Lastly, Claudin14-heterozygosity, but not complete deficiency, also enhanced endothelial cell proliferation significantly. These data establish a new role for Claudin14 in the regulation of tumour blood vessel integrity and angiogenesis that is evident only after the partial loss of this molecule in Claudin14-heterozyous mice but not in Claudin14-null mice.The maintenance of endothelial cell-cell junctions is vital for the control of blood vessel leakage and is known to be important in the growth and maturation of new blood vessels during angiogenesis. Here we have investigated the role of a tight junction molecule, Claudin14, in tumour blood vessel leakage, angiogenesis and tumour growth. Using syngeneic tumour models our results showed that genetic ablation of Claudin14 was not sufficient to affect tumour blood vessel morphology or function. However, and surprisingly, Claudin14-heterozygous mice displayed several blood vessel-related phenotypes including: disruption of ZO-1-positive cell-cell junctions in tumour blood vessels; abnormal distribution of basement membrane laminin around tumour blood vessels; increased intratumoural leakage and decreased intratumoural hypoxia. Additionally, although total numbers of tumour blood vessels were increased in Claudin14-heterozygous mice, and in VEGF-stimulated angiogenesis  Blood vessel maturation, during angiogenesis, involves establishment of endothelial cell-cell contacts, deposition of an intact basement membrane, lumen formation and the recruitment of supporting cells Endothelial cell-cell junction integrity is a determinant of vascular permeability. Tight junctions (TJs) in both epithelial and endothelial cells consist of several protein families, including JAMs, occludin and claudins Some TJ protein families have endothelial cell-specific members such as vascular endothelial junctional adhesion molecule , and endothelial cell-enriched molecules, for example Claudin5 (Cldn5) in vivo and particularly in tumour angiogenesis are unknown.Another claudin family member, Claudin14 (Cldn14), has been identified in both epithelial and endothelial cell layers. Cldn14 mutations have been identified as a key cause of autosomal recessive deafness in vivo, ex vivo and in vitro.Here we describe that, surprisingly, homozygous loss of Cldn14 has little to no effect on tumour angiogenesis. However, in Cldn14-heterozygous mice (with loss of only one copy of Cldn14) we found the following major phenotypes: (1) disruption of cell-cell junctions in tumour blood vessel; (2) abnormal tumour blood vessel basement membrane organisation and reduced supporting cell association; (3) increased intratumoural leakage and decreased tumour hypoxia; (4) enhanced tumour angiogenesis but no significant difference in the proportion of lumenated tumour blood vessels; (5) no effect on syngeneic tumour growth and (5) increased endothelial cell proliferation In short our data establish that Cldn14 heterozygosity, but not complete deficiency, can affect tumour blood vessel functionality and describe a gene dosage effect of this molecule on angiogenic processes.All animals were used in accordance with UK Home Office regulations and approved by the Queen Mary University of London and Oxford University ethics committee. License 70/7449.5′ - GGC TGC ATA ACC AGG ATA CTC - 3′] with Cldn14 WT primer [5′ - GTA CAG GCT GAA TGA CTA CGT G - 3′] for the wild-type allele (340 bp band) and Cldn14 common with Cldn14 Mutant primer [5′ - CAG CTC ATT CCT CCC ACT CAT GAT C - 3′] for the null allele (275 bp band) in two separate PCR reactions. Mice were born in normal Mendelian ratios and male:female ratios, no obvious adverse effects were observed, and proportionately decreased Cldn14 mRNA levels were confirmed in kidneys and brains of Cldn14-het and Cldn14-null animals, compared to wild-type littermates .Claudin14-null mice on a C57/bl6 genetic background 6 cells ml−1. 0.5×106 tumour cells in 100 µl PBS were injected subcutaneously in the flank. Palpable tumours were measured with callipers every other day and bisected. Half of the tumour was snap-frozen for cryosectioning, and the other half fixed in 4% formalin. Formalin-fixed tumours were embedded in paraffin for sectioning.Mouse tumour cell lines B16F10 melanoma (ATCC® Number: CRL-6475 ™) and Lewis Lung Carcinoma (ATCC® Number: CRL-1642 ™) cells were grown in DMEM (Gibco) supplemented with 10% v/v FBS  and penicillin/streptomycin (Gibco). Tumour cells were trypsinised and resuspended in PBS at a concentration of 5×104, L2895).Anti-endomucin 1∶500 (Santa Cruz: V.7C7 sc-65495); anti-α-Smooth Muscle Actin-Cy3 1∶1000 ; anti-Pimonidazole 1∶10 ; Ki67 1∶100 , anti-ZO-1 1∶100 . PE-PECAM 1∶500 , FITC-BS1 lectin . 100 µm Z-stacks  were taken using a Zeiss LSM 510 confocal microscope. LSM images were analysed using ImageJ: red (PE-PECAM) and blue (Hoechst) pixel intensities were obtained using colour thresholding to remove background noise and Hoechst staining was quantified and normalised to blood vessel density for each section to give Relative Intensity values.10 minutes prior to sacrifice, tumour-bearing mice were injected with 100 µl PE-PECAM antibody . 1 minute before sacrifice, the same mice were injected intravenously in the tail vein with 100 µl 4 µg/ml Hoechst dye , diluted in ddH2O to a final concentration of 10 mg/ml) intravenously via the tail vein 1 hour prior to sacrifice, tumour-bearing mice were injected with 60 mg/kg pimonidazole hydrochloride . (2) Deparaffinising in xylene and ethanol solutions of decreasing concentration. (3) Blocking: 30 minutes in 2% v/v Goat serum, 1% w/v Bovine Serum Albumin, 0.1% v/v Triton X-100 in PBS. (4) Washing with 0.1% w/v Bovine Serum Albumin in PBS. (5) Incubation with anti-endomucin primary antibody (diluted in wash buffer) overnight at 4°C. (6) Washing in wash buffer. (7) Incubation with 1∶100 secondary antibodies . (8) Final washing with PBS then ddH2O. (9) Mounting with ProLong® Gold Antifade with DAPI (Invitrogen: P36931).FFPE 8 µm sections were processed as follows: (1) antigen retrieval 2×10 minutes boiling in citrate buffer (0.294% w/v Tri-Sodium citrate (Fisher) in ddH2O wash. 9) Mounting with ProLong® Gold Antifade with DAPI.8 µm cryosections were processed for ZO-1 and PECAM immunostaining as follows: 1) fixation with −20°C methanol for 10 minutes. 2) Blocking: 45 minutes in 5% w/v BSA, 0.1% v/v Triton X-100 in PBS. 3) 3× PBS washes. 4) Incubation with primary antibodies for 45 minutes at room temperature, diluted in blocking buffer. 5) 3× PBS washes. 6) Incubation with secondary antibodies for 2 hours, diluted in blocking buffer. 7) 3× PBS washes. 8) Final H8 µm cryosections were processed for Ki67 immunostaining as follows: 1) fixation in −20°C acetone for 10 minutes. 2) Permeabilisation with 0.5% NP-40 for 10 minutes. 3) Blocking: 1 hour in 1% w/v BSA, 0.1% Triton X-100 in PBS. 4) Incubation with primary antibody diluted in blocking buffer at 4°C overnight. 5) Washing, secondary antibody incubation and mounting as in steps 5–9 above.et al. 2012 The aortic ring assay was performed as described in Baker −1 heparin (Sigma), 1% v/v 100× glutamine , Endothelial Growth Supplement  and 1% v/v 100× penicillin/streptomycin (Gibco)). Lungs were then minced and digested in 0.1% Type I Collagenase , passed through a 70 µm cell strainer  and the resulting single-cell suspension plated on pre-coated plastic. Macrophages were removed from the resulting cultures using rat anti-mouse FCγII/III (Pharmingen) antibody and sheep anti-rat IgG-coated magnetic bead sorting . Endothelial cell cultures were enriched by positive sort, using two rounds of anti-CD102 (ICAM2) antibody (Pharmingen) and Dynal anti-rat magnetic beads.Primary mouse endothelial cells were purified and cultured from mouse lung as described in Reynolds and Hodivala-Dilke, 2006 in vivo, we used a Cldn14 genetic ablation approach. Cldn14-heterozygous (Cldn14-het) mice were inter-crossed to produce wild-type (WT), Cldn14-het and Cldn-14 null progeny. No effects on Mendelian ratios or male:female ratios were observed in litters . qPCR analysis confirmed that levels in Cldn14-het organs were approximately half those detected in WT mice, whilst Cldn14 transcript was undetectable in Cldn14-nulls . Due to the lack of reliable reagents, we were not able to test whether Cldn14 is expressed differentially in quiescent and angiogenic blood vessels in vivo.It is conceivable that Cldn14 could affect vascular integrity, since it is a component of tight junctions. To test the requirement for Cldn14 in the maintenance of vascular integrity and in angiogenic processes 6 B16F10 melanoma cells. At 10 days post tumour cell inoculation the tumours were snap-frozen and cryosections double immunostained for the tight junction marker ZO-1 and the endothelial cell marker PECAM. ZO-1 staining was confined to a continuous pattern of expression at endothelial cell-cell junctions in tumours from both WT and Cldn14-null animals, indicating that Cldn14-deletion was not sufficient to affect the organisation of ZO-1 at cell-cell junctions. In contrast, and surprisingly, a discontinuous staining pattern of ZO-1 was observed in the majority of Cldn14-het tumour blood vessels , Cldn14 heterozygous (Cldn14-het) and Cldn14-null mice were injected subcutaneously with 0.5×10Given that extracellular matrix deposition and maintenance are crucial steps in the maturation of tumour blood vessels, we next asked if the distribution of laminin within the blood vessel basement membrane was affected by changes in stromal Cldn14 levels. Tumour sections were double immunostained for PECAM and laminin and observations showed that the pattern of laminin deposition was proximate to the blood vessel wall in sections from WT and Cldn14-null mice, indicating, again that Cldn14 deficiency was not sufficient to affect this process. In contrast, a high frequency of disorganised laminin deposition around blood vessels, with a ‘shorelining’ pattern, was observed in sections from tumours grown in Cldn14-het mice  cells. Intravenous injection of anti-PECAM (PE-PECAM) antibody and Hoechst dye into tumour-burdened mice revealed no significant difference in Hoechst leakage