Fluctuating asymmetry (FA), as an indirect measure of developmental instability (DI), has been intensively studied for associations with stress and fitness. Patterns, however, appear heterogeneous and the underlying causes remain largely unknown. One aspect that has received relatively little attention in the literature is the consequence of direct mechanical effects on asymmetries. The crucial prerequisite for FA to reflect DI is that environmental conditions on both sides should be identical. This condition may be violated during early human development if amniotic fluid volume is deficient, as the resulting mechanical pressures may increase asymmetries. Indeed, we showed that limb bones of deceased human fetuses exhibited increased asymmetry, when there was not sufficient amniotic fluid  in the uterine cavity. As amniotic fluid deficiency is known to cause substantial asymmetries and abnormal limb development, these subtle asymmetries are probably at least in part caused by the mechanical pressures. On the other hand, deficiencies in amniotic fluid volume are known to be associated with other congenital abnormalities that may disturb DI. More specifically, urogenital abnormalities can directly affect/reduce amniotic fluid volume. We disentangled the direct mechanical effects on FA from the indirect effects of urogenital abnormalities, the latter presumably representing DI. We discovered that both factors contributed significantly to the increase in FA. However, the direct mechanical effect of uterine pressure, albeit statistically significant, appeared less important than the effects of urogenital abnormalities, with an effect size only two-third as large. We, thus, conclude that correcting for the relevant direct factors allowed for a representative test of the association between DI and stress, and confirmed that fetuses form a suitable model system to increase our understanding in patterns of FA and symmetry development. After grouping reduced fluid volumes and ample fluid volumes, the interaction between age and amniotic fluid volume was statistically significant, indicating different slopes for fetuses with reduced amniotic fluid volumes compared to fetuses with ample amniotic fluid volumes (interaction amniotic fluid volume * (log-transformed) age: F1,1068 = 2.76, P < 0.01). Asymmetry decreased significantly with age for fetuses with ample amniotic fluid volume  as seen in [+ 0.73%, F1,1068 = 1.3, P = 0.17). To compare FA between AH/OH and PH/NV with the different direction of slopes, we estimated effects at the average age in our study (27.8 weeks): fetuses with AH/OH showed significantly higher asymmetry than fetuses with NV/PH . We compared levels of asymmetry between fetuses that developed with NV, PH, OH, or AH with (log-transformed) age added to the linear model . The sloes [AH N= and OH , nervous system abnormalities (NS) and urogenital abnormalities (UG), of which only UG abnormalities remained significant after Bonferroni correction , AH (N=19), OH (N=42), and PH (N=35)) in a correspondence analysis . The firrrection . Relatiorrection . Χ2 = chi-squared value, df = degrees of freedom, P = p-value, * = significant after bonferroni adjustment).The distribution of congenital abnormalities over the different groups of amniotic fluid volume are not independent for muscular abnormalities (MUS), nervous system abnormalities (NS) and urogenital abnormalities (UG), of which only UG abnormalities remained significant after Bonferroni correction. Urogenital abnormalities associate significantly with an- and olighohydramnios . Relatio+ 0.02%). Fetuses with AH/OH, but without urogenital abnormalities had significantly higher asymmetry at an average age compared to fetuses from the former group . Similarly, fetuses with ample amniotic fluid volumes but with urogenital abnormalities also showed significantly higher FA than fetuses without urogenital abnormalities and with normal amniotic fluid volumes . As expected, fetuses with both AH/OH and urogenital problems showed the highest FA (estimate: 0.82 + 0.09%), but the combined effect of the interaction between urogenital abnormalities and reduced amniotic fluid volumes was not significant , indicating that the effects reduced amniotic fluid volumes and the presence of congenital abnormalities in the urogenital system were additive. Both AH/OH and UG contributed to higher limb FA and the effects of AH/OH on asymmetry did not change conclusions on associations between urogenital abnormalities and FA.For the final analyses, we examined the effects of urogenital problems  and amniotic fluid volumes on FA, while adding (log-transformed) age to the model. We corrected the (log-transformed) age for the average age in order to compare effect sizes. Fetuses without urogenital abnormalities and NV showed the lowest FA at an average age . Reduced amniotic fluid volumes therefore have effects on fluctuating asymmetry, which is normally used to measure developmental instability. Urogenital abnormalities, which are described to increase fluctuating asymmetry in Van Dongen et.al. 2009 are found associated with reduced amniotic fluid volumes. We evaluated the significance of the direct effects of compression of the limbs on fluctuating asymmetry and the indirect effects of disturbed urogenital developments on FA in fetuses with urogenital abnormalities and found smaller effects sizes for the direct effects of reduced amniotic fluid volumes than for the indirect effects of disturbed development by urogenital abnormalities on FA. Correcting for the relevant direct factors allows representative testing of the associations between DI and stress.Fluctuating asymmetry as measure for developmental instability and its relation to stress/quality has been widely studied. The effect sizes of associations between FA and stress/quality were found to be only weak to moderate in most reviews, but there is high heterogeneity in the literature and little is known about the sources driving this heterogeneity. To evaluate the usefulness of FA as a measure of fitness and stress, it is crucial to know which factors confound associations with FA in bilateral symmetrical traits. One aspect that has received little attention is the direct mechanical effects on development instead of indirect effects of DI . Here, we examined the possibility that reduced amniotic fluid volume and the resulting mechanical pressure on the developing fetus may confound FA of the limb bones prenatally, similar to postnatal effects of mechanical loading due to lateralized behavior. Amniotic fluid volume rises progressively from 10-20 mL at 10 weeks of gestation, when the fetal kidneys start to develop, to 800 mL at 24 weeks, and then remains relatively constant until term. As expected, we find higher asymmetry for the limb bones if amniotic fluid volume is abnormally low. Fetuses that developed with (severe) deficiencies in amniotic fluid show a higher limb asymmetry at an average age of 27.7 weeks compared to fetuses with sufficient amniotic fluid volumes. This increase of subtle asymmetries was not unexpected as substantial asymmetrical development of the limbs is generally considered to be a defect due to compression -20,30. INormal amniotic fluid volumes are mainly determined by fetal urination and swallowing . AlthougThe associations between amniotic fluid deficiencies and urogenital abnormalities may imply that the increased FA with urogenital problems as found by Van Dongen et al.  are not


The order of the authors is incorrect. The correct order of authors is as follows: Mukul Joshi, Anupama Jha, Avinash Mishra and Bhavanath Jha. The correct Citation is: Joshi M, Jha A, Mishra A, Jha B(2013) Developing Transgenic Jatropha Using the SbNHX1 Gene from an Extreme Halophyte for Cultivation in Saline Wasteland. PLoS ONE 8(8): e71136. doi:10.1371/journal.pone.0071136.


The authors would like to include more information for one of the listed grants. The grant number for the grant from the Chao Family Comprehensive Cancer Center at University of California of Irvine (ZJ and DAM) is P30CA62203.


