About 90% of these lesions are benign and a reliable approach to their preoperative characterization is necessary. Unfortunately conventional thyroid scintigraphy does not allow the distinction among benign and malignant thyroid proliferations but it provides only functional information  and galectin-3 knockout tumors were used as targets in different experiments in vivo. 38 mice with tumor mass of about 1 gm were injected in the tail vein with 100 µCi of 99mTc-labeled mAb to galectin-3 . Tumor images were acquired at 1 hr, 3 hrs, 6 hrs, 9 hrs and 24 hrs post injection by using the mini-gamma camera.The galectin-3 based thyroid immuno-scintigraphy uses as radiotracer a specific Results from different consecutive experiments show an optimal visualization of thyroid cancer xenografts between 6 and 9 hours from injection of the radiotracer. Galectin-3 negative tumors were not detected at all. At 6 hrs post-injection galectin-3 expressing tumors were correctly visualized, while the whole-body activity had essentially cleared.In vivo imaging of thyroid cancer may allow a better selection of patients referred to surgery. The possibility to apply this method for imaging and treatment of other galectin-3 expressing tumors is also discussed.These results demonstrate the possibility to distinguish preoperatively benign from malignant thyroid nodules by using a specific galectin-3 radio-immunotargeting.  The high prevalence of benign thyroid nodules in adult population makes the preoperative detection of thyroid cancer comparable to ‘looking for a needle in a haystack’. The prevalence of thyroid nodules increases with age, average 4–7% for the U.S.A. adult population hot nodules), the large majority of thyroid proliferations that fail to concentrate iodide (cold nodules) are biologically benign The expression of Sodium Iodide Symporter (NIS) on the membrane of the thyroid cells allows the thyroid gland to concentrate iodide from the serum. NIS-mediated iodide uptake is required for the subsequent organification and oxidation steps, which are key events for the production of thyroid hormones. This peculiar property of the gland supports conventional thyroid scintigraphy, which uses radioiodine in defining the thyroid gland in both physiological and pathological states via specific cDNA transfection generates a transformed phenotype, blocking the apoptotic program, a feature that favors the development of cancer in vivo and ex vivo experimental models of thyroid cancer we show the possibility to obtain a reliable thyroid cancer imaging in vivo by targeting the galectin-3 lectin molecule. This diagnostic approach may be also used for imaging different galectin-3 expressing tumors in vivo.Normally thyroid cells do not express galectin-3. A forced expression of galectin-3 in vivo and provides an useful model for setting experiments of galectin-3 immunotargeting with and without galectin-3 mRNA interference. Cells were cultured in standard conditions at 37°C and 5% CO2 atmosphere in RPMI-1640 medium supplemented with 2 mM glutamine, 10% FCS, penicillin and streptomycin .Thyroid carcinoma cell lines ARO, kindly provided by Dr. Silvia Soddu  was previously described CAACAGGAGAGTCATTGTTTTCAAGAGAAACAATGACTCTCCTGTTGTTTTTGGAAA-3′5′-GATCCCC; CAACAGGAGAGT-CATTGTTTCTCTTGAAAACAATGACTCTCCTGTTGGGG-3′5′-AGCTTTTCCAAAAA  ; ACCTTACATGTGTAAAGGTTTCAAGAGAACCTTTACACATGTAA-GGTTTTTTGGAAA-3′GATCCCC ; and ACCTTAC-ATGTG TAAAGGTTCTCTTGAAACCTTTACACATGTAAGGTGGG-3′5′-AGCTTTTCCAAAAA .For galectin-3 mRNA interference three different sequences were identified and tested as reported previously In the experiment shown in Down-regulation of Galectin-3 expression was checked in western blot analysis at different time points (12–72 hrs after transfection and tumour cells injection in mice).A purified horseradish-peroxidase conjugated (HRP-conjugated) rat monoclonal antibody to galectin-3  was used in immunohisto-cytochemistry according to the manufacturer's instructions as previously reported Total cell extracts (TCEs) were obtained using a lysis buffer composed by: Tris HCl 50 mM, NaCl 150 mM, Tween 20 1%, PMSF 1 mM and 1 tablet of complete protein inhibitor cocktail (Roche). An aliquot of TCE (30–70 µg) was separated in 10% SDS-PAGE, then blotted onto nitrocellulose membrane (BIO-RAD). The following antibodies were used in immunoblotting: a purified rat mAb to galectin-3 , a mouse mAb anti-α-tubulin  and HRP-conjugated anti-mouse IgG and anti-rat IgG specific antisera (Sigma). Immunoreactivity was detected by using chemo-luminescence analysis . The molecular species resolved in western blotting were finally analyzed by densitometry, using a dedicate software .Results are expressed as relative densitometry unit (rdu) after normalization with densitometry values of the band obtained for α-tubulin.(nu/nu) mice  were used for establishing thyroid cancer xenografts in vivo. Mice were kept in cages of 4 animals each with water and food ad libitum. Galectin-3 expressing and highly tumorigenic poorly differentiated follicular thyroid carcinoma cell line (ARO) and the derived galectin-3 knockout ARO cells  were considered for these experiments.Pathogen-free 4–5-week-old nude 6 to 107 cells /0.2 ml saline solution, depending by the experiment. Injection of the cells was performed by using a standard insulin needle.Wild-type and interfered ARO cells were maintained in standard culture conditions as aforementioned, detached from tissue culture plates with trypsin 0,05%-EDTA 0,02% (Gibco), washed in sterile PBS and injected subcutaneously into nude mice at a concentration ranging from 7×10No anesthesia was necessary. Fifty animals were used in three different sets of experiments for establishing tumor xenografs. Mice were examined three times a week for signs of measurable tumor growth.in vivo imaging experiments when tumor weight