from tumour vessels between WT and Cldn14-null tumour-bearing mice relative to PE-PECAM signal. However, a significant increase in Hoechst leakage was observed in Cldn14-het mice when compared with either WT or Cldn14-null mice  into tumour-bearing WT, Cldn14-het and Cldn14-null mice showed that the relative levels of tumour hypoxia were similar between WT and Cldn14-null mice . The apparent lack of correlation between tumour size and blood vessel density suggested that the functionality of the vessels in tumours grown in Cldn14-het mice might have been affected. Tumour blood vessel functionality is heterogeneous like the tumour mass itself, including those that are functional (lumenated), and those that are not (closed). Analysis of endomucin-stained tumour sections showed that the percentage of closed vessels was elevated in Cldn14-het tumour sections .Since enhanced angiogenic responses may reflect an increase in endothelial proliferation we hypothesized that Cldn14-hetrozygosity may elevate these processes. To test this, we first measured proliferation of endothelial cells in tumours 14-nulls Figure 6These combined results demonstrate a role for the tight junction protein Cldn14 in maintenance of tumour blood vessel integrity and angiogenesis that was previously unknown. Importantly this effect is realised not by the total genetic ablation of Cldn14 but in its partial expression within the stromal microenvironment.The consequences of changes in Cldn14 expression levels on tumour blood vessel fragility and angiogenesis have not been addressed previously. Here we have shown that Cldn14 heterozygosity, but not total deficiency, induces destabilisation of tumour blood vessels, which correlates with enhanced vessel leakage and decreased tumour hypoxia without affecting tumour growth.Several papers have described how loss off cell-cell junction function can enhance blood vessel leakage Our observations that Cldn14 heterozygosity, but not total deficiency, can cause: decreased endothelial cell-cell junctional organisation; poor blood vessel basement membrane distribution; and reduced supporting cell coverage, all describe how more subtle changes in endothelial cell-cell junctions can dramatically affect vascular function.Claudins have been described to signal in co-ordination with β1-integrins. Genetic ablation of the α3-integrin subunit results in a basement membrane defect in which components of the basement membrane, such as laminin, show a disorganised expression pattern and a ‘shorelining effect’ in vivo, ex vivo and in endothelial cell cultures in vitro. It is tempting to speculate that this gives rise to an actual increase in total blood vessel numbers in Cldn14-het mice even if a significant proportion of these vessels are not properly lumenated. Cldn14 has previously been found to be downregulated in proliferating endothelial cells In addition, the increased tumour vascular fragility that we have observed in Cldn14-het mice is associated with enhanced endothelial proliferation Our work may also have some clinical relevance. It is difficult to prove that any pharmacological inhibitor has a complete blocking effect when used therapeutically, therefore understanding how partial loss, or blockade, may affect biological outcome also becomes of interest. To our knowledge Cldn14 inhibitors have not yet been tested for their effect on blood vessels, if indeed they are available, but if they were it is conceivable that partial pharmacological inhibition could promote some or all of the phenotypes that we have described in the Cldn14-het mice. For example, partial Cldn14 inhibition may decrease intratumoural hypoxia and pericyte association with blood vessels. Others have shown that decreased tumour hypoxia can sensitise tumour cells to chemotherapy and radiotherapy treatments In short, our findings highlight new roles for Cldn14 in vascular function and angiogenesis that are relevant directly to its levels of expression and not simply the presence of absence of this molecule.Figure S1Genotyping and colony statistics for Cldn14 mice. (A) A representative agarose gel is shown with separate PCR reactions, for Cldn14 WT  and Cldn14-null alleles . PCR products identify wild-type (lanes 1 and 2), Cldn14-heterozygous (lanes 3 and 4) and Cldn14-null (lanes 5 and 6) DNA samples. (B) The bar chart represent the numbers and of WT, Cldn14-Het and Cldn14-null mice at weaning from Cldn14 heterozygous breeding pairs. All genotypes developed at expected Mendelian ratios. N = 25 litters and 189 mice. (C) Proportion of male:female pups in the Cldn14 colony is normal and as expected. (D) qPCR analysis Cldn14 transcript expression from WT, Cldn14-het and Cldn14-null tissues. Cldn14 mRNA levels are shown relative to β-actin (ACTB) controls, with approximately half as much transcript detected in Cldn14-het organs and undetectable levels in Cldn14-nulls when compared with WT controls. Please see Text S1 for qPCR method details. Bars show relative transcript levels ± SEM. N = 3 separate tissue samples per genotype.(TIF)Click here for additional data file.Figure S2Vessel density in unchallenged skin is unaffected by the Cldn14 genotype. Blood vessel density was quantified in WT, Cldn14-het and Cldn14-null transverse skin sections, taken from non-tumour burdened mice. Values are given as mean number of dermal blood vessels per mm2 of dermal section. Bars represent mean ± SEM. NSD: no significant difference.(TIF)Click here for additional data file.Figure S3Migration of WT, Cldn14-het and Cldn14-null endothelial cells. Primary endothelial cells were cultured from WT, Cldn14-heterozygous and Cldn14-null mouse lungs. Cells were plated on coverslips and inverted over Dunn chamber slides filled with serum-free growth medium and medium containing 100 ng/ml VEGF to stimulate cell movement. Cells were photographed at 10-minute intervals over 16 hours to create movie files for cell tracking with Andor software and analysis using Mathematica software. (A) Raw cell tracking data with all cell starting positions at a single point of origin. (B) Speed of cellular movement (µm/min). (C) Persistence of cell movement . Please see Text S1 for Dunn Chamber Chemotaxis assay method details. Bar charts show means ± SEM. N = 12–20 fields per genotype, 280–348 cells per genotype, 2 independent experiments. * P<0.05 ** P<0.01.(TIF)Click here for additional data file.Text S1Supplementary methods. Details of methods used to produce data for Figure S1 and Figure S2: organ harvesting, RNA extraction, qPCR and Dunn Chamber Chemotaxis assays.(DOC)Click here for additional data file.


Adenosine deaminase (ADA) is useful in the diagnosis of tuberculous pleural effusion (TPE). This study aims to determine the factors affecting pleural fluid ADA levels and to establish the optimal ADA levels for diagnosis of TPE for different age groups.This was a retrospective study from January 2007 to October 2011. One hundred and sixty patients who had pleural fluid ADA performed for investigation of pleural effusion were analyzed. Variables examined included demographics, pleural fluid characteristics and peripheral blood counts. The ADA cut-offs according to age were selected using the receiver operating characteristic (ROC) curve.The mean pleural fluid ADA was significantly higher in the TPE group (100 ± 35 IU/L) compared to non TPE patients (30 ± 37 IU/L). There was significant correlation between pleural fluid ADA and age, pleural fluid protein, LDH, and fluid absolute lymphocyte count. The strongest correlation was seen with age (r = −0.621). For patients ≤ 55 years old the ROC for ADA had area under curve (AUC) of 0.887. A pleural fluid ADA of 72 IU/L had sensitivity of 95.1%, specificity of 87.5%, positive predictive value (PPV) of 95.1% and negative predictive value (NPV) of 87.5% for the diagnosis of TPE. For patients > 55 years old the AUC is 0.959. ADA of 26 IU/L had a sensitivity of 94.7%, specificity of 80.4%, PPV of 62% and NPV of 97.8%.There is a significant negative correlation between pleural fluid ADA and age. For older patients, a lower ADA cut-off should be used to exclude TPE. Pleural effusion is a common medical condition with many possible underlying etiologies. Neutrophilic predominant exudative effusions are due to acute processes e.g. pneumonia or acute pulmonary embolism  whereas Pleural fluid analysis and closed pleural biopsy are integral parts in the investigative work up of an exudative pleural effusion. For diagnosis of tuberculous pleural effusion (TPE), the yield of pleural fluid culture for mycobacteria is low at about 36% . Joint sAdenosine deaminase (ADA) is an enzyme which catalyses the conversion of adenosine to inosine and plays an important role in the differentiation of lymphoid cells. Its activity is high in diseases in which cellular immunity is stimulated . DiffereOur study therefore aims to:a) Determine whether certain patient demographic characteristics, pleural fluid biochemistry and peripheral blood leucocyte counts affect pleural fluid ADA level.b) Establish ADA cut off value for diagnosis of TPE in our study population.If pleural fluid ADA does correlate significantly with the variables studied, ADA value for patients should then be interpreted based on patient demographics and relevant laboratory investigations.This was a retrospective study of the medical records of patients investigated for pleural effusion in our hospital from January 2007 to October 2011. The study design was approved by the Institutional Review Board (IRB) and exempted from further IRB review. Based on laboratory records a list of consecutive patients with pleural fluid ADA sent was obtained. We felt that this was a reliable way of obtaining the list of patients investigated for pleural effusion as pleural fluid ADA is routinely sent in our institution for all cases of pleural effusion. Pleural fluid ADA was measured by the spectrophotometric method described by Giusti and Galanti. 