There is limited evidence that imaging biomarkers can predict subsequent response to therapy. Such prognostic and/or predictive biomarkers would facilitate development of personalised medicine. We hypothesised that pre-treatment measurement of the heterogeneity of tumour vascular enhancement could predict clinical outcome following combination anti-angiogenic and cytotoxic chemotherapy in colorectal cancer (CRC) liver metastases.Ten patients with 26 CRC liver metastases had two dynamic contrast-enhanced MRI (DCE-MRI) examinations before starting first-line bevacizumab and FOLFOX-6. Pre-treatment biomarkers of tumour microvasculature were computed and a regression analysis was performed against the post-treatment change in tumour volume after five cycles of therapy. The ability of the resulting linear model to predict tumour shrinkage was evaluated using leave-one-out validation. Robustness to inter-visit variation was investigated using data from a second baseline scan.ve), tumour enhancing fraction (EF), and microvascular uniformity  . Other variables, including baseline volume were not statistically significant. Median prediction error was 12%. Equivalent results were obtained from the second scan.In all, 86% of the variance in post-treatment tumour shrinkage was explained by the median extravascular extracellular volume (Traditional image analyses may over-simplify tumour biology. Measuring microvascular heterogeneity may yield important prognostic and/or predictive biomarkers. There is considerable interest in developing pre-treatment biomarkers of microvascular structure and function that predict subsequent therapeutic response  and dynamic contrast-enhanced MRI (DCE-MRI) offer the opportunity to study tumour pathophysiology   and therapeutic response  followed every 2 weeks by 10 mg kg–1 bevacizumab plus FOLFOX-6  for 2 months (cycles 2–5) as first-line treatment.Retrospective analysis was performed on DCE-MRI data collected between July 2006 and October 2007 in 10 patients with CRC liver metastases. Patients were originally recruited for an investigator led study that examined the temporal action of bevacizumab during a single cycle of therapy ; normal ECG.Exclusion criteria were previous treatment with vascular endothelial growth factor (VEGF) inhibitors or cytotoxic chemotherapy; contraindications to VEGF inhibitors; exposure to any other investigational drug (within the last 4 weeks) or concurrent therapy likely to influence the vasculature on imaging; pregnant or breast-feeding women; previous clinically significant haemorrhage, thrombosis, or cardiovascular disease within the last 6 months; proteinuria; contraindication to MRI.T1-weighted fast field echo images  and T2-weighted single shot turbo spin echo images  were acquired. Both sequences employed FOV 375 × 375 mm2, matrix 256 × 256 with a 4-mm slice thickness.Patients were examined on a 1.5-T Philips Intera system . Each patient was scanned twice before treatment, to allow measurement reliability to be assessed. In one patient (with three tumours), data were only available from one pre-treatment scan. The field of view (FOV) was centred on the liver. In each examination, α=20°, one signal average, FOV of 375 × 375 mm2, matrix 128 × 128; in-plane voxel size 2.93 × 2.93 mm2) following calculation of baseline T1 using the variable flip angle method . Temporal resolution was 4.97 s. On the sixth dynamic time point, 0.1 mmol kg–1 of Gadodiamide  was administered intravenously through a Spectris MR power injector  at 3 ml s–1, followed by a saline flush. Slice thickness was 4 mm for small target lesions or 8 mm for larger lesions, giving superior–inferior coverage of either 100 or 200 mm. Images were acquired during gentle free breathing.For the DCE-MRI series, 75 3D axial volumes were acquired consecutively  on each slice in the imaging volume and then by visual assessment of the dynamic time series images. In- and through-plane motion was investigated and a categorical score was assigned for each tumour based on the evaluations of bulk motion . Tumours with a motion assessment score of 3 or 4 were excluded.T1- and T2-weighted sequences. In some patients, multiple lesions were defined. To quantify microvascular characteristics, the extended Tofts version of the Kety model (Ct(t) is the concentration of contrast agent at time t in each voxel and Cp(t) is the concentration of contrast agent in the arterial blood plasma  and median fractional volume of the extravascular extracellular space .Voxel-wise analysis was performed allowing estimates of the median bulk transfer coefficient  was calculated to characterise the overall level of tumour perfusion (P<0.05 on Mann–Whitney Wilcoxon rank-sum test) were classified as enhancing , is illustrated . In addition, fractal measures based on the information dimension (d1) and correlation dimension (d2) were calculated. These latter metrics retain magnitude values of Ktrans, ve, and vp and are described in detail elsewhere  were derived from the DCE-MRI data. The calculation of one of these fractal measures, box dimension  and the remaining tumour volume (%) from baseline to EC5 was calculated by comparison with the pre-treatment tumour volume.X-ray computed tomography (CT) examination of the abdomen and pelvis was performed at baseline within 72 h of the baseline MRI and at the end of cycle 5 (EC5) to evaluate clinical response. Patients were imaged on a LightSpeed Plus CT scanner , with typical clinical helical acquisition variables . Images were acquired following intravenous injection of 200 ml Omnipaque-140  and reformatted to produce contiguous 5 mm slices with no overlap. Tumour volumes were measured . In a preliminary analysis, a mixed-effects model was used to explore potential within-patient clustering since some patients had more than one tumour. However, no statistically significant evidence for such effects was found. Subsequently, tumours were treated independently.Stepwise errors-in-variables regression was used to model the percentage of remaining tumour volume based on the pre-treatment volume, summary DCE-MRI statistics, and heterogeneity statistics using data from the first pre-treatment scan. The regression was repeated using the data from the second scan to investigate robustness to inter-visit variation. In the above analyses, the missing data for one patient's first pre-treatment scan (three tumours) were dealt with using list-wise deletion or imputation (from the second scan) as appropriate, with the aim of maximising the amount of data available, while minimising bias.tumour was left out in turn; the coefficients on each variable were computed—by applying errors-in-variables regression to the left-in tumours' data—and used to predict the response of the left-out tumour. In the second analysis, the data for each patient was left out in turn ; the coefficients on each variable were computed—by applying errors-in-variables regression to the left-in patients' data—and used to predict the responses for the tumours in the left-out patients.The ability of the linear model to predict tumour response was evaluated using two leave-one-out analyses. In the first analysis, each Prediction error was quantified using the absolute difference between the actual and predicted percentage of remaining tumour volume. A cumulative distribution function (CDF) of prediction error was plotted for each leave-one-out analysis; a CDF permits estimation of the proportion of predictions that would be expected to be less than or equal to a given prediction error. Bland–Altman plots were formed to assess the agreement between actual and predicted percentage of the remaining tumour volume.Statistical modelling was performed using Stata/IC version 10.1  and leave-one-out analysis was performed using Mathematica version 7.0.1 .The mean patient age was 68.3 years . All patients completed therapy to EC5. Two patients achieved partial responses; seven had stable disease; one had disease progression by RECIST 1.0 criteria. In all, 26 tumours were identified in the 10 patients .ve, EF, and d0 . This suggests that the identified variables and the underlying model are robust to typical inter-visit variation. Scatter plots showing the relationships between the retained variables and percentage remaining tumour volume are provided in The final errors-in-variables regression analysis modelled tumour response in terms of the following pre-treatment biomarkers: median The results of the two leave-one-out analyses suggest that tumour response can be predicted with an error of no more than 12% in 50% of cases, and with an error of no more than 31% in 80% of cases . No diffThe Bland–Altman plots for the leave-one-tumour- and leave-one-patient-out predictions were very similar, with the differences  having almost identical mean (−0.04%) and standard deviation (30%) . In one could be explained by baseline image data, but that simple measures of size or function  may lack predictive power.In this study, we have investigated the relative value of pre-treatment biomarkers of the microvasculature in explaining the percentage of remaining tumour volume resulting from combined bevacizumab and FOLFOX-6 therapy. We hypothesised that tumour size change ve, EF, and d0) were statistically significant within the model and provide complementary types of information about the tumour environment. These results are congruent with other studies, which report that multi-parametric image analyses may be better at predicting clinical outcome  was explained by combining various pre-treatment imaging biomarkers. Importantly, robustness to inter-visit variation was validated by a second data set derived from the same tumours. Of note, pre-treatment tumour volume was not found to be a statistically significant determinant of subsequent change in tumour volume following treatment. However, three variables  was associated with better tumour response. This parameter can be low if there are few enhancing voxels or if enhancing voxels are non-uniformly distributed. The proportion of enhancing voxels is already captured in EF but d0 is also significant, implying that the spatial arrangement of enhancing tissue within a tumour (and therefore the uniformity of drug delivery) is important. As used here, d0 reflects not only microvascular uniformity but also depends on tumour shape. Our data are comparable to a study of rectal carcinoma treated by cytotoxic chemotherapy, where a similar parameter calculated from pre-treatment thresholded single slice parameter maps for area under the ΔR2* curve (where R2*=1/T2*) predicted tumour regression after 8 weeks of therapy (Enhancing fraction and ve), and there were no significant differences between the coefficient or R2 values – providing internal validation.The CDFs presented in Our study has four main limitations. First, it was retrospective. Second, the imaging parameters used require significant post-processing effort to obtain and do not have accepted standardisation. Third, while the biomarkers identified appear to predict shrinkage, they require testing against survival in a larger study. Fourth, respiratory or other patient motion can complicate any image analysis, particularly those performed on a per voxel basis . Our resIn conclusion, these data provide preliminary evidence that a combination of pre-treatment measures derived from DCE-MRI parameter maps may predict tumour shrinkage in response to combined bevacizumab and cytotoxic chemotherapy in CRC liver metastases, with relatively low error. Although the applicability of these results to other tumours types and metastatic disease in other organs cannot be inferred from the data presented, this type of approach may have value in determining personalised tumour therapy regimes and patient selection. These results encourage the further evaluation of image heterogeneity in cancer studies using MRI, PET, and other techniques, to explore whether similar findings are seen in alternative combinations of patient group, therapy, and imaging technique.


The byline published in the correction is incorrect. The correct byline is: Ho Dang Trung Nghia, Le Thi Phuong Tu, Marcel Wolbers, Cao Quang Thai, Nguyen Van Minh Hoang, Tran Vu Thieu Nga, Le Thi Phuong Thao, Nguyen Hoan Phu, Tran Thi Hong Chau, Dinh Xuan Sinh, To Song Diep, Hoang Thi Thanh Hang, Hoang Truong, James Campbell, Nguyen Van Vinh Chau, Nguyen Tran Chinh, Nguyen Van Dung, Ngo Thi Hoa, Brian G. Spratt, Tran Tinh Hien, Jeremy Farrar, Constance Schultsz.


