Fluid overload is a clinical problem frequently related to cardiac and renal dysfunction. The aim of this study was to evaluate fluid overload and changes in serum creatinine as predictors of cardiovascular mortality and morbidity after cardiac surgery.Patients submitted to heart surgery were prospectively enrolled in this study from September 2010 through August 2011. Clinical and laboratory data were collected from each patient at preoperative and trans-operative moments and fluid overload and creatinine levels were recorded daily after cardiac surgery during their ICU stay. Fluid overload was calculated according to the following formula: (Sum of daily fluid received (L) - total amount of fluid eliminated (L)/preoperative weight (kg) × 100). Preoperative demographic and risk indicators, intra-operative parameters and postoperative information were obtained from medical records. Patients were monitored from surgery until death or discharge from the ICU. We also evaluated the survival status at discharge from the ICU and the length of ICU stay (days) of each patient.P = 0.002 and OR 2.91; CI: 95% 1.92 to 4.40, P <0.001, respectively). Fluid overload played a more important role in the length of intensive care stay than changes in serum creatinine. Fluid overload (%): b coefficient = 0.17; beta coefficient = 0.55, P <0.001); change in creatinine (mg/dL): b coefficient = 0.01; beta coefficient = 0.11, P = 0.003).A total of 502 patients were enrolled in this study. Both fluid overload and changes in serum creatinine correlated with mortality (odds ratio (OR) 1.59; confidence interval (CI): 95% 1.18 to 2.14, Although both fluid overload and changes in serum creatinine are prognostic markers after cardiac surgery, it seems that progressive fluid overload may be an earlier and more sensitive marker of renal dysfunction affecting heart function and, as such, it would allow earlier intervention and more effective control in post cardiac surgery patients. Cardiac surgery is the surgical procedure most frequently associated with acute kidney injury (AKI) . Kidney Creatinine is the most widely used marker of kidney function in patients undergoing cardiac surgery. According to Lassnigg, a small increase in serum creatinine 0 to 0.5 mg/dL) has been associated with 30-day mortality  mg/dL ha,7. Due tClassically, creatinine has been used to identify kidney dysfunction; however, serum creatinine may remain within the normal range until about half of the kidney function is lost . Hence, We conducted a cohort study. This study was approved by the Ethics Committees of the Instituto de Cardiologia do Rio Grande do Sul/Fundação Universitária de Cardiologia (IC/FUC), Porto Alegre, RS, Brazil and Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Brazil, and registered under the numbers 4560/10 and 1781/107, respectively. Data were collected anonymously following the clinical routine, with a waiver for informed consent. We conducted a one year cohort prospective study of all patients from the Postoperative ICU who underwent surgery involving cardiopulmonary bypass (CPB) in a tertiary cardiac referral hospital, Instituto de Cardiologia do Rio Grande do Sul/Fundação Universitária de Cardiologia (IC/FUC), beginning September 2010. Inclusion criteria were as follows: adult patients (>18 years old) submitted to the following elective surgical procedures: myocardial revascularization surgery, valve replacement surgery, valve replacement surgery plus myocardial revascularization, atrial septoplasty and ventricular septoplasty. The exclusion criteria were as follows: length of stay in the Postoperative ICU less than 24 hours, loss to follow-up and patients submitted to surgical re-intervention during the same hospital stay.The hypothesis of this study was that fluid overload is independently related to mortality and length of stay in the ICU following cardiac surgery. Thus, the purpose of this study was to evaluate the effects of fluid overload and serum changes in serum creatinine on death and length of ICU stay and mortality in patients who underwent heart surgery, regardless of other perioperative risk factors.We followed the STROBE statement of observational studies.Preoperative demographic and risk indicators, intra-operative parameters and postoperative information were obtained from medical records. The preoperative variables were age (in full years), gender, weight (kg), serum creatinine (in mg/dL), EuroSCORE risk and related diseases. Serum creatinine was measured using the automated colorimetric method , reference values from 0.30 to 1.30 mg/dL. At our hospital, the routine creatinine analysis variation was 1.4%. Cardiac function was quantified using the ejection fraction (%) obtained by the echocardiogram. During the intra-operative period, fluid balance (fluid administered - fluid excreted - in mL) and CPB time (in minutes) were evaluated.During postoperative follow up all available liquid intake and output data from surgery day until ICU discharge or death were included. Fluid balance (fluid administered - fluid excreted - in mL) and changes in serum creatinine were evaluated daily. Fluid management and all other interventions were determined by attending physicians and were not influenced by the study researchers. Patients were monitored from surgery until discharge or death during ICU stay period. We also evaluated the survival status and the total length of ICU stay (in days) of each patient.To assess the impact of fluid accumulation and acute kidney dysfunction on mortality and length of ICU stay, percentage fluid accumulation and changes in serum creatinine were measured and analyzed.For fluid accumulation, we considered the whole length of the ICU stay, including the day of surgery. We measured the 24-hour totals of fluid intake and output daily, including the trans-operative surgical period. In order to calculate daily fluid balance, the following formula was applied daily: Total fluid received (L) - Total amount of fluid eliminated (L).In order to quantify fluid accumulation, the following formula was applied: Daily fluid accumulation sum = [Total quantity of fluid received (L) - total amount of fluid eliminated (L)]/Preoperative weight (kg) × 100. This percentage of fluid accumulation was calculated according to procedures found in previous studies ,16. We uWe also evaluated age, EuroSCORE risk, preoperative creatinine, stratified changes in creatinine , stratified volume accumulation  and CPB time as potential confounding factors. We considered infection, pulmonary edema, bleeding, cardiac arrhythmia and death as combined events in the analysis of the study outcome. We did not analyze the temporal fluctuations of fluid overload in response to diuretics as a marker of mortality.The study size was determined using the computer software Programs for Epidemiologists (PEPI version 4.04x). The program indicated a base of 480 patients for the 5% (α = 0.05) minimal error type I and a 20% probability error type II to estimate the OR value = 3, mortality incidence about 5% when comparing patients with the 0 to 0.5 creatinine change in relation to decreased -0.3 to 0 values, according to Lassnigg[Quantitative data were described using mean and standard deviation or the median and interquartile range in the presence of skewness. Categorical data were expressed using counts and percentages. We excluded patients who died in the trans-operative period and in the first 24 hours after surgery.Volume accumulation and changes in creatinine were transformed to Z scores to analyze the association of unit changes in both variables. We did individual Chi-square tests to identify the potential morbidities. Due to the limited number of deaths, we analyzed the combined morbidities, including mortality, and its association with potentially predictive factors using logistic regression to obtain ORs with their respective 95% CIs.Due to the skewness, the length of ICU stay was log-transformed. For the association of volume accumulation and length of ICU stay we used a linear regression model (r Pearson coefficient). Length of ICU stay was modeled using multiple linear regression to evaluate its relationship to a number of independent factors. In this model, changes in serum creatinine were expressed as Δ creatinine, which was calculated as: Changes in creatinine = Highest serum creatinine value - Baseline creatinine. Standardized beta coefficients were used to obtain adjusted contributions of these factors in the model. Significance level was set at α = 0.05. Data were analyzed using SPSS version 18.0.Five hundred and eighty-six patients were admitted to the postoperative ICU from September 2010 through September 2011. Eighty-four patients were excluded because they had undergone other surgical procedures, surgical reintervention or were lost to follow-up. Five hundred and two patients were prospectively studied  for death at each Z score in fluid overload. For creatinine, we found a significant increase of 2.91 in the OR for death  at every creatinine Z score change (Table P = 0.009), and ≥0.6 mg/dl . When we analyzed the association of fluid accumulation and changes in serum creatinine by Z score variables with mortality, we observed that fluid overload and changes in serum creatinine were significantly associated with mortality. For fluid overload, we found a significant increase of 1.59 in the OR . We also found a significant and independent increase in combined events when the fluid accumulation increased 10%  .e et al.  and founWe have to emphasize that none of the patients who died had a decreased creatinine level. Considering this observation, we did not analyze the group separately. It was possible to demonstrate that of the 157 patients who had decreased creatinine levels, only four (2.5%) presented fluid overload ≥10%.P <0.001). We also found that patients who survived after four days in the ICU had no fluid accumulation . As the majority of patients stayed only two days in the ICU, , we analyzed these patients in relation to the upper percentile (P75). We found that only 66 patients stayed more than four days in the ICU (P75). The outcome of fluid overload is time related , and few patients required dialysis (seven patients). Also, the percentage time of the fluid overload was not considered in this study, a factor which, as is known, may interfere in the length of hospitalization and mortality . TherefoWe did not analyze the mechanisms involved in the development of fluid overload, including the contribution of volume infusion of fluids, the particular type of fluid administered or the absence of response to diuretics. These points need to be addressed in further studies.Other measurements associated with fluid overload, such as central venous pressure or pulmonary capillary pressure, and pro BNP measures could be performed. In fact, these parameters were obtained from several patients, but not in a systematic protocol. Therefore, we did not analyze and present the related data. However, our results strongly indicate that fluid accumulation control and changes in serum creatinine together may represent valuable tools to detect a population with a higher cardiovascular risk among patients treated in a 'real life' scenario.In summary, we showed a significant and independent association between fluid overload and changes in serum creatinine in relation to combined events of death, infection, bleeding, arrhythmia and pulmonary edema in postoperative cardiac surgery. We also found that fluid overload was the variable most related to length of stay in postoperative care following cardiac surgery.As such, our findings contribute towards expanding the knowledge regarding this still unclear field of the functional interaction between the kidneys and the heart. Further randomized and controlled studies are needed to define whether early fluid overload detection would be an unrecognized form of initial renal failure and a target treatment to lower mortality associated with cardiac surgery.Fluid therapy is widely endorsed for the resuscitation of critically ill patients across a range of conditions. Yet, the approach to fluid therapy is subject to substantial variation in clinical practice. Emerging evidence shows that choice, timing and amount of fluid therapy affect outcome. Further studies would need to focus on these aspects of fluid therapy by means of larger, more rigorous and blinded controlled trials.• Fluid overload is independently associated with combined events in post operative cardiac surgery.• Increase in serum creatinine is independently associated with combined events in post operative cardiac surgery.• Fluid overload is substantially more effective in accounting for ICU stay than 0.1mg/dL changes in serum creatinine.• The early identification of fluid overload is essential to establish adequate management in cardiac patients.; OR: odds ratio; P25 to P75: interquartile range.AKI: acute kidney injury; b: angular coefficient obtained in a multiple linear regression model; CI: confidence interval; CPB: cardiopulmonary bypass; Δ variation: ; EuroSCORE: European System for Cardiac Operative RiskThe authors declare that they have no competing interests.AS and MCI conceived the study, participated in study design, prepared data, performed statistical analysis and wrote the paper. LVS, CRB, WRM and JRV participated in study design, prepared data, collected data and wrote the paper. EMCP, RE, LA and FCC contributed in the writing and critical appraisal of the manuscript. All authors read and approved the final manuscript.