80 patients with TPE were identified. Another 80 patients with diagnoses other than TPE were randomly selected using a random number generator.Pleural effusions were diagnosed to be due to pleural TB (TPE) if: (i) pleural fluid smear was positive for acid fast bacilli or fluid culture was positive for mycobacteria tuberculosis; (ii) pleural biopsy histology showed granulomas with no other causes of granulomatous lung disease; (iii) pleural biopsy culture positive for mycobacteria tuberculosis; (iv) sputum culture positive for mycobacteria tuberculosis. Malignant pleural effusions were confirmed by positive pleural fluid cytology or pleural biopsy histology . Parapneumonic effusions were diagnosed based on clinical and radiographic features that were consistent with an acute pulmonary infection and exclusion of other causes of pleural effusion. Effusions were attributed to congestive cardiac failure (CCF) if the effusion was a transudate with consistent clinical features. An effusion was considered to be lymphocyte-predominant if pleural fluid lymphocyte count was > 0.5 of total fluid leucocyte count, and neutrophil-predominant if pleural fluid neutrophil count was > 0.5 of total leucocyte count. Peripheral blood counts used for analysis were most recent to the date of pleural fluid investigations.Continuous variables are expressed as mean ( SD) while categorical variables are expressed as number and group percentages. Differences in ADA levels between groups were analysed using unpaired Student t test and one way Anova. Correlation between ADA and specified variables was quantified using Pearson correlation coefficient. This was performed for the whole study population (n = 160) as well as individually for the TPE and non TPE groups. A correlation coefficient (r) of < 0.3 was considered to be negligible correlation, r 0.3-0.5 was considered weak correlation, r 0.5-0.7 was considered moderate correlation, r 0.7-0.9 was high correlation and r > 0.9 was very high correlation. The best cut off value of ADA for diagnosis of TPE was selected by receiver operating characteristics (ROC) curve. We also fitted a multivariate linear regression model and included all variables identified to be statistically significant to see which ones remain independently associated with ADA level. All statistical analyses were performed using IBM SPSS Statistics version 19. A 2 tailed p value of < 0.05 was taken to be statistically significant.A total of 160 patients were investigated in this study. The baseline characteristics of these patients are shown in Table Mean pleural fluid ADA was significantly higher in the TPE group compared to non TPE group . There was no statistically significant difference in pleural fluid ADA level between the malignant, parapneumonic and others group, with mean ADA 28 ± 49 IU/L, 40 ± 34 IU/L and 9 ± 6 IU/L respectively. Only 1 patient was HIV positive (0.6%), 88 were HIV negative (55%) and HIV status was unknown in 71 (44.4%).There was no significant difference in pleural fluid ADA level between genders. The Chinese appeared to have a lower mean pleural ADA compared to the Indians and other races, with ADA of 52 ± 48 IU/L, 94 ± 47 IU/L, and 110 ± 48 IU/L respectively. However, the difference in ADA levels seen between the racial groups was likely due to the lower proportion of TPE in Chinese compared to the Indians and other races . This explanation was supported by subgroup analyses of TPE and non TPE patients which did not reveal any difference in ADA levels between the racial groups. There was a moderate negative correlation between age and pleural ADA, r = −0.621, p < 0.001, indicating that pleural fluid ADA decreases with age. There was also a moderate positive correlation between ADA and pleural protein, r = 0.556, p < 0.001. Weakly positive correlations were seen between pleural fluid ADA and pleural lactate dehydrogenase (LDH), r = 0.427, p < 0.001, and pleural absolute lymphocyte count, r = 0.314, p < 0.001. Peripheral blood white cell counts and lymphocytes count had negligible correlation with ADA with r = −0.177, p = 0.025 and r = 0.201, p = 0.011 respectively.We proceeded to analyse pleural fluid ADA levels in the TPE and non TPE groups as shown in Table Multivariate linear regression analysis was performed and we found that the independent predictors of pleural fluid ADA were age, pleural fluid protein, LDH, and absolute lymphocyte count.The receiver operating curve (ROC) for ADA was performed for our study population. The area under curve (AUC) was 0.941 (95% CI 0.899-0.983)  Figure . A pleurOur study suggests that patient and pleural fluid characteristics need to be considered during interpretation of pleural fluid ADA as it decreases with age and increases with pleural fluid protein. This is also, to the best of our knowledge, one of the first few studies to determine the ADA level for diagnosis of TPE according to different age groups: 72 IU/L in those age ≤ 55 yrs and 26 IU/L in those > 55 yrs.This study adds to a relatively small number of papers published on pleural fluid ADA performed on an Asian population. Contrary to the study by Niwa et al.  which shFew studies have looked at the factors affecting pleural ADA. In our study age had the strongest correlation with pleural fluid ADA. We noted that the correlation between age and ADA was much weaker when examined within the subgroup of TPE patients. One possible explanation is that the number of older patients aged > 55 years was relatively small in the TPE group resulting in a less representative population. The relationship between age and ADA demonstrated in our study was similar to that of Yeon et al. from Korea . Their sLee et al.  examinedADA has greatest activity in lymphoid tissues and is responsible for the differentiation of lymphoid cells. There are 2 isoenzymes, ADA1 and ADA2, with ADA2 found only in monocytes and macrophages. The high total level of ADA in tuberculous pleural effusion is due largely to high ADA2 activity . There iOne clinical application of our study’s findings would be the interpretation of pleural fluid ADA according to patient characteristics. Pleural fluid ADA decreases with age and therefore increases the number of ‘false negative’ results for diagnosis of TPE when a fixed cut-off level is used in an older population compared to a younger population. In our study, if the widely accepted standard ADA cut-off level of 50 IU/L was used, 5 of the 19 patients (26.%) with TPE in age group > 55 yrs would have a false negative result. If the cut-off level of 26 IU/L was used, only 1 patient (5.%) would have a false negative ADA result. Similarly, caution might have to be exercised in excluding a diagnosis of TPE based on low ADA level if the pleural protein and LDH are also low.Limitations of the study include its retrospective nature but selection bias in this study would have been limited as all patients in our institution investigated for pleural effusion would have pleural fluid ADA performed. The etiologies of 3 of the pleural effusions were undetermined but none had an eventual diagnosis of TPE.In summary, we have demonstrated that pleural fluid ADA is affected by age and pleural fluid protein. Using a lower ADA level to exclude TPE in an older population can reduce the number of false negative results, and based on the pleural fluid ADA results tuberculous treatment may potentially be instituted early. Similarly, a higher ADA level in younger patients would improve the accuracy of TPE diagnosis. Future large prospective studies would be needed to validate the above findings.ADA: Adenosine deaminase; AUC: Area under curve; LDH: Lactate dehydrogenase; NPV: Negative predictive value; PPV: Positive predictive value; ROC: Receiver operating characteristics; TB: Tuberculosis; TPE: Tuberculous pleural effusion.The authors declared that they have no competing interests.TR contributed to study conception and design, data analysis and interpretation, as well as article drafting and revision, and served as principal author. AT contributed to study design, data interpretation, article revision and review of the final manuscript. Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/546/prepub


Standard anaesthetic practice in the high-risk surgical patient is to insert invasive arterial and central venous catheters and then to use ΔCVP and ΔMAP to guide fluid therapy, despite an accumulation of evidence to suggest that filling pressures are inadequate predictors of fluid status and responsiveness. Recent interest has been directed towards dynamic measures of cardiac filling such as SVV, SPV, PPV and Δdown and ΔVpeak. A number of large multicentre trials are underway using the LiDCOrapid. There is, however, little information about the utility of this device or, indeed, any other minimally-invasive cardiac output monitor in the prediction of fluid responsiveness.The haemodynamic parameters of 70 high-risk patients  undergoing major vascular surgery (mean duration 4.2 ± 1.1 hours) were evaluated retrospectively using LiDCOviewPro. All patients underwent standard induction and maintenance of anaesthesia, with propofol/remifentanil TIVA and IPPV  via a supraglottic airway. Monitoring included BIS, NICO and LiDCOrapid. Fluids were administered according to clinical assessment of need and available haemodynamic parameters. Only fluid boluses given in the absence of HRV >10%, brisk ongoing blood loss and of volume ≥250 ml were included in the evaluation. Positive response to a fluid challenge was defined as ΔSVI ≥10%. Statistical analysis was performed using SPSS 17.0.P = 0.08), -0.01 and 0.18 (nonsignificant). The AUROCs were 0.75 (95% CI = 0.57 to 0.93), 0.587 (0.36 to 0.82) and 0.67 (0.48 to 0.86), respectively. The best cut-off value for SVV using Youden's index was 13.5%, with J = 0.48. The positive likelihood ratio was 2.74 and the negative likelihood ratio 0.34, with diagnostic odds ratio 8.06 at this level.Thirty-two out of 43 valid fluid challenges were positive (74.4%). The correlation coefficients between the baseline SVV, SPV and PPV with ΔSVI were 0.27 (It has been reported that only 50% of critically unwell patients respond to fluid challenge, compared with 74.4% in this intraoperative study of noncardiac surgical patients. The SVV was an adequate predictor of fluid responsiveness. The diagnostic threshold of 13.5% was consistent with previous studies.