The ability of an Escherichia coli-produced E7-preparation, assembled into particles, to induce protective immunity against a HPV16-related tumour in the TC-1-C57BL/6 mouse tumour model, was evaluated.The HPV16 E7 protein is both a tumour-specific and a tumour-rejection antigen, the ideal target for developing therapeutic vaccines for the treatment of HPV16-associated cancer and its precursor lesions. E7, which plays a key role in virus-associated carcinogenesis, contains 98 amino acids and has two finger-type structures which bind a ZnE. coli, purified via a one-step denaturing protocol and prepared as a soluble suspension state after dialysis in native buffer. The presence in the E7 preparation of particulate forms was analysed by non-reducing SDS-PAGE and negative staining electron microscopy (EM). The Zn++ ion content was analysed by mass-spectrometry. Ten μg of protein per mouse was administered to groups of animals, once, twice or three times without adjuvant. The E7-specific humoral response was monitored in mice sera using an E7-based ELISA while the cell-mediated immune response was analysed in mice splenocytes with lymphoproliferation and IFN-γ ELISPOT assays. The E7 immunized mice were challenged with TC-1 tumour cells and the tumour growth monitored for two months.E7 was expressed in In western blot analysis E7 appears in multimers and high molecular mass oligomers. The EM micrographs show the protein dispersed as aggregates of different shape and size. The protein appears clustered in micro-, nano-aggregates, and structured particles. Mice immunised with this protein preparation show a significant E7-specific humoral and cell-mediated immune response of mixed Th1/Th2 type. The mice are fully protected from the tumour growth after vaccination with three E7-doses of 10 μg without any added adjuvant.E. coli-derived HPV16 E7 assembled in vitro into micro- and nanoparticles represents not only a good substrate for antigen-presenting cell uptake and processing, but also a cost-effective means for the production of a new generation of HPV subunit vaccines.This report shows that a particulate form of HPV16 E7 is able to induce, without adjuvant, an E7-specific tumour protection in C57BL/6 mice. The protective immunity is sustained by both humoral and cell-mediated immune responses. The  The cells were then harvested onto filters using an automatic harvester and counted in a Beta Counter . The results were expressed in stimulation index (SI), calculated by dividing the mean counts per minute (cpm) of cells exposed to the E7 peptides by the mean cpm of cells incubated only with medium. The IFN-γ ELISPOT assay was performed using commercially available reagents (Mabtech AB). T-cell enriched splenocytes were seeded in triplicate (5 × 105 cells per well) in 200 μl complete medium with the E7 stimulator peptides. After 18 h at 37°C in a humidified 5% CO2 incubator, the plates were analysed for the presence of IFN-γ as described in 6 tag and purified via a one-step denaturing protocol until a high level of homogeneity and low endotoxin content were achieved [The E7 protein was expressed in achieved ,34. The 5 particles/ml (not shown). The E7 preparations were also subjected to EM immunolabelling but neither commercial anti-E7 monoclonal nor in-house prepared polyclonal antibodies [Preparations of purified E7 were analysed by negative staining EM. Figure tibodies  revealedE. coli [++ content by high resolution mass spectrometry (HR-ICP-MS). The recombinant GST protein was used as a control. The Zn++ ion concentration in the E7 preparations was 1.13 μg/g ± 0.10 while GST showed only trace level of Zn++ (0.19 μg/g ± 0.02). The metal/protein ratio was calculated to be 0.19, therefore only about 19% of the E7 molecules were bound to Zn++.HPV16 E7 contains a Zinc finger-like domain that binds the metal even when the protein is expressed in E. coli ,11. SincE. coli-derived HPV16-E7 preparation, administrated without adjuvant, was able to induce a tumour-protective immunity, groups of mice were inoculated with 10 μg of protein per mouse, 1, 2 or 3 times, at one week intervals. As a control group, mice were inoculated with a saline solution (naïve group). Two weeks after the last immunization, some of the animals were bled and killed to analyse the immune response, in vitro. The remaining animals were challenged with the TC-1 tumour cells and the inhibition of tumour growth in these mice was monitored for 2 months.To investigate if this To quantify the humoral immune response and to compare the results obtained from several animal groups, the sera from the animals of each group were pooled, then sequentially diluted to determine the antibody titres by end-point dilution in an E7-based ELISA . The antThe therapeutic effector functions of antibodies depend on their class and subclasses . In ordein vitro, with the E7-specific CTL peptides and processed for T cell proliferation and γ-IFN ELISPOT assays. Splenocytes from naïve mice were pulsed with an unrelated mixture of peptides used as control. The results are shown in Figure To analyse the induction of the cell-mediated immune response in mice after 1, 2 or 3 doses of the E7 preparation, T-enriched splenocytes from mice of the same immunization group were stimulated To evaluate the efficacy of the E7 preparation as inductor of anti-tumour immunity, the mice immunised with 1, 2 or 3 doses of E7 were challenged with the TC-1 tumour cells, and the tumour growth was monitored for two months after the challenge. The results are shown in Figure E. coli-derived HPV-E7 preparation containing particles. E7 has been intensively studied for many years. However, this is the first time, to our knowledge, that a particulate form of E7 has been used as an immunogen and proposed as a non-adjuvated vaccine. Our results show that, the tumour-protective immunity in the mouse TC1/C57BL/6 tumour model correlates to the elicited E7-specific T cell response, and to the IgG isotype switching (IgG2b and IgG2c).This study reports the induction in mice of a tumour-protective immunity using an et al. [et al. [++ content in the E7 preparation used as immunogen in mice, considering that while Zn++ is an essential mineral in eukaryotic systems, a high quantity of the metal is also toxic [Previous studies on bacterial-derived E7 showed that Zinc has a role in E7 particle formation. Chinami et al.  obtained [et al.  obtainedso toxic . When ZiAs the aim was to obtain a highly immunogenic E7 preparation, we did not focus on obtaining identical particles, considering that particles of different size can be taken up by different types of antigen presenting cells, such as dendritic cells, macrophages and polymorphonuclear leukocytes, sustaining a more potent immune response ,40. HoweE. coli-derived E7 fused, through the N-terminus, to either HPV16 E6 or GST, was also investigated in mice. An antigen-specific immune response of Th2 polarity was obtained when the fusion proteins were administered to mice without adjuvant (data not shown). However, we were unable to observe the typical micro- and nanoparticles in these E7-fusion proteins prepared from E. coli (data not shown).The immunogenicity of Recently, the cytosolic accumulation of E7-oligomers shown in HPV16 cervical cancer cell lines and in clinical samples by indirect methods, supports a new hypothesis regarding the presence of E7 isoforms and their role in different cell compartments -43. As kE. coli-derived E7, assembled in different forms, would contribute to explaining how the different branches of the immune system in the HPV16 mouse tumour model are stimulated. Significant differences exist between the HPV16 mouse tumour model and human HPV16-dependent diseases. However, studies on IgG subclasses and their FcγR receptors between mouse and human are comparable (37). We believe that HPV16 E7 immunogenicity studies in mouse will provide insights into the understanding of the protective immunity against human HPV16 infections as well.More studies on recombinant E. coli have been described [E. coli-derived E7 in particle form in subunit vaccines. The E. coli expressed proteins represent a well-studied and cost-effective means for the production of vaccines. These methods require reduced time, costs, labour and can be easily scaled up in industrial-scale productions. A generation of new low-cost HPV vaccines could represent the only possibility for women living in developing countries to gain access to HPV vaccination programs to prevent or treat pre-cancerous lesions and cancer.The commercial preventive HPV vaccines have high production costs which has made widespread vaccination programs still not possible. Recently, new combined preventive and therapeutic HPV vaccines produced in escribed -47 and tin vitro into particulate form, to induce protective immunity against a HPV16-related tumour in an HPV16 mouse tumour model. Data show that E7 particles, used without adjuvant, are excellent stimulators of the immune system. In C57BL/6 mice, the E7 preparation induces anti-tumour immunity sustained by both humoral and cell-mediated immune responses. This E7 protein (derived from Escherichia coli) without adjuvant could represent, along with the recently proposed E. coli-derived HPV antigens [The paper describes, for the first time, the use of recombinant HPV16 E7, assembled antigens -47, a loThe authors declare that they have no competing interests.LP carried out the biochemical and immunological assays, made contribution to the analysis and interpretation of the data and helped to draft the manuscript; MGA carried out the EM analysis and made contribution in data analysis; AC performed the experiments with the animals. SC carried out the mass spectrometry experiments; FB made contribution in data analysis; CG made contribution to the analysis and interpretation of the data, in critical revision of the manuscript and in acquisition of funding. PDB conceived and designed the study, analysed and interpreted the data and drafted the manuscript. All authors read and approved the final manuscript.LP's present address: Istituto Nazionale Malattie Infettive "L. Spallanzani", Rome. CG 's present e-mail: ros.giorgi@gmail.com.


The correct grant information is: "the National Basic Research Program of China (2007CB108705,


There is a grant number missing from the Funding Statement. The following is the missing information:This work was supported by Grant No. ZR2010HM046 from the Shandong Science and Technology Committee of China.