Leiomyomas are benign tumours that originate from smooth muscles. They are often seen in the uterus, but also in the renal pelvis, bladder, spermatic cord, epididymis, prostate, scrotum or the glans penis. Leiomyomas of the tunica albuginea are extremely rare.A 59-year-old white male has noted an asymptomatic tumour on the right side of his scrotal sac for several years. This tumour has increased slowly and caused local scrotal pain. An inguinal incision was performed, in which the hypoplastic testis, the epididymis and the tumour could be easily mobilized. Macroscopically the tumour showed a solid round nonencapsulated whorling cut surface. Histologically the diagnosis of a leiomyoma was made.We report here a very interesting and rare case of a leiomyoma of the tunica albuginea. Leiomyomas can be a possible differential diagnosis in this area.http://www.diagnosticpathology.diagnomx.eu/vs/2585095378537599 Leiomyomas are benign tumours that originate from smooth muscles cells and are often found as benign lesions arising in the uterus,2. But tA 59-year-old white male has noted an asymptomatic tumour on the right side of his scrotal sac for several years. Since the size of this tumour has increased and lately sometimes even caused local scrotal pain, he was presented to the clinic of urology.Physical examination revealed a solid tumour, approximately 5 cm in diameter, on the right scrotal side. The testis on this side felt unremarkable, though it seemed to be very small. Inguinal lymph nodes were not palpable.Ultrasound of the scrotum revealed a tumour with both echogenic and cystic areas and a diameter of 4cm on the right scrotal side. The testis was hypoplastic but unsuspicious Figure. WhetherChoosing an inguinal incision, the hypoplastic testis, the epididymis and the tumour could be easily mobilized. The resection of the tumour was accomplished without harming testis and epididymis. Testis and epididymis were replaced into the scrotum. The postoperative course was uneventful.After excision, the tumour tissue was sent to the department of pathology for histological examination. In macroscopical examinantion a solid round nonencapsulated whorling tumour of white colour and a mass of 5 x 3,5x 3.5 cm was seen Figure. The tesLeiomyomas are benign tumours orginate from smooth muscles cells. Three different types respective to their origin are known; (1) derivation of arrector pili muscle (piloleiomyoma), (2) derevation of smooth muscles of blood vessels (angioleiomyoma), and (3) genital leiomyoma (p.e. from the smooth muscles of the scrotum). TumoursThis is an extremely rare and interesting case of a leiomyoma of the tunica albuginea. As far as we know only 5 cases of this tumour of the tunica albuginea have been reported. Leiomyomas in this area increasing slowly and may cause scrotal pain. Leiomyomas can be a possible differential diagnosis in this area.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.FB and FJK drafted the manuscript and were responsible for the interpretation of the data. FB and CLB were responsible for microscopic and histopathologic elements. LT, FK and EH cared for the patients. EH was responsible for the critical revision of the manuscript. All authors read and approved the final manuscript.


Mycobacterium smegmatis, like its slow-growing pathogenic counterpart Mycobacterium tuberculosis, has the ability to adapt to microaerobiosis by shifting from growth to a non-proliferating or dormant state. The molecular mechanism of dormancy is not fully understood and various hypotheses have been formulated to explain it. In this work, we open new insight in the knowledge of M. smegmatis dormancy, by identifying and characterizing genes involved in this behavior.The aerobic fast-growing M. smegmatis mutants unable to survive a coincident condition of hypoxia and low carbon content, two stress factors supposedly encountered in the host and inducing dormancy in tubercle bacilli. Two mutants were identified that mapped in the uvrA gene, coding for an essential component of the Nucleotide Excision Repair system (NER). The two mutants showed identical phenotypes, although the respective transposon insertions hit different regions of the uvrA gene. The restoration of the uvrA activity in M. smegmatis by complementation with the uvrA gene of M. tuberculosis, confirmed that i) uvrA inactivation was indeed responsible for the inability of M. smegmatis cells to enter or exit dormancy and, therefore, survive hypoxia and presence of low carbon and ii) showed that the respective uvrA genes of M. tuberculosis and M. smegmatis are true orthologs. The rate of survival of wild type, uvrA mutant and complemented strains under conditions of oxidative stress and UV irradiation was determined qualitatively and quantitatively.In a library generated by transposon mutagenesis, we searched for Taken together our results confirm that the mycobacterial NER system is involved in adaptation to various stress conditions and suggest that cells with a compromised DNA repair system have an impaired dormancy behavior. Mycobacterium tuberculosis, the etiological agent of tuberculosis, has the ability to enter human macrophages and survive inside them in a 'latent' or 'non-proliferating' form for a long period of time. This behavior is termed dormancy or latency. During their lifetime, latent bacilli can reactivate giving rise to active tuberculosis, the transmissible form of the disease . To assM. smegmatis gene pool [Ten thousand clones of a transposon library containing more than 20,000 mutants and covering the majority of the ene pool  were scrSalI restriction enzyme and used as template in Ligated Mediated (LM)-PCR reactions [uvrA gene (Table uvrA gene encodes the UvrA protein that belongs to the nucleotide excision repair system (NER). As the two mutants showed identical phenotypes, S1 was chosen for further characterization.In order to identify the sites of transposon insertions, the genomic DNA of all clones was extracted, digested with the eactions . Using tne Table . The uvruvrA mutants in our in vitro model system was a direct effect of UvrA deficiency, we performed complementation analyses. A wild type allele of the uvrA gene was PCR-amplified, cloned into the integrative expression vector pNip40-b [uvrA from M. smegmatis (here defined as S1-uvrA-Ms) fully restored the dormancy defect of the parental mutated strain. Identical results were obtained for the S2 mutant (data not shown).To verify whether the severe dormancy defect of the pNip40-b  and elecuvrA of M. smegmatis as a query showed that this gene is highly conserved in M. tuberculosis. The orthology between the M. smegmatis and M. tuberculosis UvrA proteins was verified by using the M. tuberculosis uvrA gene to complement the M. smegmatis uvrA deficient strain  was able to restore the wt phenotype in the M. smegmatis mutated strain. Our results demonstrate that UvrA is essential for M. smegmatis to enter or exit dormancy upon hypoxia. Moreover, we proved that the M. smegmatis and M. tuberculosis gene products are true orthologs.As shown in Table n Figure . The reiuvrA mutant and the complemented strains in minimal medium supplemented with the following final glucose concentration: 0.4%; 0.2% or 0.01% (w/v). As shown in Figure uvrA mutant cells grown in 0.2% glucose entered stationary phase at a lower optical density (OD600nm≈1.1) in compared to cells of the same strains grown in higher (0.4%) glucose concentration. Moreover, both wt and uvrA cell growth arrested at the limiting glucose concentrations (0.01%). Taken together these results indicate that M. smegmatis growth rate is limited by the amount of carbon available and also that absence of UvrA does not affect M. smegmatis growth under nutrient-limited conditions.In addition to hypoxia, nutrient starvation is also supposed to affect cell growth. To check whether the NER deficiency had an effect on cell growth in nutrient limitation, we monitored the growth rate of the E. coli where the uvr gene products protect bacteria from different types of DNA damages including those induced by UV radiations [uvrA (S1), the complemented derivatives of this mutant, containing the uvrA gene from M. smegmatis (S1-uvrA-Ms) and M. tubercolosis (S1-uvrA-Tb), respectively. As shown in Figure uvrA cells were unable to grow after a 15 sec exposure to UV light (λ = 254 nm), the wild type and the complemented strains were unaffected by the treatment. To further verify the importance of UvrA in preventing UV-induced DNA damages, all strains were exposed to different UV light doses. As shown in Figure 2 UV, whereas the wild type and both complemented strains showed a comparable dose-dependent sensitivity to UV irradiation with more than 60% survival after exposure to the same UV dose. Taken together these results suggest that M. smegmatis UvrA is involved in the repairing of UV-induced DNA damages as reported for other bacteria [The NER system has been extensively studied in diations . To veribacteria .It is hypothesized that inside the granuloma, dormant bacilli are continuously exposed to reactive oxygen species (ROS) and Reactive Nitrogen Intermediates (RNI) -27, lipouvrA mutant S1 and complemented strains S1-uvrA-Ms and S1-uvrA-Tb cell growth. To this purpose, cells were incubated in the presence of 5 mM H2O2 and growth (OD600nm) was monitored at 3 hours intervals for 48 h. As shown in Figure uvrA mutant strain, in contrast to wild type and complemented strains, stopped growing after three mass doubling time in the presence of hydrogen peroxide. The uvrA mutant strain reached a maximal cell density of 8 × 106 c.f.u. ml-1, which was approximately 4-fold higher than the density of the initial inoculum (2 × 106 c.f.u. ml-1) but 1000-fold less than the density of the wild-type and the two complemented strains (8 × 109 c.f.u. ml-1). Interestingly, the growth curve of the two complemented strains shows a lag-phase under normal growth conditions  [s mc2155  is the p17recA1)  was usedM. smegmatis mc2155 and derivatives were grown in LB medium containing 0,05% Tween 80 (LBT).M. smegmatis mc2155 and derivatives were grown in M9 containing 1 mM Mg2SO4 and supplemented with glucose at the following final concentrations: 0.4%; 0.2% or 0.01% (w/v).For nutrient limitation experiments, Escherichia coli strains were grown in LB medium. When required, antibiotics were added to the medium at the following final concentrations: ampicillin 100 μg/ml, kanamycin 25 μg/ml. Hygromicin was used at 200 μg/ml for E. coli and 50 μg/ml for M. smegmatis.M. smegmatis transposon insertion mutants [2SO4 and 0.2% glucose at 37°C in standing condition until OD600nm = 1.0. After incubation time, wild type and mutant strains were serially diluted 1:10 up to 10-5 and spotted on M9 agar plates containing glucose. Control plates were incubated in normal atmosphere (20% O2) for 4-5 days at 37°C, whereas experimental plates were transferred to anoxic jar (Oxoid) for 2 weeks at 37°C. Hypoxia was generated using AnaeroGen gas pack system (Oxoid) inside jars and anaerobiosis (O2 <1%) was checked by using methylene blue as indicator. Plates were finally removed from the anoxic jar and incubated in normal atmosphere to enable growth of the surviving bacteria. mutants  were thauvrA genes from M. smegmatis mc2 155 and M. tuberculosis H37Rv were PCR amplified as follow: the wild-type uvrA gene from M. smegmatis mc2155 was amplified by PCR with Pfu Turbo high fidelity DNA polymerase (Stratagene) by using chromosomal DNA as a template and oligos uvrA-Ms-F and uvrA-Ms-R (Table M. tuberculosis H37Rv was amplified using primers uvrA-Tb-F and uvrA-Tb-R and Pfu Turbo high fidelity DNA polymerase (Stratagene). PCR product was purified with the PCR purification Qiagen kit, digested with XbaI and ligated into the pNIP40b at the unique XbaI site. One clone was selected and sequenced. These plasmids were electroporated into the M. smegmatis uvrA mutant strain S1 (uvrA ::Tn611) and transformants were selected on hygromicin containing LB plates and named S1-uvrA-Ms and S1-uvrA-Tb.Plasmid and chromosomal DNA preparation, restriction digestion, ligation, bacterial transformation and agarose gel electrophoresis were performed as described ,37. For Transposon insertions were mapped by using LM-PCR as previously reported . LM-PCR M. smegmatis strains were grown in LBT medium up to exponential phase (OD600nm = 0.4-0.6). Samples from these cultures were streaked on LB agar plates. Plates were exposed to UV light during 0, 15, 30 and 45 seconds and then incubated at 37°C for 3-4 days. The percentage of survival of these strains after UV irradiation was also determined; exponential phase cultures of all strains were harvested and pellets were re-suspended in 2 mL of 1× PBS. 200 μL were exposed to UV intensities of 0, 2, 4 and 6 mJ/cm2 (as measured with a VLX 3W dosimeter). Viable counts of the cultures were determined by plating serial dilution on LB plates with appropriate antibiotics after 4 days at 37°C.M. smegmatis strains, were grown in triplicate in LBT medium up to stationary phase (OD600 = 1.5). Cultures were serially diluted 1:100 in LBT supplemented with 0 and 5 mM H2O2 freshly prepared, placed in the microtiter well plates and incubated in a Bioscreen C kinetic growth reader at 37°C with constant shaking. Growth was monitored as OD600nm at 3 h intervals for 48 h.The authors declare that they have no competing interests.AC carried out most of the experiments, contributed to experimental design and draft the manuscript; BA carried out complementation experiments and UV assay; IC carried out oxidative stress experiment; DE carried out LM-PCR experiment; JMR conceived and supervised the study. All authors read and approved the final manuscript.