Neurofibromas are uncommon benign tumours and are still rarer in intramuscular locations. They are not detected until they cause a significant damage to the neighbouring tissues. We present a case of a giant intramuscular myxoid neurofibroma of the left forearm which eroded the radius and ulna, restricting the movements at the elbow and wrist joints and causing wrist drop resulting in an above elbow amputation. It was diagnosed by histopathology and was later confirmed by immunohistochemistry. Neurofibromas are tumours of the nerve sheath origin and may arise from the Schwann cells, perineural cells, and fibroblasts. Intramuscular neurofibromas are rare  and not We present this case because being benign in nature, due to its giant size and location, this tumour caused complications like wristdrop, erosion of the bones and resulted in an above the elbow amputation. Here we present a case of 70 yrs male who presented with a painless swelling of the left forearm for the past one year, which incapacitated the movements at the elbow and wrist joints and resulted in wrist drop.On clinical examination, the tumour was deep seated and the X-ray showed erosion of the radius and ulna. A clinical diagnosis of a malignant soft tissue tumour was made.The patient was thoroughly examined for the clinical manifestations of neurofibromatosis. There were no “cafe au lait” spots, two or more neurofibromas, Lisch nodules, axillary or inguinal freckling, sphenoid wing dysplasia or thinning of the cortex of long bone, and optic glioma. At least two or more lesions are required for the diagnosis of neurofibroma . An above elbow-amputated specimen of the left upper limb was received. The dissection showed a glistening white tumour located within the forearm muscles measuring 13.5 × 10.5 cms in association with the median nerve. Cut section was ivory white and glistening with myxoid areas.Histopathological examination showed a tumour with the cells arranged in lobules of anastamosing cords, strands, and nests against a background of myxoid material and intervening fibrous septae. The cells were spindle to elongated with scanty cytoplasm and showed elongated wavy nuclei. No mitoses were seen. Areas of haemorrhage were noted. A diagnosis of myxoid neurofibroma was made. Immunohistochemistry showed expression of S100P by the tumour cells.Neurofibromas (NF) are rare tumours  and are Aggressive angiomyxoma are perineal or pelvic in location, shows prominent medium to large vessels.Low-grade fibromyxoid sarcoma shows alternating fibrous myxoid patterns with swirling and whorled appearance.Myxoid liposarcoma shows arborising vascular pattern, signet ring type of lipoblasts, and are more cellular.Nodular fasciitis moderately cellular with undulating bundles of cells, tissue culture appearance with tears, and spaces in the tissue.Synovial sarcoma with monophasic or biphasic type and myxoid areas.Cellular myxoma showing moderate cellularity (20%), arching blood vessels, and collagenous stroma.Chordoma which shows physaliphorous cells. Schwannoma shows spindle cells with Antony A and Antony B areas.Histochemical and immunohistochemical stains are useful in the final diagnosis [The other differential diagnoses are the following.iagnosis . Myxoid neurofibroma (MN) is a benign tumour of perineural origin, which is demonstrated by a positive immunohistochemical staining for S100 protein. The most common locations are the face, shoulder, anus, periungal, and in the feet. Only one case occurring in the trunk is reported .The presence of mitotic activity in neurofibroma is indicative of malignancy and is seen even in schwannoma .One case each of a giant genitourinary plexiform neurofibroma associated with lower limb gigantism , periung


Vitiligo is a chronic depigmented skin disorder with regional melanocytes depletion. The pathogenesis was not completely clarified. Recently, more and more evidence suggested that polymorphisms of some genes are associated with vitiligo risk. Here, we want to examine the association between the inducible nitric oxide synthase (iNOS) gene polymorphisms and the risk of vitiligo in Chinese populations.iNOS gene were genotyped by using the PCR-restriction fragment length polymorphism (PCR-RFLP) and mutagenically separated PCR (MS-PCR) methods, respectively. We found the iNOS-954 polymorphism was associated with a significantly higher risk of vitiligo . Furthermore, this association is more pronounced in vulgaris vitiligo, active vitiligo and vitiligo without other autoimmune diseases in the stratification study. Analysis of haplotypes showed increased risk for the C-1173C-954ExC16+14 . In addition, the serum iNOS activity is significantly associated with iNOS-954 combined genotype (GC+CC) and is much higher in vitiligo patients than in the controls (P<0.01). Logistic regression analysis of iNOS activity showed increased risk between higher activity and iNOS-954 G→C variant genotype carriers (trendP<0.001).In a hospital-based case-control study of 749 patients with vitiligo and 763 age- and sex-matched healthy controls, three polymorphisms of INOS gene polymorphisms may play an important role in the genetic susceptibility to the development of vitiligo. Vitiligo is an acquired depigmentary disorder characterized by the appearance of white patches resulting from the loss of functional melanocytes and melanin from the skin and mucous. It affects 0.1–2% of the world population 2 and NADPH. NOS family consists of three isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) et al.Nitric oxide (NO), a short-lived, free radical gas, act as an intercellular messenger in most or all mammalian organs, playing a major role in diverse physiologic processes and pathologic conditions iNOS gene is located on the chromosome 17q11.2–q12, has a genomic size of 48 kb, and encodes a protein of 131 kDa iNOS that have been reported previously, the promoter polymorphisms iNOS-1173 C→T and -954 G→C have been shown to modify iNOS transcription and mRNA levels C-to-T transition at exon 16 was located only six amino acids N-terminally from the deletion reported by Daff The human iNOS gene may be associated with the risk of vitiligo. However, to date, there has been no research about the polymorphisms of iNOS gene in the etiology of vitiligo. We genotyped the three reported functional single nucleotide polymorphisms of the iNOS gene  to test this hypothesis in our ongoing hospital-based, case-control study on vitiligo in a Chinese population.Considering the important role that iNOS plays in the production of NO and, thus, in the pathogenesis of vitiligo, we hypothesized that the functional polymorphisms in the This study included 749 Han Chinese patients with vitiligo and 763 age- and sex-matched controls. Because genotype frequencies can vary among ethnic groups, only Han Chinese patients (more than 90% of population of China) and controls were included in this analysis. All the patients had not subjected to any therapy in recent 6 months. All the healthy subjects did not show clinical evidence or family history of vitiligo or of any other autoimmune disorder. Vitiligo was clinically characterized in patients as segmental and non-segmental iNOS-954 G→C and Ex16+14 C→T polymorphisms were determined using the PCR-RFLP method, whereas the iNOS-1173 C→T polymorphism was carried out using MS-PCR. Genomic DNA was prepared from venous blood samples using a DNA extract kit . DNA purity and concentration were determined by spectrophotometric measurement of absorbance at 260 and 280 nm. PCR was used to amplify the fragments that contained the select iNOS polymorphic sites. The primers of iNOS-954 G→C were as follows: 5′- CATATGTATGGGAATACTGTATTTCAG -3′ (forward) and 5′- TCTGAACTAGTCACTTGAGG -3′ (reverse), as previously reported Ex16+14 C→T was amplified with following primers: 5′- CATATGTAAACCAACTTCCGTG -3′ (forward) and 5′- GGCAGGGCTAGGAGTAGGAC -3′ (reverse), as previously reported iNOS-1173 C→T are as follows: 5′- GACAAGAAGGAAATGAGTGGACACAGGTAGCAAAGTGTTGAGAC -3′ (MS-P2F), 5′- GCATTTTTCCATCATAAAAGTAA -3′ (MS-P3R) and 5′- GTGGTAGCAAATGTTGGAAT -3′ (MS-P4F), as previously reported iNOS-954 G→C and Ex16+14 C→T polymorphisms were 573 bp and 219 bp, respectively. Then we used BsaI and Tsp509I restriction enzymes  to delineate the iNOS-954 G→C and Ex16+14 C→T polymorphisms, respectively. Cuts by these enzymes resulted in 573-bp and 446-bp fragments in the case of the iNOS-954 G allele, 175-bp and 44-bp fragments in the case of the iNOSEx16+14 C allele. The amplified PCR products of the iNOS-1173 C and iNOS-1173 T alleles were 131 and 102 bp, respectively. About 10% of the samples were randomly selected and genotyped again with the same method to test the discrepancy rate, and the results were 100% concordant. In addition, every genotype was sequenced to confirm its authenticity.Genomic DNA was extracted from cell pellets by using a blood genomic DNA purification kit . The We used computer-generated random numbers to assign 89 vitiligo patients from experimental group and 89 normal samples from control group for serum iNOS activity analysis. The statistical analysis of the epidemiologic feature and genotype frequency between the selected 89 vitiligo patients and experimental group did not show significance. The unit of serum NOS activity is defined as: one milliliter serum generates 1 nmol NO per minute is defined as one unit of activity. NOS has three major types: neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible (iNOS). nNOS' and eNOS' existence is depended on the calcium and resided in the neuron and the endotheliocyte; however, iNOS resided in the macrophage and its existence has no relationship with the calcium. According to this principle, we could type the NOS easily. Here, we used the NOS examining kit  to detect the serum iNOS activity in differential samples, the experimental process was performed according to the instruction manual and previously reported iNOS polymorphisms and the serum iNOS activity between the vitiligo cases and controls. Unconditional univariate analyses and multivariate logistic regression analyses were performed to obtain the crude and adjusted odds ratios (ORs) for the risk of vitiligo as well as the 95% confidence intervals (CIs). The multivariate adjustment included age and gender. The D′ value for linkage disequilibrium between the three iNOS polymorphisms and the haplotypes frequencies were calculated by SHEsis  iNOS polymorphisms was evaluated by using multivariate logistic regression models. Statistical significance was established at a P value of 0.05. Two-side tests of statistical significance were performed by using the SAS software .The chi-square test was used to evaluate the differences in frequency distribution for the selected demographic variables, including each allele and genotype of the P = 0.703). The frequency distributions in men and women were 55.3 and 44.7%, respectively in the cases, and 54.1 and 45.9%, respectively in the controls (P = 0.675). Among the cases were 43 (5.8%) segmental vitiligo and 706 (94.2%) nonsegmental vitiligo patients and 623 (83.2%) active and 126 (16.8%) stable vitiligo patients. One hundred and twenty eight (17%) vitiligo patients were considered to have a family history and 28 (4.2%) cases have other auto-immune diseases. Patients were considered to have early onset vitiligo  