supportive treatments. Dengue-specific IgM and/or RNA were detected in serum samples collected 2 or 8 days after the onset of symptoms. All patients recovered and were discharged from the hospital after 8 or 10 days. Of the 5 other patients  who had hemorrhagic manifestations, all had fever, epistaxis, and melena (5/5) associated with thrombocytopenia  and China (2009) was circulating during the Senegal outbreak . Dates oAedes aegypti, Anopheles gambiae, Culex quinquefasciatus, Cx. tigripes, Cx. tritaeniorhynchus, Cx. antennatus, Cx. ethiopicus, and Cx. nebulosus. Most mosquitoes were collected as adults, but 1,675 emerged from larvae that were collected in the field. Cx quinquefasciatus mosquitoes predominated, but Ae. aegypti mosquitoes, the only dengue vector collected, were found in all sites. DENV-3 was detected from 3 pools of mosquitoes collected in 2 neighborhoods of Dakar: Plateau (2 pools) and Parcelles Assainies (1 pool).The entomologic investigation found high epidemic risk in all localities infested with DENV vectors. The Breteau index ranged from 6.6 to 195.2 in Dakar, 1.6 to 32.7 in Louga, and 1.1 to 14.9 in Thies, whereas the container indices ranged from 15 to 63.2 in Dakar, 5.3 to 15.2 in Thies, and 14.3 to 64.2 in Louga. A total of 5,730 mosquitoes were collected; these belonged to 8 species: An epidemic of DENV-3 occurred in Senegal during September–December 2009; of the 196 laboratory-confirmed cases, most (193) occurred in Dakar. This finding could indicate that transmission rates were higher in this urban area but may have been the result of bias in sample collection; dengue surveillance was less active at health facilities in in other regions, which provided only 277 (39%) of the 696 samples collected from persons who were suspected to have dengue. Fever, headache, myalgia, vomiting, thrombocytopenia, and leukopenia were the most frequent signs and symptoms among patients with confirmed dengue, as described , Lusophone , and French  communities. Moreover, 5 (83.3%) of the 6 cases with hemorrhagic manifestations occurred in the Lebanese community, which suggests that disease severity might be associated with community exposure. The percentage of confirmed dengue cases among suspected cases in different communities showed that persons in Senegalese communities were significantly less affected than those in Leebanese (χAe. aegypti mosquitoes were the most likely vector of DENV-3 transmission during this epidemic, given their association with DENV and the overlap of their distribution and abundance with the locations of dengue confirmed cases. Ae. aegypti mosquitoes are known to be a competent vector for DENV in West Africa (Ae. aegypti mosquitoes, suggests that the first reported case-patient contracted the DENV infection elsewhere, possibly in Dakar, which he had visited several times.The phylogenetic analysis of DENV-3 strains isolated during the outbreak suggests that they belong to genotype III and are closely related to DENV-3 isolated from Côte d’Ivoire and China in 2008 and 2009, respectively (Our findings suggest that increased urban dengue activity is plausible in Senegal. Given ongoing population growth, explosive urbanization, infrastructure building, and international travel, dengue surveillance and preparedness should be reinforced. Furthermore, phylogenetic studies incorporating more DENV-3 strains would shed light on the origins of this DENV-3 outbreak.