For such responses to occur, dendritic cells (DCs) have to migrate to the tumour, take up tumour antigens and respond to danger signals with maturation, before they engage in T-cell activation. Here, we have studied the effect of 5-aminolevulinic acid (ALA)-mediated PDT on DCs Spheroids of the GB cell lines U87 and U251 were treated with ALA/PDT, and effects on attraction, uptake of tumour antigens and maturation of DCs were studied. To block heat-shock protein-70 (HSP-70) on the spheroids, neutralising antibodies were used.5-Aminolevulinic acid /PDT-treated GB spheroids attracted DCs that acquired tumour antigens from the spheroids effectively. Moreover, co-culture with ALA/PDT-treated spheroids induced DC maturation as indicated by the upregulation of CD83 and co-stimulatory molecules as well as increased T-cell stimulatory activity of the DCs. Heat-shock protein-70 was upregulated on the spheroids after ALA/PDT treatment. Uptake of tumour antigens and DC maturation induced by the ALA/PDT-treated spheroids were inhibited when HSP-70 was blocked.ALA/PDT treatment of glioma spheroids promotes the three initial steps of the afferent phase of adaptive immunity, which is at least partially mediated by HSP-70. Photodynamic therapy (PDT) is a promising approach in the treatment of various tumours , danger signals have to induce DC maturation, associated with migration of DCs to local lymph nodes and mature DCs in the lymph nodes have to present the tumour antigens to the specific T cells in the context of adhesion, co-stimulatory and other accessory molecules, either directly or after antigen transfer to other DCs  is the most frequent and aggressive malignant primary brain tumour  at 37 °C and 5% CO2 in tissue culture flasks . To induce glioma spheroid formation, cells were harvested, and 2 × 106 cells were plated in 20 ml supplemented DMEM medium without phenol red in agar-coated T75 tissue culture flasks. After 3 to 4 days of culture, spheroids with a diameter of 200 μm had formed.The human GB cell lines U87 and U251 were maintained in DMEM medium  supplemented with 10% foetal calf serum, 100 U mlμg ml−1 ALA  for 4 h at 37 °C and 5% CO2 as described  using a Ceralas 633 nm PDT diode laser . For ALA-only controls, spheroids were treated with ALA, but not exposed to laser light. After laser light exposure, spheroids were used directly for the experiments. Under these conditions, viability of cells was 50% after 16 h of tissue culture as determined by WST-1 assay, and TUNEL staining indicated apoptosis in about 60% of cells  as described (+ monocytes were cultured in 24-well plates (Greiner) at 1 × 106 cells per ml and 2 ml per well in serum-free CellGroDC medium  supplemented with 1000 U ml−1 GM-CSF  and 1000 U ml−1 IL-4 (Cellgenix). After 3 days, half of the medium was replaced by fresh medium containing cytokines. Immature CD14−DCs were harvested after 6 days of culture. To induce DC maturation, 1000 U ml−1 TNFα (Cellgenix), GM-CSF and IL-4 were added to immature DCs on day 6, and cultures were continued for 3 days.Buffy coats were obtained from healthy individuals after informed consent. CD14The influence of GB spheroids on DC maturation was studied in co-cultures. In all, 25 untreated spheroids or spheroids that had been treated with ALA (ALA-only) or exposure to laser light (laser-light-only) or both (ALA/PDT) were added to the DC cultures on day 6 and cultures continued for 3 days before analysis. To study the effect of blocking HSP-70 on glioma spheroids, the spheroids were pre-incubated with goat-anti-human HSP-70 polyclonal IgG antibody  before the co-cultures were initiated.+ DCs was determined by flow cytometry on an FACS Canto using the DIVA software , identifying antigen uptake by the appearance of HLA-DR/CFSE double-positive DCs within living cells identified based on forward- vs side-scatter characteristics. At least 10 000 living cells were acquired in each experiment. To study the effect of blocking HSP-70 on antigen uptake, the spheroids were pre-incubated with goat-anti-human HSP-70 polyclonal IgG antibody before assessing antigen uptake by flow cytometry.To assess uptake of tumour material, treated and untreated spheroids were labelled with CFSE  and co-cultured with immature DCs. After 16 h of co-culture, DCs were labelled with anti-HLA-DR monoclonal antibody (mAb) and uptake of tumour material by HLA-DRThe following mAbs were used for immunostainings: PE-conjugated CD14 (RMO52), CD83 (HB15a) and FITC-conjugated CD80 (MAB104) and CD83 (HB15a) specific mAb from Beckman-Coulter ; PE-conjugated HLA-DR (G46-6) and FITC-conjugated CD40 (5C3) and CD86 (FUN-1) specific mAb from BD Biosciences; and PE-conjugated goat polyclonal IgG specific for HSP-70 (K-20) from Santa Cruz.−1 CCL3  were transferred into a 24-well plate (500 μl per well). Transwell inserts  were coated with 100 μl fibronectin  for 1 h at room temperature, placed in the 24-well plate containing the migration targets and immature DCs in CellGroDC medium (50 000 DCs/200 μl per well) were added into the insert. After 16 h of incubation at 37 °C and 5% CO2, cells were fixed with methanol and migrated DCs stained with Fields solution  before microscopically determining the number of migrating cells. Counting of cells was performed automatically using the ImageJ software (http://rsbweb.nih.gov/ij/).To assess migration of immature DCs towards spheroids, a transwell assay was used. Spheroids, CellGroDC medium (negative control) and medium containing 40 ng mlα-matured DCs or immature DCs, which had been co-cultured for 3 days with ALA/PDT-treated or untreated spheroids, were co-cultured with 105 allogeneic T cells, which were obtained by negative selection from buffy coats as described . Statistical significance was evaluated with the Student's vs 36±18 migrating cells; n=4, P=0.0079) and U87 spheroids  compared to medium controls. Control spheroids showed no or only weak attraction of immature DCs.Attraction of immature DCs to ALA/PDT-treated spheroids or control spheroids  was assessed in a transwell migration assay . The immvs 349.3±108.0 mean fluorescence intensity (MFI) FITC; n=4, P=0.0217) as well as from U87 spheroids  compared to untreated control spheroids. Spheroids that had been treated with ALA-only or laser-light-only showed no such activity. Fluorescence microscopy of DCs revealed perinuclear localisation of the fluorescent tumour material . In contrast, co-culture of immature DCs with control spheroids  did not induce DC maturation ; n=3), with a trend towards reduced allostimulatory activity of the DCs after co-culture with untreated U251 spheroids (P=0.0644). When maturation of DCs was induced with TNFα, allostimulatory activity increased significantly compared to the immature DCs (P=0.0094 for P=0.0004 for n=3). Increased allostimulatory activity of DCs was also observed after co-culture with ALA/PDT-treated U251 (P=0.0014 (ANOVA); n=3) or U87 spheroids (P<0.0001 (ANOVA); n=3) compared to co-cultures with untreated spheroids. Co-cultures of DCs with ALA/PDT-treated U87 spheroids reached allostimulatory activity levels similar to TNFα-matured DCs (P>0.05 (ANOVA); n=3). For U251 spheroids, ALA/PDT treatment could compensate the reduction of allostimulatory activity observed after co-culture of immature DCs with untreated spheroids and activity increased to levels comparable to the immature DCs (P>0.05 (ANOVA); n=3). Thus, for both cell lines an increased functional activity of DCs after co-culture with ALA/PDT-treated spheroids was observed, which is consistent with the induction of DC maturation.Functional activity of DCs matured in the presence or absence of ALA/PDT-treated or untreated spheroids of the cell lines U251  or U87  was evalHeat-shock protein-70 has been shown to be upregulated on the surface of mouse squamous cell carcinoma cells after photofrin-mediated PDT and to contribute to immune activation . TherefoWhen HSP-70 surface expression was compared for untreated and ALA/PDT-treated U251 and U87 spheroids, a clear upregulation of surface HSP-70 was evident for the treated spheroids. Heat-shock protein-70 was absent or expressed at only very low levels on the untreated spheroids (vs 769.7±46.3 MFI FITC; n=3, P=0.0014; U87, 1295.0±95.3 vs 679.0±180.5 MFI FITC; n=3, P=0.0393; + mature DCs (U251, 40.0±4.5 vs 6.7±1.3% CD83+; n=3, P=0.002; U87, 25.6±2.5 vs 3.8±0.6% CD83+; n=3, P=0.0006; + DCs was observed when ALA/PDT-treated U251 or U87 spheroids were treated with a control immunoglobulin before co-culture with the immature DCs (Blocking of HSP-70 on the ALA/PDT-treated spheroids before assessing uptake of tumour material from the spheroids inhibited tumour antigen uptake significantly (U251, 1456.0±73.7 ture DCs .+ cytotoxic T cells as main effectors and a supportive role for CD4+ T-helper cells (+ and CD8+ T-cell infiltrates have been observed in lesions after Fotolon-mediated PDT of recurrent angiosarcoma, which underwent remission after therapy (There is clear evidence for an immunological component in the anti-tumoural effect of PDT from extracranial tumours , suggestFor such effector T-cell responses to occur, immature DCs have to migrate to the tumour, take-up tumour antigens and respond to local danger signals with maturation, before they will engage in T-cell activation in the draining lymph nodes in the afferent phase of cellular adaptive immunity (in vivo. Evidence for uptake of tumour antigens after PDT also comes from the intratumoural injection of DCs following PDT treatment: improved survival of animals and induction of tumour-specific immunity indicate that the DCs have taken up tumour antigens (in vitro with CT-26 colon carcinoma cells subjected to photofrin/PDT, enhanced uptake of tumour material by the DCs was detected (The efficiency of PDT-treated tumour cells as vaccines for induction of anti-tumoural immunity has been shown for SCVII squamous cell carcinoma, Lewis lung carcinoma, EMT6 mammary sarcoma, P815 mastocytoma and C6 rat glioma models using photofrin, benzoporphyrin derivative or haematoporphyrin as photosensitisers (γ-secreting cells and the generation of tumour-specific CTL in the treated animals reported by others, it can be concluded that DCs have undergone maturation owing to PDT treatment of the tumour cells in these studies as well. This has been confirmed by in vitro analyses showing induction of MHC class II molecules, CD80, CD86 and IL-12 in co-culture experiments of DCs and PDT-treated mouse tumour cells (Attraction of DCs and uptake of tumour antigens alone, however, are not sufficient for the initiation of an immune response, but may result in immunological tolerance , and frovs necrotic cell death. The release of inflammatory mediators, damage-associated molecular patterns and alarmins like the pro-inflammatory cytokines HMGB1 and IL-1α during apoptosis may contribute to DC maturation (β, IL-6, TNFα, prostaglandins and various HSPs, and glucose-regulated proteins (κB blocked macrophage activation and TNFα release induced by photofrin-mediated PDT of SCCVII mouse squamous cell carcinoma cells. Thus, surface expression of HSP-70 appears to be a major DC maturation-inducing stimulus after PDT in mice as well as in humans. Whether other factors, including pro-inflammatory cytokines and HSP or glucose-regulated proteins, which have been reported to be upregulated on tumour cells in response to PDT (The factors and mechanisms involved in promoting attraction, antigen uptake and maturation of DCs following PDT are not well understood. Depending on the photosensitiser, its location within the cell and dose, the cell type and oxygen concentration, PDT results in apoptotic or necrotic cell death (in vitro. However, our study holds limitations: although the spheroid model may mimic microtumours more closely than monolayer cultures (in vitro or the tumour, including its vasculature and stroma in vivo, may result in a different effect of ALA/PDT. In addition, it remains to be elucidated whether the above-described effects on DCs result in cross-presentation of tumour antigens and induction of cytotoxic T-cell-mediated anti-tumoural immunity in vivo. Therefore, further studies evaluating the effect of ALA/PDT on primary tumour cells as well as in vivo studies are needed to document efficacy of PDT in GB and the contribution of an anti-tumoural immune response to it.For human GB, the immunological consequences of ALA/PDT have not been analysed so far. In this study, we could show that ALA/PDT treatment of GB spheroids attracts DCs and promotes uptake of tumour antigens as well as DC maturation In summary, like in extracerebral tissues, ALA/PDT may not only kill GB cells directly due to its phototoxic effect, but may also result in the induction of anti-tumoural immunity, mediated by the induction of HSP-70 surface expression. This immunological effect may contribute to long-sustaining responses and the potential clinical benefit of ALA/PDT for GB (