if the age of onset was prior to 20 years old The frequency distributions of selected characteristics of the controls and vitiligo patients are summarized in 2: P = 0.877 for iNOS-1173 C→T; P = 0.133 for iNOS-954 G→C; P = 0.080 for iNOSEx16+14 C→T). The differences in iNOS-1173 and Ex16+14 genotyping distributions between vitiligo cases and controls were not statistically significant . Similarly, the iNOS-1173 T allele and Ex16+14 T allele did not differ significantly between cases and controls . However, the iNOS-954 variant C allele frequency was significantly higher among cases than among controls , and the frequency of the iNOS-954 (GC+CC) combined genotype was also significantly higher in cases than in controls .As shown in iNOS-954 GG genotype as the reference group, we found a statistically significant increased risk of vitiligo was associated with the -954 combined (GC+CC) genotype . However, when iNOSEx16+14 CC and iNOS-1173 CC were used as reference groups, no significant risk was associated with any other genotypes.When we used the iNOS polymorphisms on vitiligo, we performed an additional stratification analysis with the dichotomized genotypes groups. We observed significant association with iNOS-954 combined (GC+CC) genotype in subgroups that were with active vitiligo , that were diagnosed with nonsegmental vitiligo  and that had no other autoimmune diseases  .To investigate the effects of 04–1.85) . We alsoiNOS gene were in Linkage Disequilibrium . There were eight possible iNOS haplotypes derived from the known genotypes, whereas those frequency<0.03 were ignored in analysis . As shown in iNOS C-1173C-954ExC16+14 haplotype frequency was significantly different between vitiligo cases and controls . Moreover, the haplotype C-1173C-954ExC16+14 was associated with a significantly increased risk of vitiligo .The Linkage Disequilibrium tests showed that the three polymorphisms of iNOS, we further evaluated the interaction among the iNOS polymorphisms and the association between the combined genotypes of iNOS-1173, -954, and Ex16+14 and the risk of vitiligo, with the multivariate logistic regression models. Firstly, we analyzed any two of the three polymorphisms. We found the interaction between iNOS-1173, -954, and Ex16+14 was not statistically significant . We next analyzed the combined genotypes of the three iNOS polymorphisms, but we found no statistically significant relationship between any two of the three polymorphisms or among the three iNOS polymorphisms (data not shown).Considering the potential joint effects of the three polymorphisms of vs.13.29±2.45 U/ml; u = 6.58, P<0.01) , we only examined the serum iNOS activity in iNOS-954 protective genotype GG group with the risk genotype (GC+CC) group. Our results showed that compared with the iNOS-954 protective genotype GG group, the risk genotype (GC+CC) group has the higher serum iNOS activity   . Further P<0.01) .Ptrend<0.001).As shown in iNOS-954 genotypes by iNOS activity  and 1–2 risk genotypes (-954 GC or -954 CC). When the -954 GG was used as the reference, the individuals with 1–2 risk genotypes (-954 GC or -954 CC) and higher iNOS activity (≥14.05) showed more increased risk of vitiligo . When we divided the iNOS activity into three decile according to the controls' activity, the -954 GC or CC genotype combined higher iNOS activity was associated with increased risk of vitiligo .Furthermore, we estimated the risk of vitiligo associated with activity . We diviiNOS-1173 C→T, -954 G→C, and Ex16+14C→T polymorphisms with risk of vitiligo in Han Chinese populations. We found that compared to the iNOS-954 GG genotype, the iNOS-954 combined (GC+CC) genotypes were associated with an increased risk of vitiligo. The association was more pronounced in patients with active vitiligo, nonsegmental vitiligo and vitiligo without other autoimmune disorders. This suggests that iNOS-954 gene polymorphism may have a greater effect on these subgroups. The differences maybe due to the reduction in the number of observations in some strata or may reflect true susceptible subgroups. Further exploration is needed to determine whether the effects found here are an aberration, and what the exact mechanisms underlying such effects are.In this hospital-based case-control study, we investigated the associations of the iNOSEx16+14 T allele frequencies were similar to the frequencies in other Chinese controls and Japanese iNOS-954 C allele frequency was lower in Han Chinese than in African iNOS-954 polymorphism vary with ethnicity.Compared with the previously published data, the iNOS-954 polymorphism is located in the iNOS promoter region, which could be an activator of gene expression INOS-954 C allele, but not the iNOS-954 G allele, may have a higher affinity to a DNA binding protein, thus, transcriptional activity and mRNA levels were increased in the iNOS-954 C allele. Then the NOS activity and NO production were increased. Our result certified iNOS-954 C allele can increase iNOS activity.The iNOS polymorphisms and vitiligo in the Han Chinese population. An early event in the onset of vitiligo appears to involve the overproduction of tetrahydrobiopterin, which leads to the accumulation of a potent inhibitor of melanin biosynthesis de novo attachment of melanocytes to the extracellular matrix components. Moreover, increased iNOS activity induces NO production and O2−, which result in the accumulation of hydrogen peroxide. High of hydrogen peroxide can lead to melanocytes destruct and depigmentation ultimately To the best of our knowledge, this is the first study on the association between the iNOS polymorphisms may influence the risk and clinical progression of vitiligo in Han Chinese populations. A statistically significantly increased risk of vitiligo was associated with the iNOS-954 (GC+CC) genotype compared with the -954 GG genotype, which was more pronounced among vitiligo patients with the following characteristics: non-segmental, active vitiligo and without other autoimmune diseases. But no evident risk was associated with the iNOS-1173 and Ex16+14 polymorphisms. Furthermore, we found the serum iNOS activity was significantly higher in vitiligo and was increased in iNOS-954 combined genotype (GC+CC) compared with -954 GG genotype. Nevertheless, better-designed and larger prospective studies are needed to confirm these findings, and more detailed environmental exposure data are necessary to further test potential gene-environment interactions.In summary, we provide evidence that


We report the first laboratory-confirmed human infection with O'nyong-nyong virus in Chad. This virus was isolated from peripheral blood mononuclear cells of a patient with evidence of a seroconversion to a virus related to Chikungunya virus. Genome sequence was partly determined, and phylogenetic studies were conducted. Rickettsia typhi, R. conorii, Legionella pneumophila, Bordetella pertussis, HIV, and human herpesviruses 1 and 2. Results of malaria testing were also negative. The patient received intravenous acetaminophen for 2 days, according to the protocol used by the French Armed Forces Medical Service in the event of fever occurring overseas. He recovered after 5 days without sequelae.On November 2, 2004, a febrile 19-year-old French soldier staying in Chad and returning from a mission in Sarh, in the southern part of the country, was admitted to the hospital. Clinical examination showed a high body temperature (38°C), rash, periocular erythema, and pharyngitis. Abdominal, cardiopulmonary, and neurologic functions were normal. Except for body temperature, biochemical and hematologic values were normal. Serologic results were negative for Serum samples, collected during the acute phase  and after  were transported to our laboratory and tested by ELISA for immunoglobulin M (IgM) and IgG antibodies to a battery of arboviruses by IgM-antibody capture (MAC-ELISA) and antigen-capture ELISA, respectively . The IgMAedes albopictus) and Vero (E6 clone) monolayers. After 5 days, supernatants were collected and used to infect fresh cell cultures. After 2 days, cytopathologic effects were observed in Vero monolayers; a high level of cell death was also observed in C6/36 cells. Infected Vero and C6/36 cells were then examined by indirect immunofluorescence assay (IFA) for 8 different alphaviruses with alphavirus-specific antibodies and in-house mouse hyperimmune ascitic fluids to CHIKV, Mayaro (MAYV), Tonate (TONV), Semliki Forest (SFV), and Sindbis (SINV) viruses. Results of IFA were positive when alphavirus-specific antibodies and antibodies to CHIKV, MAYV, and TONV were used; no fluorescence was observed when antibodies to SFV and SINV were used at the dilution 1:200.Results of CHIKV-specific real-time reverse transcription–PCRs (-130).CHIKV-specific real-time RT-PCRs were negative when cell culture supernatants were used as samples; this result excluded CHIKV as the etiologic agent. We next used cM3W and M2W2 alphavirus-specific primers . RT-PCR products  were sequenced  and compared with ONNV sequences available on GenBank database; alignments were performed with ClustalW 1.7 software. Comparison of partial sequences showed a high degree of homology between the virus we isolated in Chad and strains previously isolated. In all 4 regions sequenced, paired identity at the nucleotide and amino acid level ranged from 92% to 98% and from 95% to 98%, respectively. Compared with ONNV isolate Gulu, the nsP3 sequence of our isolate featured a glycine-encoding codon deletion . This deletion was also observed in ONNV strains SG650 and IbH10964, which might indicate a common lineage.Four phylograms were constructed, each based on 1 genomic region we sequenced. Among ONNV sequences, all 4 phylograms exhibited a similar pattern: ONNV isolate IbH 10964 (Nigeria) and ONNV strain SG650 (Uganda) seemed to be closely related, whereas isolates Gulu (Uganda) and IMTSSA/5163 (Chad) were placed in 2 different branches . The phylogenetic tree based on E1-encoding sequence (1080-nt long) gave the opportunity to include 2 other ONNV sequences .Togaviridae, genus Alphavirus) was first isolated from human blood and anopheline mosquitoes in Gulu, Uganda, in 1959 (,Anopheles funestus and A. gambiae have been reported in East Africa (,,ONNV (family Anopheles spp. in this region. Patient infection occurred outside any reported outbreak involving ONNV or another arbovirus. Moreover, the mission involved 9 other French soldiers whose serum specimens, collected a few weeks after their return from Sarh and transported to our laboratory, did not show serologic evidence of infection with an alphavirus.The Sarh region, where the patient was infected, consists predominantly of plains covered with a mixture of grasses and woodlands. This region receives heavy rainfall during the 6-month rainy season, from May to October. To our knowledge, no recent data are available concerning the presence of SFV antibodies failed to detect ONNV by IFA, although both viruses are members of the same antigenic complex (In the absence of virus isolation, the diagnosis of infections with ONNV is difficult because of the close antigenic relationship of this virus with other alphaviruses, especially CHIKV. Development of a specific serologic assay for ONNV within the SFV antigenic complex would be a valuable tool for diagnosis and surveillance studies.