Vibrio cholerae-induced diarrhea and to characterize its pathophysiology. Ligated ileal loops of adult mice were inoculated for 6, 9, 12 and 18 h with a classical O1 hypertoxigenic 569B strain of V. cholerae (107 CFU/loop). Time-course studies demonstrated that the optimal period for inducing diarrhea was 12 h post-inoculation, when peak intestinal fluid accumulation (loop/weight ratio of ∼0.2 g/cm) occurred with the highest diarrhea success rate (90%). In addition, pathogenic numbers of V. cholerae (∼109 CFU/g tissue) were recovered from ileal loops at all time points between 6–18 h post-inoculation with the diarrheagenic amount of cholera toxin being detected in the secreted intestinal fluid at 12 h post-inoculation. Interestingly, repeated intraperitoneal administration of CFTRinh-172 (20 µg every 6 h), an inhibitor of cystic fibrosis transmembrane conductance regulator (CFTR), completely abolished the V. cholerae-induced intestinal fluid secretion without affecting V. cholerae growth in vivo. As analyzed by ex vivo measurement of intestinal electrical resistance and in vivo assay of fluorescein thiocyanate (FITC)-dextran trans-intestinal flux, V. cholerae infection had no effect on intestinal paracellular permeability. Measurements of albumin in the diarrheal fluid suggested that vascular leakage did not contribute to the pathogenesis of diarrhea in this model. Furthermore, histological examination of V. cholerae-infected intestinal tissues illustrated edematous submucosa, congestion of small vessels and enhanced mucus secretion from goblet cells. This study established a new adult mouse model of V. cholerae-induced diarrhea, which could be useful for studying the pathogenesis of cholera diarrhea and for evaluating future therapeutics/cholera vaccines. In addition, our study confirmed the major role of CFTR in V. cholerae-induced intestinal fluid secretion.Cholera is a diarrheal disease causing significant morbidity and mortality worldwide. This study aimed to establish an adult mouse model of  Vibrio cholerae, the causative agent of cholera, colonizes the intestine and induces massive intestinal fluid secretion through actions of its enterotoxins, especially cholera toxin. We have developed a ligated ileal loop model of V. cholerae-induced diarrhea in adult mice, in which inoculation of V. cholerae (107 CFU/loop) consistently caused intestinal fluid secretion in 12 h. Interestingly, administration of CFTRinh-172, a small molecule CFTR inhibitor, completely abolished V. cholerae-induced intestinal fluid secretion, indicating that CFTR plays a pivotal role in the pathogenesis of diarrhea in this model. Furthermore, we demonstrated that intestinal fluid accumulation in this model was neither caused by increased intestinal paracellular permeability nor enhanced vascular leakage. This adult mouse model of V. cholerae-induced diarrhea could be very useful in the studies of pathogenesis of V. cholerae infection-induced diarrhea and in the evaluation of potential therapies/cholera vaccines.Cholera is a diarrheal disease causing significant morbidity and mortality worldwide.  Vibrio cholerae. It has been estimated that cholera affects millions of people and causes several hundreds of thousands of death each year, especially in developing countries V. choleraeCholera is a life-threatening disease caused by intestinal infections with a gram-negative bacterium V. cholerae-derived enterotoxins, especially cholera toxin (CT), on intestinal epithelium − secretion, which in turn provides a driving force for Na+ and water secretion in vitro and in vivo experiments demonstrated that cystic fibrosis transmembrane conductance regulator (CFTR) mediates CT-induced apical Cl− efflux into intestinal lumen and therefore represents a promising therapeutic target for treatment of cholera − channels (IRC) might help mediate cAMP-activated Cl− efflux across the apical membrane of intestinal epithelial cells V. cholerae of several other virulence factors other than CT V. cholerae and inflammatory response induced by V. cholerae independently of CT may be involved in the pathogenesis of cholera Diarrhea in cholera results from both direct and indirect (via enteric nervous system) effects of V. cholerae infection models have used both infant rabbits and infant mice V. cholerae have been shown to exhibit massive diarrhea and mucous secretion from goblet cells despite having intact microscopic architecture of intestinal epithelium, resembling pathology of cholera in human V. cholerae infection is an infant mouse model of cholera induced by oral inoculation of V. cholerae. This infant mouse model has been used extensively for studying growth and colonization of V. cholerae in the intestine V. cholerae (Inaba strain 569B) or CT in various laboratory animals including gerbil, rat, hamster, guinea pig, chinchilla, cat and mouse. It was found that other types of animals developed profuse and consistent intestinal fluid secretion, whereas mice showed little or no fluid accumulation at 16 h after intestinal injection of bacteria or CT, despite normal replication of V. cholerae in mouse intestine. Based on the fact that V. cholerae do replicate in adult mouse intestine as shown by Basu and Pickett V. cholerae might induce diarrhea in adult mice under condition optimal for V. cholerae expression of their virulence factors, especially CT. In this study, we aimed to establish an adult mouse model of V. cholerae-induced diarrhea using a closed ileal loop approach, and to use it to investigate the underlying pathophysiology of diarrhea. A closed ileal loop approach was chosen because V. cholerae has been known to preferentially colonize distal small intestine particularly ileum V. cholerae (Inaba strain 569B) challenges into the ligated 2–3 cm ileal loops of adult ICR outbred mice consistently produced intestinal fluid accumulation in 12 h after inoculation in a CFTR-dependent manner.To date, This study has been approved by the Institutional Animal Care and Use Committee of the Faculty of Science, Mahidol University (permit number 240). This study was performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.Vibrio cholerae 569B Inaba  was used in this study. Bacteria were grown in Thiosulfate-Citrate-Bile-Sucrose (TCBS) agar and then duplicated using Luria Broth (LB) plate. To prepare the inoculum, V. cholera were cultured at 37°C overnight in LB. The overnight culture were then subcultured into fresh LB at the ratio of 1∶100 and grown to a mid-log phase. V. cholerae cells were collected by centrifugation and the pellet was re-suspended in phosphate buffered saline to obtain the inoculum containing 108 CFU of V. cholera/ml. CT was purchased from List Biological Laboratories, Inc. . CFTRinh-172 was purchased from Calbiochem . Fluorescein isothiocyanate-linked dextran  and other chemicals were obtained from Sigma-Aldrich .V. cholerae (107 CFU/loop). After abdominal closure by suture, mice were allowed to recover from anesthesia. At specific time points after bacterial inoculation , the mice were re-anesthetized and ileal loops were exteriorized for measurements of weight/length ratio. Mice with intestinal weight/length ratio of al least 0.1 g/cm were considered as having positive diarrheal response. Diarrhea success rates (% diarrhea) were computed by dividing a number of surviving mice with intestinal loop weight/length ratio of at least 0.1 g/cm by a total number of surviving mice at a specific time point. In the study of CT-induced diarrhea, ileal loops were instilled with PBS or PBS containing CT (1 µg/loop), and loop weight/length ratio was quantified 4 h later. In some experiments, CFTRinh-172 (20 µg in 0.1 ml of DMSO) was intraperitoneally administered every 6 h until euthanasia. For bacterial colonization assays, ileal loops at various time points after V. cholerae inoculation were homogenized in PBS, followed by serial dilutions and plating on LB agar for colony forming unit (CFU) counting.Six-week-old ICR outbred mice (weight 30–35 g) were acquired from the National Laboratory Animal Center, Bangkok, Thailand. Before surgery, mice were fasted for 24 h and anesthetized by intraperitoneal injection of nembutal (60 mg/kg). While maintaining the body temperature at 37°C using a heating pad, a small abdominal incision was made and a loop of distal ileum was isolated by suture (2–3 cm in length). The closed ileal loop was instilled with 100 µl of phosphate buffered saline (PBS) or PBS containing 1 enzyme-linked immunosorbent assay. Briefly, 100 µl of sample, standard CT in PBS or PBS alone was added to GM1-coated 96-well plates in duplicate and incubated at 37°C for 1 h. The plate was then washed three times with PBS before addition of goat-anti CTb serum. An hour later, rabbit anti-goat globulin was added to the plate and incubated for 60 min. The substrate solution was then added and incubated for 30 min. The reaction was stopped by adding 50 µl of 3M NaOH and absorbance at 405 nm was measured using a microplate reader . The amount of CT was calculated according to the manufacturer's instructions.Amount of CT in fecal fluid was quantified using GM3, 3.3 KH2PO4, 0.8 K2HPO4, 0.5 MgCl2 and 10 glucose (pH 7.4). The solution was continuously bubbled with 5% CO2/95% O2 and maintained at 37°C. Electrical resistance of the intestinal tissues was calculated using values of passing current and transmembrane voltage recorded by a voltage/current clamp machine with Ag/AgCl electrodes and 1 M KCl agar bridges .Ileal loops were opened longitudinally, washed with PBS and mounted in the Ussing chamber system . Both apical and basolateral hemichambers were filled with Kreb's bicarbonate buffer containing (in mM) 120 NaCl, 25 NaHCOV. cholerae (107 CFU/loop) or PBS containing CT (1 µg/loop) into ileal loops, mice were anesthetized and intestinal fluid was removed and replaced with 0.2 mL of PBS containing 20 mg of FITC-dextran. Thirty min afterwards, mice were sacrificed and blood was collected by cardiac puncture. Blood was centrifuged at 3,000 g for 10 min (4°C) and the serum was collected for measurement of FITC-dextran using a fluorospectrophotometer . The amount of FITC-dextran in the sample was estimated from the standard curve generated by fluorometric measurements of FITC-dextran at known concentrations.Intestinal paracellular permeability was estimated using the measurement of FITC-dextran (molecular weight 4.4 kDa) flux from the intestine to the blood as previously described Intestinal tissues dissected from ileal loops were fixed for 24 h in 10% formaldehyde, followed by tissue dehydration using graded alcohol and paraffin embedding. The processed tissues were cut into 5 µm-thick sections and stained with hematoxylin and eosin (H&E) reagents or periodic-acid Schiff (PAS) reagent. Tissue slices were visualized under light microscope for histological analysis.Concentration of albumin in fluid in ileal loops was determined using a bromcresol purple (BCP) dye-binding method as previously described The data were presented as mean ± standard error (S.E.). Statistical analyses for two-group and multiple comparisons were performed using Student's t test and one-way analysis of variance (ANOVA), followed by Bonferroni's post-hoc test, respectively. P value of <0.05 was considered statistically significant.Vibrio cholerae 569B of classical O1 Inaba strains (107 CFU/loop), a bacterial strain known to possess high diarrheagenic potential V. cholerae-induced intestinal fluid secretion by analyzing three parameters, i.e., the amount of intestinal fluid secretion, percent of mice developing fluid accumulation (loop weight/length ratio of at least 0.1 g/cm), and survival rate of mice at different time points  after V. cholerae inoculation. As depicted in V. cholerae inoculation (loop weight/length ratio = 0.203±0.037 g/cm). This level of intestinal fluid secretion was sustained until 18 h post-inoculation (loop weight/length ratio = 0.192±0.042 g/cm). Diarrhea success rate, which indicated model reproducibility, and survival rate of mice were 90% and 80% at 12 h post-inoculation, and 68% and 50% at 18 h post-inoculation, respectively  caused lethality in more than 80% of mice within 6 h after bacterial challenge without inducing fluid secretion. We thought that circulatory failure resulting from V. cholerae-induced systemic inflammatory response might account for this death. Subsequent experiments were performed to characterize the pathophysiology of this cholera model.ectively . TherefoV. cholerae colonization and production of CT V. cholerae in the intestinal loops  and CT in intestinal fluid were determined. As shown in V. cholerae recovered from the intestinal loops at all time points  after inoculation was ∼109 CFU/gram of tissues. This amount of V. cholerae was comparable to that recovered from the intestine of infant rabbit models of V. cholerae infection-induced diarrhea V. cholerae was capable of colonizing the intestine in our mouse cholera model. Using GM1 ELISA assays, the amount of CT in intestinal fluid was found to be 1.688±0.563 µg/ml at 12 hour post-inoculation, whereas none was detected in the ileal loop instilled with PBS (no V. cholerae). These data indicated that V. cholerae established colonization and expressed CT in the closed ileal loop in this mouse cholera model.Pathogenesis of diarrhea in cholera required − secretion provides the driving force for CT-induced intestinal fluid secretion − secretion to the pathogenesis of diarrhea in this model, the effect of CFTRinh-172, a CFTR inhibitor, on V. cholerae-induced intestinal fluid secretion was investigated. In this experiment, CFTRinh-172 (20 µg) was intraperitoneally administered at the time of V. cholerae inoculation and 6 h thereafter, and mice were sacrificed for measurements of intestinal fluid secretion (loop weigh/length ratio) at 12 h after V. cholerae inoculation. This dosage regimen of CFTRinh-172 was used because it was previously shown to produce maximal degrees of inhibition of CT-induced intestinal fluid secretion in mice (∼90% inhibition) inh-172 completely prevented V. cholerae-induced intestinal fluid accumulation  to the 5×108 CFU/ml of V. cholerae in Mueller Hinton Broth did not affect V. cholerae growth as enumerated by a spread plate method (data not shown). Moreover, amounts of V. cholerae recovered from V. cholerae-inoculated ileal loops of CFTRinh-172-treated mice were not statistically different from that of control (V. cholerae inoculation with no CFTRinh-172 treatment) (data not shown). These data indicated that CFTRinh-172 inhibited V. cholerae-induced intestinal fluid secretion without affecting V. cholerae growth in ileal loops in vivo.It has been known that CFTR-mediated transepithelial Clmulation . In addiV. cholerae-induced intestinal fluid accumulation was investigated using both ex vivo measurements of transepithelial electrical resistance (TEER) of mouse intestine by Ussing chamber systems and in vivo FITC-dextran trans-intestinal flux assays. As shown in V. cholerae-inoculated intestinal tissues was not significantly different from that of saline-injected control and CT-injected groups . In order to confirm that V. cholerae infection had no effect on the intestinal paracellular permeability, in vivo FITC-dextran trans-intestinal flux assays were performed. In this experiment, FITC-dextran was injected into the intestinal loops and serum level of FITC-dextran was measured 30 min thereafter. It was found that V. cholerae infection had no effect on serum FITC-dextran compared with saline control and CT-injected groups  and V. cholerae-inoculated mice. It was found that levels of albumin in fecal fluid of both control and V. cholerae-inoculated intestinal loops were under detection limit (<0.3 g/dL). This result indicated that vascular leakage did not contribute to the development of diarrhea in this experimental model of cholera.To determine the contribution of vascular leakage to V. cholerae, the V. cholerae-infected and normal saline-injected control loops were subjected to histological examination. As shown in V. cholerae-infected tissues showed diffuse edema of the submucosal area and marked enlargement of the internal structures of intestinal villi compared to control. Furthermore, vascular congestion  staining, which detects mucin in tissues. As depicted in V. cholerae-infected intestine showed a marked reduction in PAS-positive goblet cells (arrows) compared with control, indicating empty goblet cells after secreting mucin in response to V. cholerae infection. These findings indicated that this animal model resembles human cholera in a histological context.To investigate the microscopic changes of the intestinal tissues after infection with  arrows,  and infl arrows,  were notV. cholerae or its virulence factors especially CT. The only V. cholerae infection model that manifests severe watery diarrhea was the infant rabbit model V. cholerae infection in adult mice. In this model, inoculation of 107 CFU of V. cholerae into closed ileal loops of 2–3 cm length led to massive intestinal fluid secretion, with the optimal period for induction of fluid secretion being 12 h after inoculation. Importantly, CT at the amount reported to elicit intestinal fluid secretion was detected in the intestinal fluid. Furthermore, we provided the evidence that this model might be beneficial for studying pathophysiology of fluid secretion during V. cholerae infection and for evaluation of anti-secretory therapy of cholera.To date, several animal models have been developed for cholera research. Most of these models are generated using both live V. cholerae inoculation (5×107 CFU/loop) into 10 cm-long ileal loops failed to cause fluid secretion V. cholerae, but shorter ileal loops, we successfully established a reproducible V. cholerae-induced diarrhea model in adult mice. Successful establishment of diarrhea model in the present study may be due to 1) optimal proportion of the amount of inoculated V. cholerae and length (i.e. volume) of ileal loops, and 2) optimal environment in distal ileum. Proportion between numbers of V. cholerae and volume of ileal loops determines the density of V. cholerae, which in turn affects V. cholerae expression of virulence factors including a colonizing factor toxin-coregulated pilus (TCP) and CT via quorum sensing mechanisms V. cholerae expression of virulence factors. These environmental factors include concentrations of bile and HCO3− in the ileal loops. V. cholerae expression of TCP and CT has previously been shown to be repressed and enhanced by unsaturated fatty acids in bile  and HCO3−, respectively V. cholerae to multiply and express CT in ileal loops in our study suggests the existence of environmental signals suitable for virulence expression of V. cholerae.More than four decades ago, it was reported that V. cholerae-induced diarrhea. In this study, we found that 12-h period of V. cholerae incubation was required to obtain maximal intestinal fluid secretion. Since it was previously reported that maximal fluid secretion of mouse ileum required at least 6 h of exposure to CT V. cholerae colonized and expressed CT in the ileal loops within 6 h after inoculation in our model of study. In support of this notion, previous study demonstrated that V. cholerae expression of virulence factors including colonizing factors (e.g. TCP) and CT occurred within 4 h after bacterial inoculation V. cholerae (∼109 CFU/g tissue) at 6 h post-inoculation in our study affirmed that V. cholerae colonization and virulence expression had occurred within the first 6-h of V. cholerae exposure. Interestingly, time course of diarrhea in our model was similar to that in canine cholera model, in which diarrhea developed at 6–12 h after inoculation of V. cholerae into duodenal lumen Time course of intestinal fluid secretion in the established cholera model is in accord with the prior knowledge regarding pathogenesis of V. cholerae-induced diarrhea. In fact, pathophysiology of diarrhea induced by CT and V. cholerae might be different, as V. cholerae can express several enterotoxins other than CT, which may participate in development of profuse diarrhea in cholera V. cholerae-induced diarrhea models 2+, which thus raised the possibilities that calcium-activated Cl− channel (CaCC)-mediated transepithelial Cl− secretion may contribute to fluid secretion in cholera inh-172, a small molecule CFTR inhibitor which inhibits CFTR but not CaCC V. cholerae-induced intestinal fluid secretion. This finding indicates the predominant role of CFTR-mediated Cl− secretion in providing the driving force for intestinal fluid secretion during V. cholerae infection. In addition, we provided evidence that paracellular and vascular leakage were not involved in the pathogenesis of V. cholerae-induced intestinal fluid secretion in this cholera model. This observation, together with the inhibitory effect of CFTRinh-172 on V. cholerae-induced intestinal fluid secretion, suggests that CFTR-mediated Cl− secretion is the major pathophysiological event leading to secretory diarrhea at least in the early phase (∼12 h) of V. cholerae infection and could be the important target of anti-secretory drug for cholera. In addition, this diarrhea model might be beneficial for cholera research, especially for further investigation of the underlying pathophysiology of secretory diarrhea in cholera, and for evaluation of anti-secretory therapy of cholera. Importantly, due to the maturation of immune system in the adult mice, this cholera model could be used for evaluating preventive efficacy of cholera vaccineInvestigation of pathophysiological mechanisms underlying diarrhea in this new cholera model has highlighted CFTR as a therapeutic target for treatment of cholera. A number of previous investigations have demonstrated that CFTR inhibitors reversed CT-induced intestinal fluid secretion in both rats and mice V. cholerae-induced diarrhea in adult mice. Pathogenesis of diarrhea in this cholera model results from CFTR-mediated transepithelial Cl− secretion with no involvement of disrupted intestinal barrier function or vascular leakage. This animal model may be beneficial for studying pathogenesis of diarrhea and evaluating potential anti-secretory therapies as well as vaccines for cholera.In summary, we have established the ligated ileal loop model of