The actin bundling protein fascin 1 is not expressed in adult epithelial tissues, but during development it is transiently expressed in many different cell types, and later in adults it is expressed in a subset of immune cells, nervous tissues, endothelial cells, smooth muscle cells and pericytes. In contrast to the wealth of knowledge about the role of fascin 1 in cancer cell migration and invasion, little is known about the involvement of fascin 1 in angiogenesis. We speculated that as angiogenesis involves migration and invasion of tissues by endothelial cells, fascin 1 might have a role in both normal and tumour angiogenesis. Here, we provide evidence that loss of fascin 1 causes relatively minor reductions to angiogenesis during embryonic, postnatal and cancerous development by examining E12.5 hindbrains, postnatal retinas and B16F0 tumour cell allografts in fascin 1-null mice. We also find that in fascin 1 null tissues, endothelial cells display reduced filopodia formation during sprouting. We thus propose that fascin 1 expression promotes angiogenesis via filopodia formation, but is largely dispensable for both normal and tumour angiogenesis. Angiogenesis, the formation of new capillaries from existing vessels, is a fundamental process in development and tumour growth. It involves multiple cell types in sequential controlled steps. Various growth factors such as vascular endothelial growth factors (VEGFs) , Notch pThe actin bundling protein fascin 1 is associated with the formation of actin-based cell membrane protrusions such as filopodia  and invain vivo angiogenesis models. Fascin 1 has been extensively studied in cancer cells and its role in promoting invasion and migration in vitro is well established, but its potential role in developmental angiogenesis or in tumour angiogenesis has not been explored. We suggest that fascin 1 facilitates angiogenesis via its well-known effects on filopodia formation and migration, but that overall the role of fascin in angiogenesis is not greatly limiting for development or tumour formation.Here, we demonstrate the effect of loss of fascin 1 on angiogenesis and endothelial cell morphology with various supplementary material Fig. S1A) and the surviving fascin 1-null pups showed retarded growth in their early life. The weight of fascin 1-null pups at day 7 and day 19 is approximately 60–90% of fascin 1+/− or fascin 1+/+ pups . Fascin 1 was reported previously to be expressed in endothelial cells, pericytes and smooth muscle cells and might be involved in the cardiovascular system .Fascin 1-null C57BL/6 mice display partial neonatal death and retarded growth in early stages . Consistr system . Immunofsupplementary material Fig. S1A) and showed no apparent hemorrhage or prenatal death (data not shown), we wondered whether non-optimal angiogenesis might contribute to abnormal brain development and retarded growth . For quantification of vascular complexity, embryonic hindbrains are ideal tools to study the potential role of fascin in angiogenic sprouting and vascular remodeling . Reduced branching complexity was observed in hindbrains of fascin 1−/− embryos, as measured by number of branch points per area . The retinal vessel network was examined for vascular sprouting at the periphery of the vessel plexus and remodeling at the center between arteries. Fascin 1−/− retinas showed less radial vascular outgrowth at P6 . Tip endothelial cells in fascin−/− hindbrains also exhibit fewer filopodia  guide sprouting angiogenesis by extending long filopodia extensions in response to angiogenic factors . These nilopodia . Also, andbrains  as well ndbrains . Fascin ndbrains , but thed length .−/− mice showed no obvious difference to wild type mice . Next, we quantified the vessel density in the tumour periphery (defined as the area 1 mm close to the tumour edge) or vessel density per entire tumour area. The number of blood vessels per cm2 area in B16F0 allografts in fascin 1−/− mice were significantly reduced compared to that in fascin 1+/+ and fascin 1+/− mice as measured by the endothelial cell marker PECAM-1   and recruitment of macrophages and CD3 positive cells in tumours , indicating that loss of fascin 1 in the host has no detectable effect on immune cell recruitment during angiogenesis and tumour growth. We thus speculate that while fascin expression potentiates angiogenesis, it is not essential in vivo and these tumours can be sufficiently vascularized to support normal growth, just as mouse development can proceed in the absence of fascin.We next asked whether fascin 1 was important for the formation of the tumour vasculature by transplanting B16F0 melanoma cells into fascin 1-null and wild-type mice and examining subsequent tumour growth and vascularization . Fascin-1, CD31) , endomuc1, CD31) . Adult fin vivo, but has not been previously studied in the context of endothelial tip cells or angiogenesis in vivo. Our observations from allografts of B16F0 melanoma also reflect that fascin contributes to, but is not limiting for tumour vascularization. As fascin inhibitors are developed with the eventual hope of targeting tumour metastasis, it is increasingly important to know what effects fascin inhibition would have on the host. We show that while fascin is abundantly expressed in endothelial and mural cells of blood vessels in vivo, its presence is not crucial for vasculature to form relatively normally, but can be limiting for branching and extent of vascularization. In addition to filopodia, fascin 1 also localizes in cell invasive structures, such as podosomes in endothelial cells and smooth muscle cells. Fascin 1 contributes to podosome formation, cell migration and basement membrane degradation both in vitro and in vivo in some specific organs and conditions  mice  were mair assays .5 µm-depth sections were deparaffinised, rehydrated and treated in sodium citrate buffer (pH 6.0). After blocking with peroxidase blocking solution, samples were incubated with the following primary antibodies: rabbit anti-PECAM1 , mouse anti-fascin 1 , rabbit anti-CD3  for 1 hour. DAB-chromogenic detections were carried out using peroxidase labeled polymer (Envision kits) and detected with substrate-chromogen (Envision kits) followed with hematoxylin counterstain. For immunofluorescence staining, after deparaffinisation and rehydration, antigen retrieval using 20 µg/ml proteinase K in TE buffer (pH 8.0)  was performed. After blocking, sections were incubated with the following primary antibodies : endomucin , rat anti-mouse F4/80 antibody  and respective species-specific secondary antibodies . After PBS washes, the sections were mounted with ProLong Gold Antifade reagent with DAPI (Invitrogen).E12.5 embryos were dissected as previously described  and compThe eyes of postnatal day 6 pups were fixed in 4% PFA in PBS and washed with PBS. Retinas were dissected as previously described  and perm+/+, fascin 1+/−, fascin 1−/− were injected subcutaneously into the scruff with 1×106 of mouse melanoma B16F0 cells in 100 µl PBS. 12-days post-inoculation, the mice were sacrificed and the tumours were removed, weighed and photographed before fixation with 10% neutral formalin. Tumours were dissected and stained with hematoxylin and eosin. Size-matched tumours (6–8 per genotype) were immunostained with endothelial cell markers to quantify tumour blood vessel density. Tumour volume (v) was calculated as v = 0.5(l×w2), where l is tumour length (longest diameter) and w is tumour width (shortest diameter) (13–16 C57BL/6 mice (2–6 month old) of fascin 1iameter) .+/+ vs. fascin 1−/−) and one-way ANOVA followed by Mann-Whitney test for selected pairs of genotypes  (GraphPad Prism5). P<0.05 was considered statistically significant. *** denotes P<0.001, ** denotes P<0.01 and * denotes P<0.05.Statistical significance was calculated using the Mann-Whitney test for comparison of two groups of large datasets (fascin 1


Many cancer research efforts focus on exploiting genetic-level features that may be targeted for therapy. Tissue-level features of the tumour microenvironment also represent useful therapeutic targets. Here we investigate the presence of low oxygen tension and sensitivity to NOS inhibition of tumour vasculature as potential tumour-specific features that may be targeted by hypoxic cytotoxins, a class of therapeutics currently under investigation. We have previously demonstrated that tirapazamine (TPZ) mediates central vascular dysfunction in tumours. TPZ is a hypoxic cytotoxin that is also a competitive inhibitor of NOS. Here we further investigated the vascular-targeting activity of TPZ by combining it with NOS inhibitor L-NNA, or with low oxygen content gas breathing. Tumours were analyzed via multiplex immunohistochemical staining that revealed irreversible loss of perfusion and enhanced tumour cell death when TPZ was combined with either low oxygen or a NOS inhibitor. Tumour growth rate was reduced by TPZ + NOS inhibition, and tumours previously resistant to TPZ-mediated vascular dysfunction were sensitized by low oxygen breathing. Additional mapping analysis suggests that tumours with reduced vascular-associated stroma may have greater sensitivity to these effects. These results indicate that poorly oxygenated tumour vessels, also being abnormally organized and with inadequate smooth muscle, may be successfully targeted for significant anti-cancer effects by inhibition of NOS and hypoxia-activated prodrug toxicity. This strategy illustrates a novel use of hypoxia-activated cytotoxic prodrugs as vascular targeting agents, and also represents a novel mechanism for targeting tumour vessels. Identification of tumour-specific, targetable features for which effective anti-cancer therapeutics can be generated is an important focus in cancer research. The variable tumour microenvironment presents opportunities for chemotherapeutic damage, with targets including hypoxic cells and the abnormal tumour vasculature.in vitro and enhances cell kill by radiotherapy and cisplatin in vivoThe presence and importance of hypoxia in tumours has been recognized for more than 50 years The pattern of central vascular damage and persistence of a viable rim caused by TPZ is similar to that seen for vascular disrupting agents (VDAs). Distinct from anti-angiogenic agents, VDAs are a class of compounds in clinical trial that aims to target the aberrant nature of existing tumour blood vessels , HT29  and SCCVII  were implanted subcutaneously into the sacral region of mice. HCT116 and HT29 cells were purchased from the ATCC; SCCVII, murine squamous carcinoma cells, were obtained in 1983 from Dr. M. Horsman (Stanford University) and held in liquid nitrogen storage. For tumour mapping experiments mice were randomly assigned to experimental groups when average tumour volumes reached 100 to 150 mm3 as measured using calipers of three orthogonal diameters  where volume  = π/6 (abc). Average tumour mass at time of excision for each model and group was a minimum of 100 mg and a maximum of 200 mg. For growth assessment tumours were randomly assigned for treatment with 40 mg/kg TPZ, 180 mg/kg L-NNA or a combination of both drugs at 14 days post-implantation when tumours measured 59.0±7 mm3; repeat measurements were performed blind.Ethics Statement: Experiments described in this manuscript were approved by the Animal Care Committee of the University of British Columbia in accordance with the Canadian Council on Animal Care guidelines. Female NOD/SCID and C3H/HeN mice were bred and maintained in our institutional SPF animal facility. Hypoxia was induced in mice breathing humidified 7 or 10% O7(3) (Molecular Probes), administered as 35 µl of 0.6 mg/ml dissolved in 75% (v/v) dimethyl sulfoxide/25% sterile H2O, or 500 kDa FITC-labeled dextran (Sigma), administered as 50 µl of 40 mg/ml solution in NaCl 7(3)) or 2–20 (FITC-dextran) min prior to euthanasia.Tirapazamine was synthesized by Dr. L.A. Huxham 7(3) or FITC-dextran fluorescence and fixed in 50% (v/v) acetone/methanol for 10 min at room temperature. Vasculature was identified using a MAb to PECAM/CD31 (BD PharMingen), bound pimonidazole using HypoxyprobeTM-1 MAb1, eNOS using a mouse MAb (Pharmingen), non-isoform specific NOS using a rabbit PAb to universal NOS ; basement membrane using a rabbit PAb to CIV (NeoMarkers); smooth muscle using a mouse MAb to αSMA (NeoMarkers) and incorporated BrdUrd using a mouse MAb to clone BU33 (Sigma), with light counterstaining using 25% hematoxylin (Sigma).Immediately after excision, tumours were frozen on an aluminum block held at −20°C, embedded in cutting medium  and stored at −20°C until sectioning. The general immunohistochemical procedure used has been previously reported 7(3) images were thresholded and a composite colour image was produced  in dark blue prioritized over DiOC7(3) alone in cyan) using Adobe Photoshop CS (version 8.0). The combined vascular image was then overlaid onto grayscale images of BrdUrd and hematoxylin, where converted grayscale images of pimonidazole (green channel) were then added using the multiply mask. Similarly, grayscale images of CD31 were thresholded and prioritized as an overlay (red) on grayscale images of FITC-dextran. CIV and αSMA are shown as black in original grayscale images with a grey hematoxylin counterstained background.The imaging system has been previously described 7(3), CD31, pimonidazole, eNOS, uNOS, BrdUrd and hematoxylin images from each tumour section were aligned, cropped to tumour tissue boundaries and staining artifacts removed. Necrosis was cropped away based on hematoxylin stained sections and the remaining viable fraction (VF) of tumours was calculated based on the ratio of the total number of pixels in necrosis-cropped images by the total number of pixels in whole tumour areas. Percent positive staining was obtained using the proportion of pixels at intensities meeting or exceeding a threshold value above background. Average intensity values represent the average pixel intensity for the whole tumour cropped to viable tissue boundaries. For distribution analysis of pimonidazole or FITC-dextran relative to vasculature, each pixel in an image was sorted based on its distance relative to the nearest CD31-positive vessel and the average intensity in 1.5 µm increments from vasculature was determined. For dual positive staining analysis of CD31 in combination with additional markers, thresholds were set to identify staining above background and a minimum 20% overlap was required to classify CD31 objects as dual labeled. The proportion of perfused (PF) and eNOS +ve vessels was obtained by dividing the total number of CD31 objects also positive for DiOC7(3) or eNOS respectively by the total number of CD31 objects.Using the ImageJ software application and user-supplied algorithms, fluorescent images were inverted and combinations of FITC-dextran, DiOCmin calculated as the control mean plus two standard deviations of the mean (2× SD). This comparison allows for an objective and quantitative detection of unperfused vessels and necrotic tissue significantly greater than that seen in control tumours. Tumours that both scored higher than their control VDSmin and showed focused areas of vascular dysfunction in tumour maps were considered positive for vascular dysfunction.The VDS score has previously been reported 5 cells/well) in 1.5 ml MCDB-131 media (Sigma) supplemented with 10% FBS, 10 µg/ml EGF (Sigma) and 10 ng/ml glutamine (Sigma). Cells formed tubular structures by 24 h post-seeding and were subsequently treated at indicated concentrations of TPZ; minimum 5 wells per treatment. Plates were brought to indicated gas concentrations in 10.3 min using a custom built aluminum chamber sealed and equilibrated via 8×1 min cycles of evacuation and pressurization and final pressure was maintained for 1 h at 37°C. Wells were then rinsed and the media replaced with supplemented MCDB-131 and were incubated at 37°C in 5% O2/5% CO2/90% N2. Fluorescent dye cell viability indicator Calcein AM (Invitrogen), 10 µM in media, was added 1 h prior to endpoint and was rinsed with phosphate buffered saline  prior to fixation in 10% formalin (Sigma) overnight.Plates  were coated in 200 µl of 50% Matrigel (BD Pharmingen) in MCDB-131 media and allowed to set at 37°C for 1–2 h. Human microvascular endothelial cells . Nonparametric Mann-Whitney U tests were used for comparisons between groups; p values *<0.05, **<0.01 and **<0.001 are reported. Where appropriate, charts display values for individual tumours as means for analysis of whole tumour sections; combined means are reported for 4–8 tumours per group ± standard error (s.e.).min (0.505) , although no significant differences were found in PF and VF values between groups (HT29 xenografts treated with TPZ show no difference in their proportion of perfused vasculature (PF), however a small but significant decrease in the proportion of viable tissue (VF) is seen ; Table 1 (0.505) . HT29 ture shown . TPZ tren groups , Table 1lower vascular density  and low (40 mg/kg) dose TPZ and L-NNA alone or in combination were assessed; VDS scores as well as perfusion and necrosis values are shown in L-NNA in combination with 40 mg/kg TPZ also results in reduced tumour growth rates relative to that seen for either treatment alone . Repeat SCCVII murine syngeneic tumours have a high vascular density with variable perfusion that results in intermittent hypoxia characterized by the presence of perfused vessels overlapped with hypoxia label pimonidazole. Treatment of SCCVII murine tumours with either L-NNA or TPZ causes a drop in both the PV and VF and produces some focused areas of vascular dysfunction; quantified data for all SCCVII treatment groups are shown in min  have proposed that TPZ may potentiate itself via this mechanism In addition to the importance of oxygen, we have demonstrated greater anti-cancer activity as well as enhanced vascular damaging effects of TPZ combined with a NOS inhibitor, including when TPZ is administered at lower than its maximum dose . TPZ is 2•− as a by-product. In the absence of oxygen, the TPZ radical undergoes further metabolism. The radical intermediate responsible for mediating hypoxic cytotoxicity is thought to be an oxidizing hydroxyl (OH•) or benzotriazinyl radical (BTZ•) The explicit mechanism by which co-administration of a NOS inhibitor enhances the anti-vascular effects of TPZ is beyond the scope of this work. There are several possibilities that all may have contributions to the effect. TPZ produces oxidizing species under both aerobic and anaerobic conditions, as cycling of the prodrug in the presence of oxygen results in Oin vivo vascular damage. Indeed, we suggest that it may be desirable to screen for and identify those agents most capable of causing vascular damage. The fragile, hypoxic tumour vasculature may represent a specific and targetable feature of the tumour microenvironment.Assessment of an anti-vascular effect is challenging, as shown by the inability of even the VDS, which accounts for both perfusion & necrosis data, to quantitatively identify those tumours with sustained anti-vascular effects. The gold standard efficacy experiment for hypoxic cytotoxins is to combine them with radiation, which preferentially kills oxygenated cells, to produce greater cell kill for the combination treatment. However, this approach is not able to discriminate between cells that die due to their hypoxic status far from vasculature and consequent toxicity of TPZ, and those that may have died due to loss of blood supply. Hypoxic cytotoxicity would kill cells promptly and therefore experiments typically harvest tumours for colony formation assay plating at <12 h. An anti-vascular effect may not be measurable unless excision occurs at >24 h, as any earlier excision would “rescue” the cells that would otherwise have died due to nutrient deprivation. There are some unexplained data that suggest this effect has occurred in other laboratories. Clonogenic survival studies of hypoxic cytotoxin RB6145 in combination with NOS inhibitor L-NNA found several magnitudes greater cell kill than expected for a hypoxic cytotoxic effect when tumours were excised at 24 h Despite some dramatic increases in vascular damage and even eradication of the viable rim in HCT116 and SCCVII tumours, NOS inhibition combined with TPZ still did not produce vascular damage in HT29 tumours . HCT116 2 (2%), and at a clinically relevant concentration (25 µM) Finally, we demonstrate effects of TPZ on human microvascular endothelial cells . The datAlthough TPZ has failed to show a significant clinical benefit, the concept of hypoxia-activated cytotoxic agents represents an active area of research, including other agents that are also inhibitors of NOS