It is a widely accepted fact that severe fluid loss is the greatest problem faced following major burn injuries. Therefore, effective fluid resuscitation is one of the cornerstones of modern burn treatment. The aim of this article is to review the current approaches available for modern trends in fluid management for major burn patients. As these current approaches are based on various experiences all over the world, the knowledge is essential to improve the status of this patient group. Appropriate fluid management of major burns directly improves the survival rates of burn patients. Despite the vast array of experience, there are still controversies regarding the best type of fluid management in major burns in the first 24 hours after injury. Currently, fluid resuscitation formulas which were developed over 30 years ago, have been accepted as guidelines, but ongoing studies are focussed on the growing concerns that burn patients are being over-or under fluid-resuscitated, often with indistinct and inappropriate end-point targets. The aim Major burn injuries result in an area of necrotic zone, beneath this lies the zone of stasis and results in release of inflammatory mediators  that increase capillary permeability and lead to localised burn wound oedema.3 This oc27As mentioned above, during this early period in which various pathopysiological changes take place, appropriate fluid management plays a fundamental role.The goal of fluid management in major burn injuries is to maintain the tissue perfusion in the early phase of burn shock, in which hypovolemia finally occurs due to steady fluid extravasation from the intravascular compartment.Burn injuries of less than 20% are associated with minimal fluid shifts and can generally be resuscitated with oral hydration, except in cases of facial, hand and genital burns, as well as burns in children and the elderly. As the total body surface area (TBSA) involved in the burn approaches 15–20%, the systemic inflammatory response syndrome is initiated and massive fluid shifts, which result in burn oedema and burn shock, can be expected. The route for fluid management is of importance in these instances. Although enteral resuscitation has been attempted for even major burn injuries, vomiting has been a limiting problem for this route. Current 10Formal fluid resuscitation formulas which were introduced in the 1960s and 1970s have been used effectively all over the world. The “ParIn centres experienced with paediatric burns, formulas which are sufficient for paediatric fluid management have been developed, as the body surface area to mass ratio in children is higher than in adults and hepatic glycogen stores in young children are depleted after 12–14 hours of fasting.16Baxter found that patients with inhalation injury required additional fluid when compared to others. Pruitt r19The most commonly used formulas are the Parkland, modified Parkland, Brooke, modified Brooke, Evans and Monafo’s formulas. These formulas take into account the body weight and the burn surface area. Several 17Initial 24 hours: Ringer’s lactated (RL) solution 4 ml/kg/% burn for adults and 3 ml/kg/% burn for children. RL solution is added for maintenance for children:4 ml/kg/hour for children weighing 0–10 kg40 ml/hour +2 ml/hour for children weighing 10–20 kg60 ml/hour + 1 ml/kg/hour for children weighing 20 kg or higherThis formula recommends no colloid in the initial 24 hours.Next 24 hours: Colloids given as 20–60% of calculated plasma volume. No crystalloids. Glucose in water is added in amounts required to maintain a urinary output of 0.5–1 ml/hour in adults and 1 ml/hour in children.Initial 24 hours: RL 4 ml/kg/% burn (adults)Next 24 hours: Begin colloid infusion of 5% albumin 0.3–1 ml/kg/% burn/16 per hourInitial 24 hours: RL solution 1.5 ml/kg/% burn plus colloids 0.5 ml/kg/% burn plus 2000 ml glucose in waterNext 24 hours: RL 0.5 ml/kg/% burn, colloids 0.25 ml/kg/% burn and the same amount of glucose in water as in the first 24 hoursInitial 24 hours: No colloids. RL solution 2 ml/kg/% burn in adults and 3 ml/kg/% burn in childrenNext 24 hours: Colloids at 0.3–0.5 ml/kg/% burn and no crystalloids are given. Glucose in water is added in the amounts required to maintain good urinary output.First 24 hours: Crystalloids 1 ml/kg/% burn plus colloids at 1 ml/kg/% burn plus 2000 ml glucose in waterNext 24 hours: Crystalloids at 0.5 ml/kg/% burn, colloids at 0.5 ml/kg/% burn and the same amount of glucose in water as in the first 24 hoursMonafo recommends using a solution containing 250 mEq Na, 150 mEq lactate and 100 mEq Cl. The amount is adjusted according to the urine output. In the following 24 hours, the solution is titrated with 1/3 normal saline according to urinary output.The formulas developed for children are as fInitial 24 hours:For older children:2 total Lactated Ringer’s (RL) solution 4 ml/kg/% burn +1500 ml/mFor younger children:2 total, in the first 8 hours4 ml/kg/% burn +1500 ml/m3RL solution + 50 mEq NaHCORL solution in the second 8 hours5% albumin in LR solution in the third 8 hours2 burn + 2000 ml/m2 total Initial 24 hours: RL 5000 ml/mThe ideal burn resuscitation is the one that effectively restores plasma volume, with no adverse effects. Isotonic crystalloids, hypertonic solutions and colloids have been used for this purpose, but every solution has its advantages and disadvantages. None of them is ideal, and none is superior to any of the others.D-lactate in RL solution containing a racemic mixture of the D-lactate and L-lactate isomers has been found to be responsible for increased production of ROS.[in vivo dilution with crystalloids  resulted in a hypercoagulable state.[Crystalloids are readily available and cheaper than some of the other alternatives. RL solution, Hartmann solution (a solution similar to RL solution) and normal saline are commonly used. There are some adverse effects of the crystalloids: high volume administration of normal saline produces hyperchloremic acidosis, RL incren of ROS. RL used le state.–41Despite these adverse effects, the most commonly used fluid for burn resuscitation in the UK and Ireland is Hartmann’s solution . Another et al. were able to achieve effective physiological resuscitation with a lower total volume when compared to isotonic solutions in the initial 24 hours.[et al. found that after 48 hours cumulative fluid loads of the patient groups who were treated with hypertonic solutions or RL were similar. They also demonstrated that hypertonic sodium solution resuscitation was associated with an increased incidence of renal failure and death.[The importance of sodium ions in the pathophysiology of burn shock has been emphasised in some previous studies. The sodium shift into the cell results in cellular oedema and hypo-osmolar intravascular fluid volume. Rapid infusion of hypertonic sodium solutions has proven to increase the plasma osmolality and limit cellular oedema. Using solutions with a concentration of 250 mEq/l, Moyer 24 hours.29 But Hund death. Currentlet al., found that albumin extravasation stops 8 hours after injury. According to Demling, capillary leakage of protein ceases significantly about 12 hours following the burn.[et al. recently showed that endothelial dysfunction and capillary leakage are present within 2 hours after burn injury and last for a median of 5 hours, much shorter than that previously described.[et al. have recently demonstrated decreased mortality in patients who received albumin. Additionally, some burn clinicians reported successful resuscitation including albumin in the early post burn period with decreased volume requirements and low weight gain compared with pure crystalloid resuscitation.[et al. demonstrated decreased fluid requirements and lower intraabdominal pressures with use of fresh frozen plasma in the first 48 hours following large burns (>50%).[et al. have found that the addition of colloid to Parkland formula rapidly reduced hourly fluid requirements, restored normal resuscitation ratios, and ameliorated fluid creep.[Leakage and accumulation of plasma proteins outside the vascular compartment contributes substantially to oedema formation. The time at which the protein leakage stops has been found to differ by various authors. Baxter’s early work showed that capillary leak may persist for 24 hours post burn. Carvajalthe burn. Vlachou escribed. Colloidsescribed.48 The cocitation.50 O’Maras (>50%). Most recid creep.In our burn centres located in two different regions of Turkey , we avoid using human albumin solution unless blood albumin levels are under 2 g/dl. If necessary, albumin administration is started at least 5 hours after the injury. The preferred dose of albumin after the first 24 hours is 0.5–1 g/kg/% burn. In the following days, the albumin support is continued until the blood level of albumin is 3 g/dl. But decisions for each individual patient are made according to current data from monitoring parameters such as existence of oedema, urine output, central venous pressure, pulse rate, pulse oximetry, and so on.53in vitro study, we found that the addition of the membrane-stabilising vitamin E, zinc and selenium prevented the increase of acid phosphatase  significantly (P < 0.01).[The membrane lipid peroxidation and ROS are the main components of burn shock. In addition, it is well known that the changed permeability of leukocyte membranes due to thermal injury causes an increase in serum enzyme levels. As such, it has been assumed that membrane-stabilising agents such as zinc, selenium and vitamin E could help in the recovery of burned patients. Antioxid54 < 0.01). In a pro < 0.01). Recently < 0.01).et al. compared burn patient groups treated in 1975–1979 with similar patients treated in 2000.[Opioids have been the mainstay of pain control in burn patients. These drugs have a significant effect on the cardiovascular system. Use of these drugs is associated with decreased blood pressure. In a recent study, Sullivan et al. suggested that measuring the hourly ratio of fluid infusion (ml/kg/% TBSA/kg) and urine output (ml/kg/hour) was an effective means of expressing and tracking fluid requirements.[All resuscitation formulas are meant to serve as guides only. Consequently, fluid management in major burns should be monitored using clinical and laboratory parameters. In severe burns, if peripheral intravenous access cannot be achieved, central venous catheterisation or surgical vascular access must be considered. After the venous line is in place, a urinary catheter and a nasogastric tube should be inserted to control and monitor the patient’s fluid balance. Hypotensirements.The treatment of all patients begins at the time of hospitalisation. Following a routine examination, IV fluid  is administered, and following the results of the electrolyte measurements, provided potassium levels are normal, the solution is changed to Ringer’s lactate. The rate of administration is adjusted according to urine output of at least 50 ml/hour. If the patient is oliguric and acidotic, sodium bicarbonate, 20–40 g of mannitol and 40–100 mg furosemide are given. If the patient still remains oliguric and potassium, blood urea nitrogen, and creatinine levels are rising, peritoneal or haemodialysis using a double lumen subclavian catheter is resorted. We think that this system is very easy to use for both haemodialysis and parenteral nutrition. A urinary catheter and a central venous pressure line are used only in severe cases or if clinical evaluation so indicated.Following initial stabilisation, the patients are taken to the dressing room for re-evaluation, and if necessary, debridement, escharotomy, and fasciotomy are preformed.61 EscharPruitt reported that patients with electrical burns required additional fluid. In our pet al. have also advocated that CVVH is an appropriate tool for treating ARF, with a lower incidence of vascular complications than continuous arteriovenous haemodialysis.[ARF is a severe complication of burns, which occurs in 0.5–30% of burn patients. ARF has 6264dialysis.et al. reviewed recent data in the literature to support an appropriate fluid management in burn patients, but they found that there are insufficient data in the literature for this purpose. So, they recommended a rational approach for the initial treatment of burn patients in the light of their investigations. The following are the practice guidelines for burn shock resuscitation, recommended by the American Burn Association.[Pham ociation.Adults and children with burns greater than 20% TBSA should undergo formal fluid resuscitation using estimates based on body size and surface area burned.Common formulas used to initiate resuscitation estimate a crystalloid need for 2–4 ml/kg body weight/% TBSA during the first 24 hours.Fluid resuscitation, regardless of solution type or estimated need, should be titrated to maintain a urine output of approximately 0.5–1.0 ml/kg/hour in adults and 1.0–1.5 ml/kg/hour in children.Maintenance fluids should be administered to children in addition to their calculated fluid requirements caused by injury.Increased volume requirements can be anticipated in patients with full-thickness injuries, inhalation injury and a delay in resuscitation.The addition of colloid-containing fluid following burn injury, especially after the first 12–24 hours postburn, may decrease the overall fluid requirements.Oral resuscitation should be considered in awake and alert patients with moderately sized burns and is worthy of further study.Hypertonic saline should be reserved for providers experienced in this approach. Plasma sodium concentrations should be closely monitored to avoid excessive hypernatraemia.Administration of high-dose ascorbic acid may decrease the overall fluid requirements, and is worthy of further study.et al. emphasised that the guidelines they had designed could aid especially the physicians who were responsible for the triage and initial treatment of burn patients.[In the above-mentioned study, Pham patients.Several studies have supported that patients who receive larger volumes of resuscitation fluid are at higher risk for injury complications and death. In the light of this prediction, the chosen types and rates of the fluid administration in major burns are at the focus of controversy. It must be kept on minds that these debates look for a rational approach for an adequate fluid resuscitation. Currently used guidelines are based on the various experiences all over the world, and the developing experiences will bring a new approach. So, clinicians must be aware of this vast experience and ongoing literature debates in order to improve the status of this patient group.