Static cardiac filling volumes have been suggested to better predict fluid responsiveness than filling pressures, but this may not apply to hearts with systolic dysfunction and dilatation. We evaluated the relative value of cardiac filling volume and pressures for predicting and monitoring fluid responsiveness, according to systolic cardiac function, estimated by global ejection fraction  from transpulmonary thermodilution.n = 18) or major vascular (n = 14) surgery in the intensive care unit. We evaluated 96 colloid fluid loading events , divided into groups of responding events (fluid responsiveness) and non-responding events, in patients with low GEF (<20%) or near-normal GEF (≥20%). Patients were monitored by transpulmonary dilution and central venous (n = 9)/pulmonary artery (n = 23) catheters to obtain cardiac index (CI), global end-diastolic volume index (GEDVI), central venous (CVP) and pulmonary artery occlusion pressure (PAOP).We studied hypovolemic, mechanically ventilated patients after coronary (P = 0.002 or lower), a low PAOP was of predictive value particularly when GEF was <20% (P = 0.004 or lower). The baseline CVP was lower in responding events regardless of GEF. Changes in CVP and PAOP paralleled changes in CI particularly when GEF was <20%, whereas changes in GEDVI paralleled CI regardless of GEF.Fluid responsiveness occurred in 8 (≥15% increase in CI) and 17 (≥10% increase in CI) of 36 fluid loading events when GEF was <20%, and 7 (≥15% increase in CI) and 17 (≥10% increase in CI) of 60 fluid loading events when GEF was ≥20%. Whereas a low baseline GEDVI predicted fluid responsiveness particularly when GEF was ≥20% , PAOP is more useful than GEDVI for predicting fluid responsiveness, but when GEF is near-normal (≥20%) GEDVI is more useful than PAOP. This favors predicting and monitoring fluid responsiveness by pulmonary artery catheter-derived filling pressures in surgical patients with systolic left ventricular dysfunction and by transpulmonary thermodilution-derived GEDVI when systolic left ventricular function is relatively normal. Statiiography . We alsoiography . Howeveriography ,17. Neveiography .We hypothesized that during fluid loading in patients with reduced systolic cardiac function as compared to those with normal function, filling pressures may be superior to filling volumes  for predicting and monitoring of fluid responsiveness, and vice versa. We thus measured prospectively cardiac filling pressures and volumes in hypovolemic patients following cardiovascular and major vascular surgery, using the pulmonary artery catheter and transpulmonary thermodilution technique, prior to, during and following colloid fluid loading.n = 18) or major vascular surgery (n = 14). Colloid fluid loading was given with modified fluid gelatin 4%, hydroxyethyl starch (HES) 6% or albumin 5%, all of which have similar oncotic properties and hemodynamic responses [This is a substudy of a prospective, non-randomized, single-center clinical trial, investigating the volume expanding effects of various resuscitation fluids . The stuesponses . We only®, Marquette, GE, Milwaukee, WI, USA). For the measurements of cardiac output and GEDV, the transpulmonary thermal-dye indicator dilution technique was used [The protocol was started upon arrival of the patients in the intensive care unit (ICU). Demographic characteristics were recorded, including the Acute Physiology And Chronic Health Evaluation II (APACHE-II) score and transesophageal echocardiographic findings prior to surgery. At baseline (t = 0 minute), hemodynamic measurements were performed. Heart rate (HR) and mean arterial pressure (MAP) from a radial artery were recorded at t = 0 and 90 minutes. The HR was taken from the continuously recorded electrocardiogram. The mean pulmonary artery pressure (MPAP) was measured at t = 0 and 90 minutes. Cardiac output, GEDV, CVP and PAOP were measured every 30 minutes, from t = 0 to 90 minutes. Pressures were measured with patients in the supine position after calibration, zeroing to atmospheric pressure and, for PAOP, after proper wedging, at the midchest level at end-expiration , cardiac index  and global end-diastolic volume index , respectively. Cardiac distensibility was determined as the surrogate for cardiac compliance and was calculated by GEDVI/(CVP + PAOP)/2 (mL/m2/mmHg), or GEDVI/CVP if PAOP was not available [2 and Fisher exact tests were used. Since continuous data were normally distributed , they were summarized by mean ± standard deviation (SD) and parametric tests were done. Paired and unpaired t-tests were used to compare data in time and between GEF groups .The groups to be analyzed were divided into low GEF <20%) and near-normal GEF (≥20%). The cutoff of 20% approximately reflects a cutoff of 40% ejection fraction of the left ventricle, the lower limit of normal, as measured by echocardiography, provided that there is no right ventricular dysfunction ,25. We available . Fluid rvailable ,17, betw0% and neP = 0.44 for response, P = 0.99 for GEF). Similarly, the MAP at t = 90 minutes in the low GEF group was 95 ± 16 and 86 ± 25 mmHg and in the near-normal GEF group 83 ± 7 and 85 ± 12 mmHg, in responders and non-responders, respectively .Table P = 0.031). The amount infused was somewhat lower in non-responding than in responding events when GEF was low (<20%),  decreased from t = 0 to 90 minutes  and PAOP did not  in the low GEF group. Changes in CI correlated to changes in both CVP and GEDVI in the near-normal GEF group . Changes in PAOP correlated better to CVP in the near-normal GEF group  than in the low GEF group .For the low GEF group, baseline PAOP and CVP inversely correlated to changes in CI, irrespective of amount and type of fluids , while in the low GEF group baseline PAOP and CVP had predictive value Table . This taOur study suggests that in patients after coronary and major vascular surgery the predictive value of cardiac filling pressures and volumes for fluid responsiveness depends on GEF, as calculated by transpulmonary dilution-derived parameters.In patients with low GEF indicating systolic cardiac dysfunction, PAOP has a greater predictive value than GEDVI for fluid responsiveness, whereas in patients with near-normal GEF, GEDVI is superior to PAOP. This suggests the increasing value of filling pressures over volumes for predicting fluid responsiveness in patients with left ventricular systolic dysfunction. Indeed, the suggestion that a low GEF reflects systolic dysfunction of the left ventricle is supported by the fact that changes in PAOP did not correlate with changes in CVP, as reported by others -31. Furtet al. data in non-surgical patients with reduced left ventricular systolic function due to dilated and ischemic cardiomyopathy [The frequency of fluid responsiveness generally agrees with the literature, utilizing various loading protocols, and also involving variable amounts of fluid, in cardiac surgery patients -17,19,20et al. and Preisman et al. [et al. [Reuter n et al. ,17 did n [et al. . The cur [et al. , showing [et al. .2 when GEF was <20%) in responding versus non-responding fluid loading events. The fluid loading protocol guided by changes in filling pressures was used to prevent deleterious fluid overloading rather than to guide treatment on the basis of fluid responsiveness, as recently advocated to ensure safety [Our study has some limitations. Since our analyses adjusted for amount and type of fluids, it is unlikely that small differences in the amounts of fluids  rather than differences in cardiac preloading, were responsible for different increases in CI  is ≤15% or >15%. Table S2. Summated fluid loading responsiveness, defined as ≥15% increase in cardiac index, when global ejection fraction (GEF) is <20% or ≥20%. Table S3. Areas under the receiver operating characteristic curve  for prediction of fluid responsiveness (increase in SVI ≥10% from t = 0 to 90 minutes (A) or ≥15% (B)) by baseline values at t = 0, according to global ejection fraction (GEF).Click here for file