Background. Goal-directed fluid therapy reduces morbidity and mortality in various clinical settings. Respiratory variations in photoplethysmography are proposed as a noninvasive alternative to predict fluid responsiveness during mechanical ventilation. This paper aims to critically evaluate current data on the ability of photoplethysmography to predict fluid responsiveness. Method. Primary searches were performed in PubMed, Medline, and Embase on November 10, 2011. Results. 14 papers evaluating photoplethysmography and fluid responsiveness were found. Nine studies calculated areas under the receiver operating characteristic curves for ΔPOP  and seven for PVI . Correlations between ΔPOP/PVI and ΔPP/other dynamic variables vary substantially. Conclusion. Although photoplethysmography is a promising technique, predictive values and correlations with other hemodynamic variables indicating fluid responsiveness vary substantially. Presently, it is not documented that photoplethysmography is adequately valid and reliable to be included in clinical practice for evaluation of fluid responsiveness. Conventional static variables include central venous pressure (CVP) and pulmonary artery wedge pressure (PAWP), but these variables have proven less reliable than initially assumed to evaluate fluid responsiveness [Whether or not to administer intravenous (iv) fluid is a common, difficult, and controversial challenge in clinical practice. The main aim of fluid therapy during surgery or critical illness is to provide adequate tissue perfusion by increasing stroke volume (SV) or cardiac output (CO). Goal-directed fluid therapy aiming to increase oxygen  as a noninvasive tool in evaluation of fluid responsiveness was first described by Partridge  and has This paper is based on searches performed in PubMed, Medline, and Embase on November 10, 2011 with the following search criteria: “(pulse oximetry OR plethysmographic OR Pleth variability index OR PVI) AND ((fluid responsiveness) OR (volume status)).” The searches generated 217 hits. Papers were checked for relevant references and 22 –34 paperreporting predictive values of ΔPOP and/or PVI after fluid challenges and/or reporting correlations between ΔPOP, PVI, and ΔPP,mechanically ventilated patients,written in English.n = 6) [n = 3) [n = 2) [n = 2) [n = 8–43). Registration periods were, with some exceptions [n = 9) [n = 3) [n = 2) [n = 2) [n = 5) [n = 1) [n = 1) [n = 2) [n = 1) [n = 1) [n = 1) [n = 5) [n = 6) [n = 1) [n = 1) [14 studies performed fluid challenges and these are summarized in n = 6) , 30–32,  [n = 3) , 33, 34, [n = 2) , 27, and [n = 2) , 24. One [n = 2) . The pulceptions , 33, shoceptions –31, 34. ceptions , 33 and ceptions , 31, 32.ceptions , 30, 34. [n = 9) , 28–31,  [n = 3) , 33, 34, [n = 2) , 29, inc [n = 5) , 30, ΔPP [n = 1) , increas [n = 5) , 26, 30, [n = 1) .Nine studies calculated areas under receiver operating characteristics curves (ROC curves) for ΔPOP , 32, 33.Seven studies calculated ROC curves for PVI –32, 34, r > 0.84) [r < 0.78) [r = 0.72, 0.46 and 0.78) [r = 0.85) [r = 0.39) [r = 0.92) [ΔPP is considered to be a good predictor of fluid responsiveness . Thus, o > 0.84) , 24, 27. < 0.78) , 32, 33. < 0.78)  concludend 0.78) , 20, 32, = 0.85) . Three p = 0.85) , 26, 32. = 0.92) , 26. ThePhotoplethysmography is applicable on most patient categories and is noninvasive, simple, widely available, and without risk of complications. Several physiological, clinical, and practical factors must be taken into account when evaluating whether or not it is a noninvasive alternative to evaluate fluid responsiveness.Firstly, there are several physiological prerequisitions for using dynamic variables.Mechanical ventilation provides the stable and predictable variations in intrathoracic pressure required for photoplethysmography to be accurate. A large mechanical tidal volume will influence intrathoracic pressure to a greater extent than a small tidal volume. It is presumed that the influence of tidal volume reaches significance at >8 mL/kg. It is a challenge that the accuracy of photoplethysmography increases with larger tidal volumes, whereas it is clinically desirable to minimize the tidal volume. The accuracy of photoplethysmography relies on a continuous beat-to-beat-analysis. Thus, patients need to have stable heart rate. Additionally, decreased RV ejection fraction can lead to false-positive variations in pulse pressure . These rSecondly, the complex network of correlations between ΔPOP/PVI and ΔPP/other hemodynamic variables varies greatly between different studies. The best correlations are found in studies where short registration periods (3–5 respiratory cycles) have been used and in patients under stable pre- and postoperative conditions. These conditions do not reflect genuine intraoperative instability, the setting where precise guidance of fluid therapy is perhaps most important. The correlations are poorer with longer periods of registration , in heteFinally, a number of additional factors must be considered. Variations in total peripheral resistance and vasomotor tone increase under the influence of general anesthesia , 42, witA threshold value refers to a value of ΔPOP, ΔPP, or PVI that separates responders from nonresponders. Failure to agree upon a threshold value in clinical settings does not necessarily make the parameters  less valuable. Different patient groups may well present with different threshold values. A septic patient may have a threshold value different from that of a hemodynamically stable patient undergoing surgery. In the same way, threshold values may also change pre-, peri-, and postoperatively. Cannesson et al.  discusseCut-off values for increases in SV/CO/CI are defined to separate reponders and nonresponders. These thresholds are based on the variability and errors in the chosen measuring technique as well as what change is believed to be clinically important. These thresholds may be more or less arbitrarily chosen and differ between the studies. Level of intra-abdominal pressure may influence ΔPP and ΔPOP and is relevant in three of the articles included , 28, 33.In theory, a number of potentially confounding factors exist. Different pulse oximeter-technology, errors due to software autogain features which filter and amplify the raw signal , atherosclerosis, type of fluid, skin pigmentation, saturation, movement artefacts, statistical weaknesses, variations in pleural and transpulmonary pressures, and venous components of the pulsatile signal may affect measurements.We conclude that although photoplethysmography is a promising technique, predictive values and correlations with other hemodynamic variables indicating fluid responsiveness vary substantially. Based on studies using short registration periods photoplethysmography might seem promising for evaluation of volume status. However, in studies using longer registration periods it has been shown that intra- and interindividual variability for ΔPOP is greater than for ΔPP, leading to poor agreement between ΔPOP and ΔPP. Thus, it is not presently evident that photoplethysmography is adequately accurate, valid, and reliable to be included in clinical practice for evaluation of volume status. In future studies it is important to evaluate new hemodynamic methods in clinically relevant settings and to test their reproducibility in clinically relevant time frames. Relatively poor predictive values during ongoing major surgery further underscore this point and results vary in different patient groups. The greatest potential for photoplethysmography in evaluation of volume status might be in settings where invasive monitoring is not indicated.


The gestational age (mean ± SD) at which the amniotic fluid sample was obtained was 16.13 ± 1.87 weeks. Magnesium levels were determined by colorimetric assay with chlorophosphonazo-III using the the Cobas c 501 analyser (Roche Diagnostics). Statistical treatment of data was performed using the SPSS program, version 15.0.Results revealed a mean magnesium value of 1.65 ± 0.16 mg/dL in amniotic fluid and 1.97 ± 0.23 mg/dL in serum.It would be interesting to extend the study to a larger number of pregnant women to determine variations in normal magnesium values in the three trimesters of pregnancy. Amniotic fluid increases in volume as the foetus grows and peaks at an average of 800 mL at approximately 34 weeks of gestation. Approximately 600 mL of amniotic fluid surrounds the baby at full term (40 weeks). This fluid is circulated constantly by the baby inhaling and swallowing existing fluid and replacing it through exhalation and urination . AmniotiLow levels of magnesium in amniotic fluid are associated with pregnancy complications such as preeclampsia  and diabConsequently, knowledge of the values for this ion in normal amniotic fluid may provide a preventive and early diagnosis of certain maternal and foetal pathologies. Amniocentesis is performed, if indicated, from the second trimester of pregnancy. Although magnesium values in amniotic fluid -11 are rMagnesium in serum is reduced during pregnancy and so additional magnesium is required in the diet ,12,13. TBecause variations in magnesium values could be useful as diagnostic and therapeutic guidelines for possible complications, our aim was to determine magnesium levels in amniotic fluid so as to establish normal values for the 14-to-18-week interval of the second trimester of pregnancy.The study design and protocol were reviewed and approved by the "Ethics Committee of Hospital Universitario Miguel Servet" in accordance with the Declaration of Helsinki and the Nuremberg Code. All the patients in the study granted their informed consent.th and 18th week of pregnancy. The average gestational age at which amniotic fluid samples were obtained by amniocentesis was 16.13 (±1.87) weeks. The extracted amniotic fluid samples were sent immediately to the laboratory for centrifugation and processing to determine magnesium levels, or, if immediate processing was not possible, they were stored at -80°C. The reasons for performing amniocentesis on the patients in our sample are summarized in Figure Included in the study were 92 pregnant women aged between 21 and 44 years old. Sample size was established on the basis of the mean number of pregnant women undergoing amniocentesis between the 14Magnesium levels were analysed for both the amniotic fluid and serum of the study population, by colorimetric assay with chlorophosphonazo-III using the Cobas c 501 analyser (Roche Diagnostics). The measurement interval was 0.24-6.08 mg/dL and the lower detection threshold was 0.24 mg/dL. The reference serum value for the normal adult population (21-59 years) was 1.6-2.6 mg/dL. Statistical analysis was performed using the SPSS program, version 15.0.Results revealed a mean amniotic fluid value of 1.65 ± 0.16 mg/dL--with the value 1.6 mg/dL repeated in many samples--and a mean serum value of 1.97 ± 0.23 mg/dL. Amniotic fluid values for magnesium were slightly lower than serum values. No variations in magnesium levels were detected in patients referred for amniocentesis to diagnose disorders that could imply a risk for the foetus. Similarly, despite variations in amniotic fluid volume during pregnancy [These results are consistent with previous studies -11 analyStaphylococcus aureus was maximal when magnesium concentrations were low, while higher concentrations suppressed toxin 1 production. On the other hand, magnesium sulphate, when given in high dosages to obstetric patients, can cause significant maternal morbidity and rare instances of maternal mortality [Falls in amniotic fluid magnesium levels are associated with pregnancy complications such as preeclampsia  and diabortality .Despite the risk associated with amniocentesis, it would be interesting to extend the study to a larger number of pregnant women and determine magnesium values for the first, second and third trimesters in both normal and abnormal pregnancies, so as to establish normal values and critical ranges and better evaluate complications and their management.The authors declare that they have no competing interests. All authors read and approved the final manuscript.JPBT conceived the study, participated in its design and coordination and helped draft the manuscript. SIA carried out laboratory tests, participated in designing the study and performed the statistical analysis. JLBF participated in the sequence alignment and helped draft the manuscript. AGL carried out the assays and participated in designing the study. JFEM helped draft the manuscript, revised it critically for intellectual content and gave final approval of the version to be published.