These findings reveal a novel autocrine pathway involving VEGF/NRP2, α6β1 and GLI1 that contributes to the initiation of TNBC. They also support the feasibility of NRP2-based therapy for the treatment of TNBC that targets and impedes the function of TICs.The characterization of cells with tumour initiating potential is significant for advancing our understanding of cancer and improving therapy. Aggressive, triple-negative breast cancers (TNBCs) are enriched for tumour-initiating cells (TICs). We investigated that hypothesis that VEGF receptors expressed on TNBC cells mediate autocrine signalling that contributes to tumour initiation. We discovered the VEGF receptor neuropilin-2 (NRP2) is expressed preferentially on TICs, involved in the genesis of TNBCs and necessary for tumour initiation. The mechanism by which NRP2 signalling promotes tumour initiation involves stimulation of the α6β1 integrin, focal adhesion kinase-mediated activation of Ras/MEK signalling and consequent expression of the Hedgehog effector GLI1. GLI1 also induces BMI-1, a key stem cell factor, and it enhances NRP2 expression and the function of α6β1, establishing an autocrine loop. NRP2 can be targeted  The hypothesis that breast tumours harbor a population of cells that can initiate tumourigenesis is supported by strong experimental evidence  and neuropilins (NRPs), another family of VEGF receptors. NRP1 and NRP2 were identified initially as neuronal receptors for semaphorins, which are axon guidance factors that function primarily in the developing nervous system  because they are characterized by a high frequency of TICs   . Importantly, mammosphere formation was inhibited by a NRP2 blocking antibody  and a NRP2 Ab blocked mammosphere formation ), and they develop highly penetrant, metastatic adenocarcinomas with a triple-negative phenotype  at the time of pregnancy, which triggers tumour onset in this transgenic model. As shown in The involvement of NRP2 in tumour initiation was evaluated pulation . A signiow cells . We madehigh and NRP2low populations sorted from SUM1315 cells revealed elevated expression of BMI-1, OCT-4 and SOX-2 in the NRP2high population, although BMI-1 exhibited the largest difference in expression between NRP2high and NRP2low populations  than did the SUM1315 NRP2low population , a negative regulator of GLI1. This possibility is supported by the observations that the regulation of GLI1 by NRPs is dependent on SuFu  to impact established tumours. Treatment of mice harboring orthotopic SUM1315 tumours with Anti-Nrp2B reduced tumour size significantly  was provided by Genentech  and laminin-1 (LN-1 or LN-111) was purchased from Invitrogen ; PF573228 (Tocris). The following Abs were used: α6 ; NRP2 ; actin (Sigma); p-FAK Y397  and FAK (Santa Cruz); EpCAM (AbCaM); Flag (Sigma); ki-67 (NovoCastra); HA (Roche); CD31 (Santacruz); p-ERK, ERK and GLI1 ; BMI-1  and VEGF . A function-blocking NRP2 Ab  for their function. Furthermore, we define a signalling cascade initiated by VEGF/NRP2 signalling that culminates in the regulation of a key stem cell factor BMI-1 that is critical for tumour initiation. This signalling cascade involves the ability of VEGF/NRP2 signalling to activate the a6b1 integrin and, consequently, FAK. FAK-mediated activation of Ras/MEK signalling enhances expression of the Hedgehog effector GLI1 that can induce BMI-1. Importantly, GLI1 also stimulates NRP2 expression, establishing a positive feedback loop that sustains this autocrine pathway. The significance of these data is evidenced by the fact that inhibition of NRP2 prevents tumour initiation and it causes regression of established tumours.The data presented provide a novel mechanism that contributes to the initiation of aggressive breast carcinomas. This mechanism integrates the function of several key molecules that have been implicated in the function of tumour stem cells. Importantly, these findings provide a strong rationale for the use of anti-NRP2 therapy for the treatment of aggressive breast carcinomas.SUM1315, SUM149 and SUM159 cells were provided by Dr. Steve Ethier (Wayne State University School of Medicine). MCF-10A and MCF-10AT were obtained from the Barbara Ann Karmanos Cancer Institute . HMECs were purchased from Lonza Bioscience and HMLE-PR cells were provided by Dr. Weinberg (Whitehead Institute).Lentiviruses containing shRNAs specific for NRP2 , α6 integrin ; GLI1 ; Smoothened (TRCN0000014366 and TRCN0000014367) or GFP (RHS4459) were obtained from Open Biosystems . A lentiviral plasmid (FUGW) expressing BMI-1 was obtained from Addgene. A constitutively active FAK construct was provided by Jun-Lin Guan . Wild-type and dominant negative Ras constructs were described previously  and stem-like (CD44+/CD24−/EpCAM+) populations in Cell adhesion assays were performed as described previously , EGF (20 ng/ml), basic fibroblast growth factor , and heparin (10 µg/ml) in Costar 3471 six-well plates at a density of 2 × 10Cells were extracted in either a Triton-X-100 buffer  or RIPA . The proteins were separated by SDS–PAGE and immunoblotted using Abs as specified in the figure legends. For qPCR, RNA was isolated using RNeasy kit (Qiagen) and converted to cDNA using Transcriptor First Strand cDNA Synthesis kit (Roche). We performed qPCR with a SYBR Green PCR master mix using an ABI Prism 7900HT instrument (Applied Biosystems).PrkdcscidIL2rgtm1Wjl (abbreviated as NSG) mice. Tumour onset was determined by palpation. In some experiments, SUM1315 xenografts were treated intraperitoneally (i.p.) with either control IgG or a NRP2 Ab (Anti-Nrp2B) at dose of 40 mg/kg (twice weekly). In some experiments, NRP2low population of SUM1315 cells expressing either a control vector or GLI1 construct were injected in the mammary fat pad of NSG mice and tumour onset was determined. To study the role of NRP2 in MMTV-Gli tumours, primary tumour cells from MMTV-GLI1 mammary tumours were infected with either NRP2 or GFP shRNAs and injected orthotopically in NSG mice. Tumour onset was used as the endpoint for these studies.SUM1315 cells were mixed with Matrigel at a 10-fold dilution and injected into a mammary fat pad of immunocompromised NOD.Cg-MMTV-PyV-MT mice transgenic for the polyoma virus middle T (PyV-MT) antigen under the control of the MMTV promoter (Jackson Laboratory) were obtained from Dr. Leslie Shaw. Cells from these mice are isolated and maintained as described before  model was generated and described before (Kumar et al, The MMTV-GLI1 transgenic mouse model was generated and described before (Fiaschi et al, in vitro cell experiments; HLG, BP and JHN were involved with in vivo experiments on mouse models; JM assist in GLI1 in vitro experiments; CWVK was involved in peptide design and synthesis. LMS, RT, PK and KS were involved in vivo transgenic mouse models; LDS and DLG were involved in xenograft experiments; HLG, CK and AMM wrote the manuscript. HLG and AMM carried out the data analysis.HLG and AMM designed experiments and interpreted data; HLG, BP and CC carried out


There was an error in funding information regarding the year the PRIN grant was granted to FS. The correct financial disclosure should be: "This work was supported by the National Institutes of Health Conte Center grant MH77970 (M.M.M.), the John C. and Edward Coleman Memorial Fund (M.M.M.), and the Italian Ministry of University and Research, PRIN 2008 (F.S.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."