The authors with to add the following funding source to their financial disclosure information: The work was partly supported by Scott & White RGP Award (R8000 R3536) to SG.


The hypothesis that the overexpression of miR-21 activates the signal transducer and activator of transcription 3 (STAT3) signaling pathway was validated by measuring the mRNA expression levels of STAT3 and the anti-apoptotic protein survivin in human malignant melanoma (MeWo) and human embryonic lung fibroblast (HELF) cell lines transfected with miR-21-mimic and comparing them with those in cells transfected with miR-control. To further study the interaction of miR-21, STAT3 and VZV replication, the effects of miR-21 overexpression and STAT3 knockdown were evaluated. Higher virus titers were detected when miR-21 was upregulated in vitro. Moreover, it was identified that significantly lower virus titers were present in MeWo cells in which STAT3 was knocked down. In addition, the overexpression of miR-21 did not stimulate VZV replication in the MeWo cell line when the STAT3 gene was silenced. Therefore, the observations of the present study indicate that the enforced expression of miR-21 promotes the replication of VZV by activating STAT3 in vitro.Varicella-zoster virus (VZV) causes chronic pain and serious complications, including zoster paresis. However, the mechanism of VZV replication, a critical part of VZV pathogenesis, remains largely unknown and was investigated in the present study. The upregulation of microRNA-21 (miR-21) was identified following VZV infection  Varicella-zoster virus (VZV) belongs to the α-herpesvirus family and causes varicella (chickenpox) with primary infection and zoster (shingles) during reactivation from a latent state. VZV causes chronic pain or postherpetic neuralgia in patients, but may also cause zoster paresis in the arm, leg, diaphragm or abdominal muscles ,2. The lMicroRNAs (miRNAs) consist of ~22 nucleotides and are small, noncoding and functional RNAs. Due to the numerous functions of miRNAs, they play crucial roles in the pathogenesis of cancers and infections and in particular viral infections –8. It haA major miRNA that plays a crucial role in cancer, cardiovascular diseases, inflammation and virus infection is miRNA-21 (miR-21) . It has STAT3 is one of seven STAT transcription factors that comprehensively affects cell survival, cell cycle progression and homeostasis ,20. MoreIn the present study, by ectopically increasing miR-21 and knocking down STAT3, the effect of miR-21 expression and STAT3 on the replication of VZV was investigated.2 at 37°C. The parental Oka strain of VZV was acquired from Wuhan Institute of Virology, Chinese Academy of Sciences . MeWo cells were used to propagate VZV by co-cultivating infected cells with uninfected cells at a ratio of 1/5.Human malignant melanoma cells (MeWo) and human embryonic lung fibroblasts (HELF) were HELF were purchased from Type Culture Collection of the Chinese Academy of Sciences  and grown in Dulbecco’s minimal essential medium  supplemented with 10% fetal bovine serum in a humidified incubator containing 5% CO6 plaque forming units (PFU)/ml. The cells were infected with VZV at a multiplicity of infection (MOI) of 1 × 10−3.Cell-free VZV was provided by Beijing Wantai Biological Pharmacy Enterprise Co., Ltd. . VZV-infected cells were suspended in a cryoprotective solution . Cells were lysed by shaking vigorously with 1-mm glass beads. Next, the lysates were centrifuged at 1,240 × g for 10 min at 4°C. The supernatant was stored at −70°C. Viral titers were determined using a standard VZV plaque assay with MeWo cells. The titer of the VZV stock was 7.9×10mirVana miRNA detection kit  and qPCR primer sets. U6 small nuclear RNA was used as an internal control. The mRNA expression levels of STAT3 and survivin were assayed using GAPDH as an internal control. The ΔΔCt method was used for relative quantification. The primers were as follows: Survivin, forward, CAT GGG TGC CCC GAC GTT G; and reverse, GCT CCG GCC AGA GGC CTCA A; STAT3, forward, GGG TGG AGA AGG ACA TCA GCG GTA A; and reverse, GCC GAC AAT ACT TTC CGA ATC C; and GAPDH, forward, GGA GTC AAC GGA TTT GGT C; and reverse, GGA ATC ATT GGA ACA TGT AAA C.DNA and RNA were extracted from cells using DNeasy and RNeasy kits . The expression levels of cellular miR-21 were detected with a Following seeding or transfection, western blotting of the cells were performed by a standard protocol. Briefly, cells were washed with PBS and lysed. Next, the lysates were centrifuged at 13,000 × g at 4°C for 30 min. The supernatants were subjected to 10% SDS-PAGE. Following electrophoresis, proteins were transferred onto nitrocellulose membranes and detected using human monoclonal STAT3 antibodies  or β-actin antibodies . Secondary antibodies conjugated to horseradish peroxidase were goat anti-mouse IgG or goat anti-rabbit IgG . Enhanced chemiluminescent detection systems  were used for detection.EcoRI/HindIII . pSUPERSTAT3si was used to conduct the knockdown of the human STAT3 gene . The pSUPERSTAT3si plasmid was constructed with a STAT3-specific RNAi sequence (GAT CCC CTT CAG ACC CGT CAA CAA ATT CAA GAG ATT TGT TGA CGG GTC TGA AGT TTT T) which was cloned to the pSUPERneo vector with AT3 gene . STAT3 dThe data are normalized as mean ± standard deviation. Student’s t-tests were conducted to compare differences in two groups. Comparisons among multiple samples were made by ANOVA. SPSS 17.0 software  was utilized to perform the statistical analysis. P<0.05 was considered to indicate a statistically significant result.in vitro following VZV infection.miR-21 has a multifunctional role in virus infection. To demonstrate the effect that miR-21 has on VZV infection, the expression of miR-21 in HELF and MeWo was examined by relative qPCR following VZV infection. The results showed that miR-21 was significantly upregulated in MeWo cells following VZV infection, compared with that in MeWo cells without viral infection (P=0.03). However, the results indicated that the expression level of miR-21 was not significantly higher in VZV-infected HELF cells compared with that in HELF cells without VZV infection. The mean expression level of miR-21 in HELF cells infected with VZV was higher than that without VZV infection , as shown in in vitro. These observations indicate that the upregulation of miR-21 is associated with activation of the STAT3 signaling pathway in vitro.The ectopic expression of miR-21 was increased by transfection with an miR-21-mimic to investigate whether or not there was an association between miR-21 and STAT3. The expression of miR-21 was confirmed, which certified the efficiency of transfection, as shown in −3. Detection of miR-21 expression confirmed the efficiency of transfection, as shown in 6 PFU/ml in HELF cells transfected with miR-21-mimic, compared with 3.55×103 PFU/ml in cells transfected with the miR-control . Comparable results were obtained in HELF cells P=0.016; .2 PFU/ml (21 + shSTAT3), 1.48×107 PFU/ml (21 + shcontrol), 1.82×102 PFU/ml (control + shSTAT3) and 3.23×104 PFU/ml (control + shcontrol), as shown in in vitro.To further investigate the associations among miR-21, STAT3 and VZV infection, the expression levels of miR-21 were increased by transfection with miR-21-mimic and STAT3 genes were silenced following VZV infection in MeWo cells. The four groups were designated as follows: 21 + shSTAT3, cells co-transfected with miR-21-mimic and STAT3 knockdown; 21 + shcontrol, cells co-transfected with miR-21-mimic and control knockdown; control + shSTAT3, cells co-transfected with miR-control and STAT3 knockdown; and control + shcontrol, cells co-transfected with miR-control and control knockdown. The expression of miR-21 was confirmed in the in MeWo cells of the four groups. Higher miR-21 expression levels were observed in the 21 + shSTAT3 and 21 + shcontrol groups compared with those in the other two groups, as shown in et al revealed that STAT3 promoted VZV replication (MiRNAs, including miR-21, have important effects on viral replication, and function as inhibiting or enhancing factors . Moreovelication . HoweverIn the present study, the expression of miR-21 was observed to be upregulated significantly in MeWo cells following infection with VZV. Upregulation of miR-21 also occurred in HELF cells following VZV infection; however, the increase in miR21 levels in HELF cells was not found to be significant. To confirm the effect of miR-21 on VZV replication, MeWo and HELF cell lines were transfected with miR-21-mimic. The transfection was shown to stimulate VZV replication, which indicates that miR-21 plays an important role as a promoter in VZV replication.STAT3 responds to a variety of signals, including growth factors, cytokines and oncogenes. STAT3 is modulated by the miR-17-92 cluster, which affects tumorigenesis ,27. FurtIn the present study, miR-21 was overexpressed in MeWo and HELF cell lines by transfection with miR-21-mimic. The upregulation of STAT3 occurred concurrently with miR-21 overexpression in these cell lines. This was further supported by evaluating miR-21 expression in cells with silenced STAT3 genes, as shown in Several viruses have been shown to directly or indirectly decrease the expression of factors associated with the host innate immune system, including proteins, genes and cell signaling pathways, in order to enhance their own replication . VZV repin vitro. This study increased the understanding of the mechanism of VZV replication, a critical part of the VZV pathogenesis.In conclusion, cellular miR-21 was found to be a promoting factor of VZV replication. The study also demonstrated that STAT3 was involved in the mechanism by which miR-21 modulates VZV replication. In addition, the enforced expression of miR-21 was shown to promote the replication of VZV by activating STAT3