Solitary fibrous tumour is a soft tissue tumour composed of a subset of fibroblast-like cells and frequently needs immunohistochemical staining for final diagnosis. Epithelioid angiomyolipoma is a variant of angiomyolipoma but characterized by the absence of both adipocytes and abnormal blood vessels. We introduce a very rare case with the combination of these two tumours. A Japanese female patient without significant symptom was hospitalized and operated due to multiple uterine leiomyomas. During the operation, the surgeons found another tumour attaching to serosa of sigmoid colon. This tumour was resected and interpreted as solitary fibrous tumour, suspicious of malignancy. After 13 months of treatment, she was hospitalized again due to hematuria. The doctors detected a tumour in her right kidney. After consultation, laparoscopic right nephrectomy was done. The pathological result of this tumour was epithelioid angiomyolipoma. This is the first report on this very rare combination of tumours with extensive immunohistochemical demonstration of both tumours. Hereby, we review clinical information and histopathological findings together with discussion on each tumour. Among the renal tumours, angiomyolipoma is widely known as a benign mesenchymal tumour composed of a variable proportion of adipose tissue, spindle and epithelioid smooth muscle cells, and abnormal thick-walled blood vessels . HoweverSolitary fibrous tumour is a soft tissue neoplasm, formerly thought to be of mesothelial nature and limited to mesothelium-covered surfaces, and is now known to be composed of a subset of fibroblast-like cells and to be quite ubiquitous . ExtraplRenal epithelioid angiomyolipoma and intra-abdominal solitary fibrous tumour have some similar features which need differential diagnoses. Both of them are very uncommon. Here we report a very rare case of both renal epithelioid angiomyolipoma and intra-abdominal solitary fibrous tumour.A 54-year-old Japanese woman was hospitalized with many tumours in the uterus consistent with multiple leiomyomas and cysts in both ovaries. She had not had a significant symptom or history of disease. The operation was done in February 2012. However, when the patient was operated, the surgeons accidentally detected a tumour attaching to the serosa of sigmoid colon. The surgeons resected uterus, bilateral adnexa, and the unidentified tumour. All of specimens were sent to the diagnostic pathology center. Afterward, the diagnosis of specimen of uterus and bilateral adnexa was multiple leiomyomas of the uterus and endometriosis, hemorrhagic follicular cysts of both ovaries. Malignant findings were not seen in these lesions.The incidentally identified tumour of the colon  was alsoMicroscopically, the tumour was composed of alternating hypercellular and hypocellular areas. Most of the cells were spindle-shaped with indistinct cytoplasm and oval-shaped nuclei with dispersed chromatin. Some tumour cells were multinucleated or had large and hyperchromatic nuclei . MitoticThirteen months after the operation, the patient returned to the hospital due to hematuria. By contrast enhanced computed tomography, a tumour in the lower portion of the right kidney was detected . After cMicroscopically, the tumour cells had a wide variety of appearances. Most of the cells were polygonal with abundant eosinophilic granular or clear cytoplasm. In some areas, the tumour contained spindle-shaped cells and multinucleated giant cells. The nuclei showed pleomorphism with prominent nucleoli and scattered mitoses . The celUsually, the diagnosis of solitary fibrous tumour was not made easily. At the first glance by H&E staining, many lesions with one or some features similar to solitary fibrous tumour should be thoroughly reviewed such as fibromatosis, fibrosarcoma, gastrointestinal stromal tumour (GIST), leiomyoma, leiomyosarcoma, and schwannoma. Compared with these tumours, the distinct features of solitary fibrous tumour are patternless arrangement of spindle cells, branching vessels, and parallel strands of collagen.Immunohistochemically, CD34 is the most important marker for the diagnosis of solitary fibrous tumour.90 to 95% of cases of solitary fibrous tumour are positive for CD34 . In compThe prognosis of solitary fibrous tumour was difficult to be assessed. Although majority of solitary fibrous tumours are benign, 10 to 15% of cases behave aggressively . The criThe second tumour in her right kidney which was interpreted as epithelioid angiomyolipoma also needed the differential diagnoses. Firstly, the component of epithelioid tumour cells with clear or eosinophilic granular cytoplasm and prominent nucleoli was found in clear cell renal cell carcinoma. However, the tumour cells were not arranged into typically acinar and alveolar pattern of clear cell renal cell carcinoma. The typical network of small thin walled blood vessels of clear cell renal cell carcinoma could not be found in the tumour. Secondly, the differential diagnosis of sarcomatoid renal cell carcinoma should be noted. The sarcomatoid component of sarcomatoid renal cell carcinoma is also composed of spindle, multinucleated giant cells, and usually high nuclear grade . BesidesThe immunohistochemical staining is necessary for distinguishing epithelioid angiomyolipoma from clear cell renal cell carcinoma and sarcomatoid renal cell carcinoma. In the literature, 94% of clear cell renal cell carcinomas show positivity for CD10 . The tumUntil now, there is no large study for Ki-67 expression of epithelioid angiomyolipoma. However, a study of Ooi et al. concluded that the Ki-67 was a useful marker which distinguishes the malignant epithelioid variant of angiomyolipoma from classic angiomyolipoma. In their study,all cases of epithelioid angiomyolipoma were strongly positive for Ki-67, while all cases of classic angiomyolipoma were completely negative . AnotherWe were also concerned about tuberous sclerosis because the patients of epithelioid angiomyolipoma usually have a history of this genetic disorders. However, except angiomyolipoma, the patient did not have the symptoms, family history, and history of the disease of tuberous sclerosis in other organs. The imaging findings of the patient in other organs were not suggestive of tuberous sclerosis.The occurrence of both solitary fibrous tumour and angiomyolipoma was very rare in the literature. We just found a case report of solitary fibrous tumour of the adrenal gland with ipsilateral renal cell carcinoma and angiomyolipoma . This paAs the patient appears to lack any genetic background and due to the complete difference of immunophenotypes between these two tumours, we think the solitary fibrous tumour and the epithelioid angiomyolipoma are two unrelated separate tumours occurring in the same patient. Further genetic analysis may disclose the basic mechanism for this very unusual combination of two different tumours.


In contrast, Egr-1 overexpression appeared to make tumour cells more susceptible to GD-induced necrosis. Finally, Egr-1 shRNA suppressed the growth of MTSs. These findings demonstrate that Egr-1 is implicated in GD-induced necrosis and tumour progression.Necrosis is commonly found in the core region of solid tumours due to metabolic stress such as hypoxia and glucose deprivation (GD) resulting from insufficient vascularization. Necrosis promotes tumour growth and development by releasing the tumour-promoting cytokine high mobility group box 1 (HMGB1); however, the molecular mechanism underlying necrotic cell death remains largely unknown. In this study, we show that early growth response 1 (Egr-1) is induced in a reactive oxygen species (ROS)-dependent manner by GD in several cell lines such as A549, MDA-MB-231 and HepG2 cells that exhibit necrosis upon GD. We found that Egr-1 short hairpin RNA (shRNA) prevented GD-induced necrosis and HMGB1 release. Necrosis-inhibiting activity of Egr-1 shRNA was also seen in multicellular tumour spheroids (MTSs), an  Unlike tumour suppressive apoptosis and autophagic cell death, necrosis promotes tumour growth, progression, and aggressiveness ,2. The tEarly growth response 1 (Egr-1) is a 3 Cys2-His2 type COOH-terminal zinc-finger transcription factor that binds to GC-rich recognition motifs (5′-GCGT/GGGGCG-3′ or 5′-TCCT/ACCTCCTCC-3′) ,7. Egr-1in vitro tumour model system. Taken together, these results demonstrate that Egr-1 plays an important role(s) in GD-induced necrosis and tumour progression.In this study, we tried to identify the mechanism underlying metabolic stress-induced necrosis. Previously, we showed that GD induced necrosis in several tumour cell types including A549, MDA-MB-231, and HepG2 cells and activation of protein kinase C by treatment of phorbol 12-myristate 13-acetate (PMA) switched GD-induced necrosis to apoptosis in A549 cells . By cDNA2. The cells were treated with GD, reactive oxygen species , or other chemicals as described previously  heat-inactivated fetal bovine serum  and 1% penicillin-streptomycin (Hyclone) in a 37°C humidified incubator with 5% COeviously . Multiceeviously  and MTSsMicroarray was performed to screen the differentially expressed genes using Operon Human Whole 35K Oligo chips  . The AffWestern blotting were performed using the following antibodies: Egr-1 ; α-tubulin ; HMGB1 ; CuZnSOD ; ERK1/2 . The HMGB1 release assay was carried out as described previously ,22. Tran5 cells/ml in 35-mm dishes. After 24 h, the cells were treated with GD for the indicated times and then stained with HO (1 μg/ml) and PI (5 μg/ml) for 15 min. In 3D culture, equal numbers of spheroids were transferred to 1.2% agarose-coated 60-mm dishes and trypsinized and then stained with HO/PI, The stained cells were observed under a fluorescence microscope and apoptotic and necrotic cells were scored. Intracellular H2O2, O2- and mitochondrial ROS measurement were conducted as described previously  and propidium iodide (PI) double staining was performed ,22. In 2eviously ,22.pcDNA3.1-Egr-1, constructed by inserting the Egr-1 open reading frame into plasmid pcDNA3.1/NEO expression vector (Invitrogen), was provided by Dr Thomas E. Eling . The vectors pcDNA3.1 and pcDNA3.1-Egr-1 were transfected into MCF-7 cells using jetPEI (Polyplus transfection) according to manufacturer’s protocol. Egr-1 shRNA target sequences were designed and verified as specific for Egr-1 by Blast search against the human genome and real-time PCR, respectively . The vecData were analyzed by the Student’s t-test and P<0.05 was considered statistically significant.2O2 and menadione (an O2- generator) increased Egr-1 mRNA expression in MCF-7 cells as determined by real-time PCR  of Egr-1 in necrosis, we examined the effects of Egr-1 shRNA, directed to the C-terminal region of human Egr-1 mRNA sequences (position from 1630 to 1648 in human cDNA, 2- is induced upon GD and mediates GD-induced necrosis and cytotoxicity (2-, and intracellular H2O2, as revealed by staining with three different fluorogenic probes including MitoTracker Red CM-H2XRos, HE, and DCFH-DA, respectively. Egr-1 interference blocked GD-induced production of mitochondrial ROS, O2-, and intracellular H2O2 (Mitochondrial Otoxicity –28. As slar H2O2 , indicatTo further examine the role of Egr-1 in necrosis, we overexpressed Egr-1 in MCF-7 cells. Egr-1 is involved in HGF-induced cell scattering, migration, and invasion via Snail activation . Egr-1 oin vitro model of solid tumours. MCF-7 cells formed tightly packed spheroids of a homogeneous size and as the MCF-7 MTSs grow, a proliferation gradient is observed, with proliferating cells at the periphery, cell cycle arrested cells in the inner regions, and necrotizing cells in the core regions (We examined the effects of Egr-1 shRNA on necrosis using MTSs. MTSs closely resemble poorly vascularized solid tumours, and thus are used for an  regions ,32. Cont regions ,33. Alth regions ; Egr-1 i regions , indicat regions . These fAn immediate early gene Egr-1 is implicated in tumour cell biology. While Egr-1 is known to induce apoptosis in tumour cells, it also promotes tumour progression, survival, and angiogenesis –13. In t