Tumor initiating cells  are a subset of cells with the ability to self-renew and differentiate into the heterogeneous populations of the original tumour  regulating the biology of tumour initiating cells.Goel et al. have identified a novel cascade downstream of the VEGF receptor Neuropilin-2 (NRP2) regulating the biology of tumour initiating cells . NRP2, ae.g. tumour initiating cells) to defined stimuli (e.g. chemotherapy). Increased understanding of the mechanisms maintaining cancer cell stemness and controlling the differentiation of tumour initiating cells will be important for future translational efforts. In particular, the molecular mechanisms regulating chromatin and DNA-based epigenetics are clinically relevant because drugs targeting different complexes regulating these events are currently undergoing clinical trials (e.g. histone deacetylase or methyl transferase inhibitors; Lee et al, …the discovery of the zinc finger transcription factor GLI1 as a central player in the regulation of stemness should fuel future efforts investigating the epigenetic mechanisms underlying the regulation of tumour initiating cellsCollectively, these findings define several novel mechanistic aspects of the biology of tumour initiating cells as well as provide the foundation for new therapeutic approaches targeting this cellular compartment. However, the study also defines new gaps in our knowledge as well as future directions that investigators in this field of study should consider. First, at the molecular level, the discovery of the zinc finger transcription factor GLI1 as a central player in the regulation of stemness should fuel future efforts investigating the epigenetic mechanisms underlying the regulation of tumour initiating cells, especially their role in cell differentiation, a key cellular process in the biology of these cells. In contrast to genetics, epigenetic processes are highly dynamic, reversible (McCleary-Wheeler et al,


The role that the peritumoral stroma plays in the growth of tumours is currently poorly understood. In this manuscript the morphometric characteristics of basal cell carcinoma subtypes and their associated peritumoral stromas are presented.Ninety eight digitized basal cell carcinoma histology slides were categorized as infiltrative, nodular, or superficial subtypes, and were analysed using a combination of manual and computer-assisted approaches. The morphometric characteristics of the tumour nests and their associated peritumoral stroma were quantified, and the presence of a marked immune reaction or elastosis was noted.The tumour to stroma ratio was different among each tumour subtype. Elastosis was identified in a greater proportion of the infiltrative tumours.Quantitative differences exist between the peritumoral stroma of basal cell carcinoma subtypes. Future work exploring the relation between these morphometric differences and biochemical variations in peritumoral stroma may further our understanding of the biology of carcinoma development.Not applicable. Carcinomas  are accompanied by a specialized peritumoral stroma which plays an important but poorly understood role in the growth and metastasis of the tumour -4. The pModifications to the peritumoral stroma include alterations in the secretion of extracellular matrix proteins and growth factors by fibroblasts. Particularly important are the stromally expressed matrix metalloproteinases (MMPs), which may have wide-ranging effects on tumor growth, angiogenesis and metastasis . A numbeIn addition to the paracrine factors secreted by fibroblasts, inflammatory cells in the peritumoral stroma also secrete growth factors, as well as reactive oxygen species which may further effect cancer growth . LikewisAs a model for the interaction between epithelial tumor cells and stroma, basal cell carcinomas (BCC) offer a number of unique opportunities to study these processes. In addition to being the most common malignant tumor, at least in Caucasians , BCC arePrevious work on BCC peritumoral stroma has defined some qualitative features of this tissue. Work by Humphreys et al. has shown an increased number of mast cells and dermal dendrocytes expressing CD34 and GP1b adjacent to BCC tumor nests, although no differences were noted among tumor subtypes . In contIn addition to biochemical factors, some morphometric characteristics of BCCs have been explored. Studies performed by Dixon et al.  and JacoThe purpose of this study is to employ advanced image analysis techniques to look for patterns in the quantitative relationship between different BCC histology patterns and the BCC peritumoral stroma.Cases were selected from the routine clinical caseload of the senior author. Images of 98 hematoxylin and eosin stained slides were digitized using a commercial Aperio CS-O slide scanner at 80 × magnification, and sections containing BCC were stored as JPEG images (1072 × 902 pixels). To avoid possible confounding effects of biopsy site changes and wound healing on the morphological assessment of tumour subtype ,31, we eThe study protocol was reviewed by the Calgary Laboratory Services Research Committee and deemed to represent a method development project not requiring further ethics approval (CLS Study Code RS-11-504).Printed copies of each of the 98 BCC images were initially evaluated manually. In each BCC image five neoplastic regions of interest (ROI) were identified, and their stromal diameters and tumour radii were delineated. ROIs were selected based on the following criteria: (i) the area must be free of technical artefacts such as variations in staining and cutting of slides and must not include borders of the tissue sample or image, and (ii) ROIs must include only one subtype of tumor . When possible each ROI was selected from a separate tumour nest. Multiple ROIs were identified in the same tumour nest only when the total number of tumour nests was fewer than five. The tumour radii and stroma diameters were measured directly from the printed image for each ROI. A limitation to this approach arose where tumour nests were close together and the intervening space was entirely composed of peri-tumoural stroma. In this instance stroma attributable to each nest was arbitrarily defined as the stroma between them divided by two. Finally, the presence of inflammation or elastosis was noted, and the tumour subtype for each slide was categorized as nodular, superficial, or infiltrative.In addition to the manual evaluation, the open source digital imaging and analysis software ImageJ (version 1.44p) was used to quantify the morphology of the tumour nests in each slide. By measuring the circularity of the tumour nest associated with each of the ROIs, the tumour nest roundness was quantified. The circularity measurement in ImageJ is calculated as follows:A circularity measurement of 1 indicates a perfect circle, while more elongated shapes result in lower values . In ordeImages were classified into tumor subtypes according to the definitions provided by Crowson . The 98 Data analyses were performed using IBM SPSS Statistics v19. Box plots of the morphometric statistics and bar plots of the categorical statistics were created using R v2.13.0. Non-parametric tests were employed, as initial calculations indicated non-normal distributions. An analysis of variance was performed using the Kruskal-Wallis test, and Mann-Whitney tests were employed when significant differences between the groups were found. Categorical variables were analysed using the Chi-squared test. Interaction effects between continuous and categorical variables were tested using binary logistic regression.Examples of the different basal cell carcinoma subtypes included in this study are illustrated in Figure The presence of elastosis was correlated with the tumour subtype (p = 0.033), as eight of the fifteen (53%) infiltrative, twelve of the sixty 20%) nodular, and six of the twenty-three (26%) superficial tumours contained elastosis. The presence of an immune reaction was not found to be significant (p = 0.1). The categorical statistics are summarized in Table 0% nodulaThis study quantified the morphometric relationships between basal cell carcinoma subtypes and their peritumoral stroma. Manual and computer-assisted methods were employed to quantify size and shape statistics for infiltrative, nodular, and superficial BCC subtypes. The presence of inflammation and elastosis was also noted for each subtype.Significant differences were found between the median tumour radii and stroma diameters of the infiltrative and nodular subtypes. The infiltrative subtype was associated with thicker stroma, but smaller tumour radii, with a correspondingly smaller tumour-to- stroma ratio. Therefore the biochemical differences previously described for infiltrative versus other BCC subtypes may be accompanied by quantitative changes in the peri-tumoral stroma.Another noteworthy finding was that elastosis was present in a significantly higher proportion of the infiltrative subtype images. One possible explanation for this finding is that the presence of extensive sun damage contributes to the phenotype of BCC, predisposing to an infiltrative growth pattern. Possible mechanisms for such a relationship are speculative but may include differences in diffusion characteristics or paracrine factor expression in sun damaged skin. Similar arguments could be made for the presence of a chronic immune infiltrate associated with infiltrative BCC: lymphocytes could be recruited in response to some characteristic of the tumor or they may themselves secrete factors that influence the BCC phenotype.Unexpectedly, the circularity of the tumour nest was not found to influence the tumour subtype. This may have resulted from the presence of nodular tumour nests within some of the infiltrating subtype slides, resulting in higher mean and more variable circularity measurements. Alternatively, variance of the circularity measurements among tumor nests on the same slide may provide a means of distinguishing the tumour subtypes based on their shape. Moreover, the use of different shape metrics may provide a superior means of distinguishing tumour subtypes based on their morphology. These questions could be explored in future studies.This study was unique in several ways. First of all, a greater emphasis was placed on the relationship of the tumour morphometry and its stroma. Previous studies have focused largely on the tumour itself, therefore ignoring an important factor in tumour growth. Another benefit was that the shape statistics were quantified using an open-source digital imaging and analysis program. This facilitates replication, as ImageJ is freely available and is capable of providing circularity measurements with the default package. A further benefit of using software to quantify the tumour nest shape is that it eliminates the subjectivity resulting from categorizing the tumour shape qualitatively. Further research could improve upon this study in a couple of ways. One limitation was that rarer BCC subtypes were not included. Also, as mentioned earlier, inherent in an observational study is the fact that it was not possible to determine whether stromal differences influenced the tumour architecture or if they resulted from the architecture itself. Another limitation of this study concerns the applicability of our findings to other malignancies. Basal cell carcinomas differ from many other malignancies in that they exhibit limited metastatic potential and often lack features common to other malignancies such as aneuploidy .Previous studies that have quantified the morphometry of BCC have focused on the relationship of the tumour's characteristics and its aggressiveness. Furthermore, these studies have focused mainly on the tumour, rather than its relationship to its stroma. As a result, it is not possible to directly compare the findings presented here with those of the previous BCC morphometry studies. That aside, previous authors have found that aggressive tumours have different morphometric characteristics than non-aggressive ones. This may be related to the different morphology found between the different subtypes in this study. The results obtained are also consistent with the presence of biochemical differences that were previously reported. As discussed, Humphreys et al.  found thIn this study we demonstrated that there are quantitative differences in the peritumoral stroma among BCC subtypes. Future work should focus on integrating these morphometric differences with the previously described biochemical differences in stromal microenvironments among BCC subtypes.The authors declare that they have no competing interests.CN devised the original study design. KL obtained the morphometric measurements. Both authors participated in drafting the manuscript. Both authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-5945/12/1/prepub


The aim was to evaluate whether breast magnetic resonance imaging (MRI) following mid-cycle chemotherapy was influencing the neoadjuvant chemotherapy (NAC) regimen.Thirty-two patients with breast malignancy receiving NAC were included in this retrospective study. Baseline and mid-cycle MRI were performed. NAC regimen and breast MRI reports were obtained from an online database. Tumour response was assessed by calculating percentage of volume reduction between interval scans. The response was categorised arbitrarily as good, moderate and poor with >50%, 20 to 50% and <20% reduction in tumour volumes respectively.n = 32) had showed good response in 23 (71.9%), moderate response in four (12.5%) and poor response in five (15.6%) on their mid-chemotherapy MRI. The five poor responders had good response in either the axillary node or in the second tumour. The second tumours (n = 9) showed good response in six (66.7%), moderate in two (22.2%) and poor in one (11.1%). No changes were made to chemotherapy regimen based on the MRI findings and these percentage tumour volume reductions.Nine patients had second tumours detected on the baseline MRI. The index tumours (Our study shows that the percentage reduction in the tumour volumes demonstrated by the interval MRI scans are an important determinant of response to NAC. It is possible to obtain similar information from newer 4D sonography, which is cheaper and quicker to perform than MRI, and is better tolerated. In our practice, assessment at the mid-chemotherapy point is now undertaken with clinical assessment and ultrasound, which have provided adequate information regarding tumour responses to chemotherapy.


The following grant information is missing from the Funding Statement:US National Institutes for Health, National Cancer Institute for support (Grant # 1R03CA156667 to RM), the National Institute of General Medical Sciences (Grant #’s GM087221 and 2P50 GM076547 to RM) and the Luxembourg Centre for Systems Biomedicine and the University of Luxembourg (for support to RM).