Although several histopathological and clinical features of canine mammary gland tumours have been widely studied from a prognostic standpoint, considerable variations in tumour individual biologic behaviour difficult the definition of accurate prognostic factors. It has been suggested that the malignant behaviour of tumours is the end result of several alterations in cellular physiology that culminate in tumour growth and spread. Accordingly, the aim of this study was to determine, using a multivariable model, the independent prognostic value of several immunohistochemically detected tumour-associated molecules, such as MMP-9 and uPA in stromal cells and Ki-67, TIMP-2 and VEGF in cancer cells.Eighty-five female dogs affected by spontaneous malignant mammary neoplasias were followed up for a 2-year post-operative period. In univariate analysis, tumour characteristics such as size, mode of growth, regional lymph node metastases, tumour cell MIB-1 LI and MMP-9 and uPA expressions in tumour-adjacent fibroblasts, were associated with both survival and disease-free intervals. Histological type and grade were related with overall survival while VEGF and TIMP-2 were not significantly associated with none of the outcome parameters. In multivariable analysis, only a MIB-1 labelling index higher than 40% and a stromal expression of MMP-9 higher than 50% retained significant relationships with poor overall and disease-free survival.The results of this study indicate that MMP-9 and Ki-67 are independent prognostic markers of canine malignant mammary tumours. Furthermore, the high stromal expressions of uPA and MMP-9 in aggressive tumours suggest that these molecules are potential therapeutic targets in the post-operative treatment of canine mammary cancer. Mammary tumours are the most prevalent neoplasms in intact female dogs  and it hSeveral studies reported that factors such as tumour size ,5, histoIn human breast cancer, molecular markers (oestrogen and progesterone receptors and c-erbB2) are routinely used for prognostic and predictive purposes . In vetePreviously published studies investigated either the characteristics of the tumour cells or those of the entire tumour population (tumour cells and stroma), seldom considering the tumour-stroma crosstalk by the individual characterization of the distinct cell types. However, it has been demonstrated, in human breast cancer, that the interactions between cancer cells and stroma are critical for tumour growth and invasion . There aIn this context, this study aimed to evaluate the prognostic value of several clinical, histological and molecular features of canine MMTs, including angiogenic factors (VEGF), matrix degrading proteins (uPA and MMP-9), and proliferation markers (Ki-67), in a prospective two-years follow up study. Factors significantly related to outcome, identified in univariate analysis, were included in a multivariable study in order to identify independent prognostic factors that may be adopted in routine practice and constitute potential targets of adjuvant therapies.The mean ± SD age of the dogs at the time of surgery was 10.3 ± 2.9 years (range 5–15). Thirteen dogs were spayed before or at the time of surgery. Only 8 had received hormonal therapy for oestrous prevention.The tumour histological types and the corresponding development of recurrences and/or distant metastases are presented in Table Overall, 30% of the dogs developed recurrences or distant metastases and 25.8% died or were euthanized due to this feature during the follow-up period. The mean ± SD survival time for dogs with recurrence/distant metastases was 11.96 ± 7.58 months (range 2–24) and the mean time for the detection of recurrence/metastasis was 5.29 ± 5.68 months (range 1–21). The overall 2-year survival was 48%. Within patients that developed recurrences or distant metastases, 38.5% had been spayed and 25% had been treated in the past with progestagens-based therapy for oestrus control.Host factors were not associated with disease-free survival (DFS) or overall survival (OS) Table , but theP = 0.025) and related death .Animals with complex carcinomas presented longer survival times and lower risk of death caused by MMT, when compared to the “others” group but not to those with simple carcinomas Tables  and 3. NThe mean ± SD MIB-1 labelling index (LI) was 39.6 ± 2.0 percent (range 6.5- 84.2). Factors significantly related to shorter DFS, shorter OS, tumour-related death and higher risk of recurrences/distant metastases included high MIB-1 LI and high uPA and MMP-9 expressions by tumour-adjacent fibroblasts  and high MMP-9 expression by stromal fibroblasts (≥ 50%) were the only factors that retained statistical significance as independent predictors of shorter DFS  cases. Apparently, as demonstrated by previous studies, this variable fails to maintain its prognostic significance when included in multivariable models ,6.In human breast cancer studies, it has been proposed that the expression of VEGF by cancer cells is a poor prognostic factor for survival , but thiThe protein TIMP-2, initially described as an inhibitor and regulator of the activity of matrix metalloproteinase-2 (MMP-2), has recently been proven to stimulate cell growth and angiogenesis, as well as inhibit apoptosis, hence contributing to tumour aggressiveness . AlthougAn increasing number of studies over the last decade identified the tumour stroma as a major player of the carcinogenic process reviewed by . During The results of this study demonstrate that the expressions of MMP-9 by stromal cells and of Ki-67 by cancer cells are independent prognostic factors in canine MMTs, that may be used for the selection of those animals that should be considered for post-operative ancillary treatments. These factors exhibited a stronger prognostic value than clinical or histological parameters traditionally used to predict patient’s outcome, such as tumour size, histological grade and mode of growth. Hence, we suggest that they should be considered in the routine prognostic assessment to help practitioners in the prediction of patients’ outcome.In addition, our findings highlight the role of tumour stroma in the biological behaviour of canine MMTs and suggest that uPA and MMP-9 may be potential targets for post-operative therapies.Eighty-five female dogs with spontaneous MMTs underwent surgical treatment and were enrolled, with owners consent, in a 2-year post-operative follow-up study. The study protocol was performed in compliance with the European Union Directives for the protection of animals used for scientific purposes (1999/575/CE and 2010/63/UE) and approved by the Ethics Committee of the Biomedical Sciences Institute of Abel Salazar, University of Porto.Animal data such as weight, age, reproductive status and previous administration of progestagens for oestrus control, were obtained and registered and, for statistical purposes, dogs were grouped into three categories: small (<10 Kg), medium (10–23 Kg), and large breeds (>23 Kg) according to the criteria of the Féderation Cynologique Internationale (FCI). The clinical staging was assessed in each animal, based in a complete physical examination, three views thoracic radiographies, and a complete abdominal ultrasound evaluation. Inclusion criteria were animals with stage I to IV MMTs whose owners declined post-operative adjuvant therapies. Exclusion criteria were animals with stage V MMTs and animals with a previous history of neoplastic disease.Removed tumours were fixed in 10% neutral buffered formalin for 48 h., measured in their largest diameter and grouped in two categories: < 3 cm or ≥ 3 cm for statistical purposes. Tumours < 1 cm were paraffin-embedded in one block, while larger tumours were cut sequentially at 5 mm intervals to provide tissue blocks representative of the entire lesion. After dehydration and embedding in paraffin wax, 3 μm sections were obtained from each block. Slides were stained using haematoxylin and eosin (HE) and the histological classification was performed by two pathologists (FG and IA) using the criteria of the World Health Organization for the histological classification of mammary tumours of domestic animals [When available, local and regional lymph nodes were processed and examined as previously described . The mitWhen more than one malignant neoplasm were diagnosed , the tumour with the more aggressive clinical and histopathological features  was selected.Serial tumour sections were immunostained for Mindbomb homolog 1 (MIB1); VEGF; uPA; and TIMP-2 as described, respectively, by Matos et al. (2006a)  and SantTo determine the MIB-1 labelling index, the areas of highest expression were selected and 1000 tumour cells nuclei were counted in these areas at 400× magnification with the help of a microscopic grid. The proportion of stained nuclei was recorded as a percentage  and tumoSections were examined independently by two observers (A.S. and A.M.) and when there was disagreement (less than 5% of the cases) a consensus was reached using a multi-head microscope.Dogs were evaluated before surgery, 3 weeks after surgery and every 3 months thereafter for a 2-year period. Owners were instructed to report and discuss with the researchers any detected abnormalities, even if not obviously related to the mammary gland tumours, at any time. Each evaluation included a thorough physical examination, thoracic radiographs (three views) and complete abdominal ultrasound. Complete necropsies were performed, after owner consent, in all dogs that died spontaneously or were euthanized, and suspected metastases, when present, were histologically confirmed. Overall survival was calculated from the date of surgery to the date of animal death/euthanasia due to tumour metastasis. Disease-free survival was calculated from the date of surgery to the date of detection of the first local recurrence or distant metastases.P < 0.05. Statistical analysis was performed with the statistical package PASW Statistics 18.0.Survival curves were calculated using the Kaplan-Meier method and the log-rank test was used to analyse the significance of the differences between the groups defined for each variable . In the BSA: Bovine serum albumin; CMT: Canine mammary tumour; CI: Confidence interval; DFS: Disease-free survival; HE: Haematoxylin and eosin; HPF: Higher power field; HR: Hazard rate; IHC: Imunohistochemistry; MIB-1 LI: Mindbomb homolog-1 labelling index; MMP: Matrix metalloproteinase; MMT: Malignant mammary tumour; OS: Overall survival; RLN: Regional lymph node; SD: Standard deviation; TIMP-2: Tissue inhibitor of matrix metalloproteinase-2; TBS: Tris-buffered saline; uPA: Urokinase-type plasminogen activator; VEGF: Vascular endothelial growth factor.The authors declare that they have no competing interests.AS and AM conceived the study, participated in its design and coordination, data aquisition and analysis, and manuscript elaboration. LM, JS, JR participated in data aquisition. AS and CL carried out the immunohistochemistry assay. FG participated in the study design and coordination, performed the histopathological evaluation and helped with the interpretation of data. IA participated in the histopathological evaluation. All authors read and approved the final manuscript.