A mastoid cavity resulting from a canal wall down mastoidectomy can result in major morbidity for patients due to chronic otorrhea and infection, difficulty with hearing aids and vertigo with temperature changes. Mastoid obliteration with reconstruction of the bony external ear canal recreates the normal anatomy to avoid such morbidity. Few have the studied the quality of life benefit that this procedure confers.This retrospective observational study was conducted to determine if mastoid obliteration with autologous cranial bone graft following mastoidectomy improves quality of life (QOL). Patients with cholesteatoma who had mastoidectomy with primary or secondary mastoid obliteration by a tertiary otologist were surveyed using the validated Glasgow Benefit Inventory (GBI), our primary outcome measure.Fifty-eight patients were interviewed. Forty-six were primary obliteration after canal wall down mastoidectomy of a primary cholesteatoma. Twelve were secondary obliteration of an existing canal wall down mastoid cavity. Overall GBI scores were improved, with average scores of 22. Average general subscale scores were 23, physical health scores were 25, and social health scores were 22. The primary obliteration group had average scores of 19, general subscale scores of 20, physical health scores of 21, and social health scores of 22. Those with secondary obliteration scored higher, with average scores of 31, general subscale scores of 34, physical health scores of 39, and social health scores of 25.This study shows that mastoidectomy with obliteration using autologous cranial bone graft offers a significant QOL benefit. The GBI scores compare favourably with other otorhinolaryngology procedures. Secondary obliterations after revision mastoidectomy scored much higher than primary obliterations. This is currently the only QOL study comparing these two patient groups. A canal wall-down mastoid cavity constitutes a major morbidity to patients with chronic ear disease. The consequences include susceptibility to infection with any water exposure, recurrent otorrhea, the need for frequent cleaning, difficulty with the use of conventional hearing aids and vertigo caused by warm or cold air or water exposure .Mastoid obliteration with reconstruction of the bony external ear canal  is a proPalva  introducVery few studies have been published regarding the quality of life (QOL) change due to primary or secondary mastoid obliteration. Dornhoffer et al.  studied The GBI was first developed by Robinson at al.  to measuIn otolaryngology, the GBI showed improvement in quality of life after different procedures such as intratympanic dexamethasone  and intrThe aim of this study is to describe the change in health status in patients after mastoidectomy and mastoid obliteration. It also looks at the change in QOL between mastoidectomy with primary mastoid obliteration and secondary obliteration.This is a retrospective QOL study. Ethics approval was obtained at University of Alberta Research Ethics Office (Pro00026634). Seventy-two patients who had undergone canal wall down mastoidectomy with mastoid obliteration using autologous cranial bone graft from 2009 till 2012 were identified as possible candidates. Patients with primary cholesteatoma who had mastoidectomy and mastoid obliteration were defined as having primary obliteration. Secondary obliterations were those with an existing canal wall down mastoidectomy who were having otorrhoea and who had revision mastoidectomy and mastoid obliteration. All patients were operated on by one tertiary otologist, therefore standardizing technique.Twenty-four patients who had primary obliteration were contacted over the phone by the interviewer who went through the GBI with these patients. Forty-eight patients were identified as requiring an interview in person during their clinic visit. This group consisted of those who had secondary obliterations, multiple ear surgeries, and patients who had primary surgery but had a language barrier that hindered communication over the phone. Two patients out of this last group were excluded from the study due to non-compliance with postoperative treatment and follow up. In addition, one patient just had surgery recently and it was too soon to interview him. Another 12 could not be contacted either because they were out of country or had changed addresses. Ultimately, only 34 of these 48 patients completed a GBI in clinic with the help of an interviewer. 58 of the 72 patients, 81% of the originally identified patients, completed their GBI questionnaires.In the primary procedure, a post-auricular incision was performed. A U-shaped superiorly pedicled temporalis muscle flap was raised off the mastoid bone consisting of temporalis muscle, temporalis fascia and some pericranium. The temporalis muscle was dissected from the underlying mastoid bone from the mastoid tip superiorly to the temporal line. The muscle was then retracted superiorly to expose 1 to 2 cm of the pericranium above the temporal line. Bone chips were harvested with a large chisel and mallet from the healthy, disease-free cortex of the mastoid bone before the mastoidectomy was performed so that the cranial autologous bone graft was not contaminated by disease. The bone chips were laid in cool normal saline to keep fresh. Bone pate was then collected in a special bone dust collector from the lateral mastoid cortex and laid to dry. The mastoidectomy was begun after harvesting the bone graft. At the end of the canal wall down mastoidectomy, the bone chips were used to obliterate the mastoid cavity and the epitympanum. The bone pate was used to resurface and reconstruct the external ear canal. Temporalis fascia was used to provide soft tissue cover over the bone graft onto which the remaining skin flaps of the ear canal were placed.In the secondary procedure the only difference was that the pre-existing mastoid cavity was already present before the harvesting of the bone graft. Again, like the primary procedure the autologous cranial bone graft, in the form of bone chips and bone pate were harvested in the same way prior to breaking into the mastoid cavity. All the skin lining the mastoid cavity was removed so as to not risk burying a cholesteatoma during the mastoid obliteration. After the completion of the revision mastoidectomy, the task of obliterating the mastoid cavity with bone chips and resurfacing the bony external ear canal with bone pate progressed in just the same way as the primary procedure.QOL of our 58 patients were measured using the Glasgow Benefit Inventory (GBI) survey described by Robinson et al. . Figure A chart review was done to obtain demographic and additional information such as the duration of symptoms before the obliteration was attempted, date of surgery, and whether the patient had other middle ear surgeries. Statistical analysis was performed using SPSS.Fifty-eight patients were interviewed in person or via telephone from October 2012 to January 2013. Average patient age was forty years , and thirty-one patients were male. Twenty-eight right ears (48%) were operated on. Forty-six were primary cases (79%), while twelve (21%) were secondary, with average time to obliteration of existing cavity being 2.9 years  , with three recurrences in the epitympanum and one in the oval window region. Recurrence was never in the obliterated mastoid cavity and no cases required the mastoid obliteration to be taken down.Evaluating our entire cohort of 58 patients, overall GBI scores were improved, with average overall scores of +22. The subgroup with primary obliteration had an average total GBI score of +19. Primary canal wall down mastoidectomy with mastoid obliteration with autologous cranial bone graft confers improvement in QOL. The subgroup with secondary obliteration of an existing cavity had an average total GBI score of +31. Comparing the primary and secondary obliteration subgroups, the difference in overall GBI score was not statistically significant (Mann Whitney U test p = 0.14). A summary of the raw GBI questionnaire data is provided in Table After primary obliteration, 33 of 46 (72%) reported improved QOL compared to 10 of 12 (83%) after secondary obliteration. With regard to the general, physical, and social subscales, while both primary and secondary obliteration patients reported an improved quality of life, those with secondary obliteration reported a more dramatic change in quality of life. Revision canal wall down mastoidectomy with secondary mastoid obliteration to reconstruct the bony external ear canal with autologous cranial bone graft confers great improvement of QOL. A comparison between subgroups is summarized in Table For the entire cohort, the most positive changes in health status were seen in change in overall effect on life (Question 2), their optimism about the future (Question 3), feeling better about themselves (Question 14), and in the things that the patient did (Question 1). For the primary obliteration group, the most positive effects were reflected in the answers from the same four questions. For the secondary obliteration group, the most positive changes in health status were seen in things that the patient did (Question 1), their optimism about the future (Question 3), feeling better about themselves (Question 14), being less inconvenienced by their ears (Question 16), and having more self-confidence (Question 5).This study is the first study reporting significant improvement in QOL with mastoidectomy, mastoid obliteration and external ear canal reconstruction using autologous cranial bone graft in a Canadian population.The GBI captured just a few QOL factors that experienced most change in health status for our cohort of patients. Questions regarding change in what things patients did, overall effect on life, optimism about the future, patients’ self-opinion were reported as clinically significant positive improvements by our entire cohort. In the secondary obliteration group positive change in self- confidence and being less inconvenienced by their ears were also reported as significant changes. Dornhoffer et al.  also repOur GBI results compare favourably with the GBI scores for general otorhinolaryngology procedures such as tonsillectomy +19 , rhinoplOur GBI scores are consistent with the current literature reporting improved health status after surgery in patients suffering from chronic otitis media. Robinson et al.  reportedThe GBI scores of our patients were similar to other otology procedures such as ossicular manipulation +16.6 , stapedeTo date, our study is the first and largest study using a validated change in QOL tool to compare the change in QOL between primary and secondary mastoid obliteration with autologous cranial bone graft. One recent study comparing change in health status between primary and revision mastoidectomy groups  reportedMastoidectomy with obliteration using autologous bone graft is one of the treatment options for patients with chronic otitis media. Mastoidectomy with obliteration provides a quality of life benefit to patients, and this appears to be more pronounced in those with secondary obliteration.The authors declare that they have no competing interests.GK worked on literature review, data collection and analysis, and manuscript preparation. KG worked on data collection, chart review, and manuscript preparation. NG worked on literature review, data collection, chart review, and manuscript preparation. AH worked on study proposal, data collection, manuscript preparation, and coordination. All authors read and approved the final manuscript.