There is an error in the number of the first author's (JFP) NIH grant. The correct grant number is: GM086416.


Intubation of patients with laryngeal granulomas on the vocal folds are sometimes difficult to manage because of potential airway obstruction. Laryngeal granulomas usually have flexible stalks where they attach to the vocal folds. We report a worst-case scenario of dislocation of the laryngeal granuloma during induction of anaesthesia.We present a case of laryngeal granulomas on the posterior vocal fold. A 20-year-old woman had an approximately 10-mm tumour in the laryngeal arytenoid region. Manual ventilation resulted in the tumour lodging in the subglottis and the inflated cuff of the intubation tube successfully returned it to its original position during tube withdrawal. Images were obtained using an Airway Scope®  and a video laryngoscope.In our case, the tumour was benign and relatively small in size; therefore, we did not select tracheotomy as an airway management strategy. The case had a granulomatous tumour arising from the posterior vocal folds on the right side, and the tumour was very flexible. To promptly gain control of the airway in such a case using direct laryngoscopy, thus avoiding tracheotomy, other strategies are suggested, such as bronchoscopic visualization with awake or semi-awake intubation. Intubation of patients with laryngeal granulomas on the vocal folds are sometimes difficult to manage because airway obstruction can occur. Laryngeal granulomas usually have flexible stalks where they attach and compared with polyps or laryngeal cancer, which are tightly attached to the larynx, intubation in patients with laryngeal granulomas must be performed carefully to avoid disrupting the tumour ,2. Altho2O. Because it was not feasible to perform tumour resection using a laryngomicroscope under these conditions, otolaryngologists were consulted, and extraction of the tumour to the supraglottic area was attempted. To obtain an adequate visual field of the right vocal fold, the tracheal tube was first moved to the left side of the mouth, and laryngoscopy was performed using the Airway Scope® from the right side of the tracheal tube. The tube was slowly withdrawn along the left side of the mouth, 1 mm at a time, until the cuff was visible distal to the vocal fold, while maintaining cuff inflation. With this manoeuver, the tumour was successfully repositioned supraglottically (Figures A 20-year-old woman, 157 cm tall, weighing 49 kg, complained of hoarseness after extraction of four wisdom teeth under general anaesthesia at another hospital 1 month earlier. She visited our hospital for consultation and treatment because the frequency and duration of the hoarseness had increased, causing intermittent, complete loss of vocalization. She had no symptoms of dyspnoea or discomfort in the larynx and no history of smoking, alcohol abuse, or other significant issues. She presented without trismus and was assessed as Mallampati Class I. Laryngeal endoscopy, performed at the otolaryngology department of our hospital, demonstrated a laryngeal tumour with a diameter of approximately 10 mm (Figure The patient’s symptoms developed following general anaesthesia for dental extractions. Based on the clinical course of increasing frequency and duration of hoarseness, it was reasonable to presume that she had post-intubation granuloma. In this case, the location of the tumour changed after manual ventilation, becoming partially lodged in the glottis by the positive pressure ventilation. Therefore, ideally, awake or semi-awake intubation should have been performed. In cases with large or malignant tumours that can obstruct the glottis and trachea, tracheotomy can be performed, but the tumour was benign and relatively small in size in this case; therefore, we did not select tracheotomy as an airway management strategy. Also, tracheotomy is contraindicated in cases of laryngeal papilloma caused by human papillomavirus, which could not be ruled out preoperatively in this case .Our case had granulomatous tumour arising from the posterior vocal folds on the right side, and the tumour was benign and very flexible. To promptly gain control of the airway in such a case using direct laryngoscopy, thus avoiding tracheotomy, other strategies are suggested. Awake or semi-awake intubation can be performed to prevent tumour disruption, and intubation can be performed by advancing the endotracheal tube along the opposite side from the tumour under bronchoscopic visualization. If the tracheal tube is advanced along the tumour side, the tumour becomes unobservable. Creative strategies, such as modifying the angle of the stylet, are necessary. In cases of dislocation and lodging of laryngeal tumours, the most appropriate device for visualization, such as a bronchoscope, should be prepared.We report a worst-case scenario of dislocation of a laryngeal granuloma during induction of anaesthesia. Advanced devices should be equipped and creative strategies for each case are needed.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.JN performed the anaesthesia and all authors discussed each case both preoperatively and postoperatively. JN and TS were major contributors to writing the manuscript, and SM and TM helped create the figures. All authors have read and approved the final manuscript.


Non-Hodgkin’s lymphoma (NHL) can commonly present as chylothorax and rarely as chylopericardium. Here we are presenting a case of a 21-years-old female with bilateral chylothorax, chylopericardium and chylous ascites all together finally diagnosed to have NHL as the etiology. To the best of our knowledge, it has been reported very infrequently. Chylothorax is an uncommon condition characterized by accumulation of fluid containing chylomicrons within the pleural cavity. Simultaneous accumulation of chyle in all serous cavities, though rare, can be associated with nontraumatic etiologies. By far, malignancy  represents the most frequent underlying cause. Management is mainly conservative with dietary modifications along with the treatment of underlying etiology.A 21-year-old female was admitted in the department of pulmonary medicine of Dayanand Medical College and Hospital with the complaints of dry cough, progressive breathlessness and orthopnea for last 20 days. She was also having throbbing headache along with slowly increasing facial and neck swelling for last 10 days.1 and S2 were diminished without any murmur. Per abdomen, shifting dullness was present without any organomegaly.General physical examination showed a pale female with gross facial and neck swelling associated with dilated superficial veins on neck and chest. There was no pulsus paradoxus and Kussmaul’s sign was absent. She was also having bilateral axillary lymphadenopathy . Respiratory examination revealed grossly decreased movements bilaterally with stony dull percussion note. Breath sounds were absent in bilateral basal regions. On cardiovascular examination, S3, differential leukocyte count: neutrophils 85%, lymphocytes 15%, protein: 4.7 g%, sugar: 139 mg%, pleural fluid triglyceride: 725 mg %, pleural fluid cholesterol: 57 mg % on right side. Pleural fluid of left side revealed total leukocyte count 5800 cells/mm3, differential leukocyte count: neutrophils 60%, lymphocytes 40%, protein 4.8 g%, sugar 50 mg%, pleural fluid triglyceride: 426 mg%, pleural fluid cholesterol: 44.8 mg%. Fluid from both sides did not reveal any abnormal cell. Serum triglyceride and serum cholesterol were 129 mg% and 99 mg% respectively. The level of adenosine deaminase in pleural fluid was 32 IU/L. Pleural fluid culture was sterile in nature for pyogenic organisms. Bactec culture also did not reveal any growth of Mycobacterium tuberculosis.Routine blood investigations and coagulation profile were within normal limits. Chest radiograph was suggestive of bilateral pleural effusion and cardiomegaly . As the Echocardiography was done which revealed moderate amount of pericardial effusion without any significant cardiac tamponade. Pericardial aspiration was done which was milky and exudative with high cholesterols. Biochemistry was in favor of chylopericardium. Abdominal ultrasound was suggestive of minimal ascites without any lymphadenopathy. In the light of confirmed bilateral chylothorax and chylopericardium, ascites was also thought to be chylous in origin. So, a diagnosis of superior vena cava syndrome, bilateral chylothorax, chylopericardium and chylous ascites was established but etiology remained unknown. She was put on conservative management along with low lipid diet but effusion re-accumulated and repeat thoracentesis was performed.Bone marrow aspiration was suggestive of normocellular, normocytic marrow. Fine needle aspiration cytology of left axillary lymph node was inconclusive. On symptomatic improvement, computed tomography of thorax was performed showing bilateral pleural effusion with large anterior mediastinal mass and superior vena cava thrombosis  and bilaChylothorax and chylopericardium are characterized by fluid with a milky appearance due to high triglyceride content and the presence of chylomicrons as detected by lipoprotein electrophoresis. The etiology of chylothorax has been divided by Light into fouDiagnosis of chylothorax is established by direct analysis of the pleural fluid. The fluid is characteristically “milky” in appearance, although not all milky effusions are chylous in nature and not all chylous effusions are milky. The triglyceride concentration greater than 110 mg/dl (in our case it was 725 mg%), a ratio of pleural fluid to serum triglyceride of greater than 1.0 (in our case it was 5.6), and ratio of pleural fluid to serum cholesterol of less than 1.0 (in our case it was 0.58) usually confirms chylothorax. Chylothorax will be excluded if pleural fluid triglyceride concentration is less than 50 mg/dl. However, in case of levels in between 50 and 110 mg/dl, a lipoprotein analysis of pleural fluid should be performed and demonstration of chylomicrons in fluid confirms the diagnosis of chylothorax.[Chylous ascites is very rare and it occurs approximately in 1 in 20000 cases.7 Chylope8The first step in the management of chylothorax demands a review of the history and physical examination. Since lyIn conclusion, early recognition of chylous leakage, prompt treatment with fat free and a medium chain triglycerides (MCT) - rich diet and good compliance are still found to be effective in the initial management of chylothorax and chylopericardium leading to favorable outcome without complications. Also, one should look for other potential sites of chylous origin apart from pleural cavity as appropriate and timely management is sometimes life saving.