was around 0.5–1 gm. To determine the in vivo expression of galectin-3 in tumor xenografts, some of the tumors were surgically excised and used for galectin-3 expression analysis as reported above. This work was performed according to the specific guidelines provided by the Italian Ministry of Public Health for the animal experimentation. This study has been approved by the Institutional Ethical Committee for animal experimentation at the National Cancer Institute Regina Elena of Rome. The animal facility at the NCI of Rome is certified by the Italian Ministry of Public Health.Mice were selected for 99mTcO4− freshly eluted from (99Mo/99mTc) generator . Labeling efficiency (LE) was assessed by using Instant Thin Layer Chromatography  with 0.9% NaCl as mobile phase.Affinity purified and highly concentrated (5 mg/ml) monoclonal antibody to human Galectin-3  was radio labeled by using the method previously reported ).The strips were analyzed by a computerized mini-radio scanner . LE, was >95% with a final specific activity of 70 µCi/µg (99mTc for labeling (range 250.000∶1 to 4.500.000∶1).In order to optimize the efficiency of antibody labeling several experiments were performed varying the molar ratio 2-mercaptoethanol/mAb (range: 1.000∶1 to 4.000∶1) and the ratio mAb / 99mTc gave the highest LE of 99mTc-anti-Galectin-3 and were used for all further experiments .The reduction of disulfide bridges at a molar ratio of 2145∶1 (2-mercaptoethanol /mAb) and a molar ratio of 1,500,000∶1 for Ab/).Specific stability studies were performed incubating fresh radio labeled antibody in saline or human plasma at 37° C for 24 hrs and evaluating the radiopharmaceutical purity by ITLC-SG at different time points . The stability in saline and plasma was high during the first 6 hours  with a slight decrease at 24 h  Position Sensitive Photo Multiplier Tube (PSPMT), charge readout electronics and a data acquisition system.The High Resolution gamma Camera (HRC) , shown in 2. The crystal dimensions are 2.05×2.05×5.0 mm3 and each crystal is covered by100 µm white reflective epoxy on his five blind surfaces. The crystal-collimator structure is coupled to the PSPMT via optical grease.The patented parallel hole collimator, already described 3 with an active area of 49.0×49.0 mm2. The photocathode is bi-alkali and the multiplication system, composed by 12 metal channel dynodes, provides a gain of 3×106 @ −1000 V.The H8500 PSPMT has an external size of 52.0×52.0×18.0 mmThe multiplied charge is collected by an array of 8×8 anodes.3) front-end electronics, and the signals are sampled with a dedicated compact USB ADC board. The acquisition system provides 4 channels at 20 M Samples/sec. Data acquisition and processing is performed on a Linux Embedded System equipped with ARM CPU PXA255 with 10″ touch screen. The system control and data processing software are proprietary applications developed in C++ language. The system allows performing a real-time acquisition with a refresh time of 0.5 sec. The system is conceived to be a battery operated portable device; as a consequence the detector head weights about 2 kg while the overall weight is about 4.5 kg.The read-out is a miniaturized (52.0×52.0×5.0 mm99mTc) over the whole FOV. The sensitivity is 210 cps/MBq and the uniformity is ±5% while it provides 2.2 mm intrinsic spatial resolution suitable for our imaging experiments in vivo with tumour xenografted mice, and ex-vivo with surgical specimens derived from human patients.The HRC energy resolution is about 20% FWHM @ 140 keV  in saline solution with 1% HSA in presence or absence of 100 µg of unlabelled anti-Gal-3 antibodyAs a proof of concept for imaging differentiated thyroid cancer in human, we performed 99mTc-labeled mAb to galectin-3 in vivo, six animals bearing galectin-3 positive carcinoma xenografts  were considered in a preliminary experiment.In order to evaluate the binding specificity of a 99mTc-labeled mAb to galectin-3 . Images were acquired at 1 hr, 3 hrs, 6 hrs, 9 hrs and 24 hrs post-injection by using the mini-gamma camera. Galectin-3 immunotargeting of ARO tumors was highly efficient in xenografted mice. A central area of tumor necrosis  with impaired binding of the radiotracer was correctly depicted in one of the instances . Preliminary experiments with different galectin-3 positive tumors including melanomas, lymphoma and breast carcinomas were also considered for this purpose with overlapping results (data not shown). As expected, the majority of galectin-3 radio labeled antibody was concentrated in the liver according to the clearance of exogenous mAbs from the blood ). In order to optimize the imaging capture and to demonstrate the binding specificity of the galectin-3 mAb a larger in vivo experiment (repeated in triplicate) was considered.Mice with tumor mass of about 1 gm were injected in the tail vein (i.v.) with 100 µCi of 6 cells/0.2 ml saline solution/mouse; the second group of mice was transplanted with galectin-3 knockout ARO cells , obtained by using a specific galectin-3 RNA interference (iRNA) The experiment included 12 xenografted mice, two groups of six animals each. The first group was transplanted with galectin-3 positive ARO cells at concentration of 5–8×107cells/0.2 ml saline solution/mouse, in order to promote a rapid tumor growth in vivo in about 4 days.ARO-Gal3 negative cells were injected subcutaneously at concentration of 10in vivo in absence of puromycine as selective antibiotic. Galectin-3 down-regulation in stable interfered ARO cells was checked in western blot at different time points (12–72 hrs). After 4 days from cell injection all the mice developed a visible subcutaneous tumor with a median diameter size of 0.5 cm. The animals were then injected in the tail vein with 100 µCi of 99mTc-labeled mAb to galectin-3 (30 µg protein) and whole body images were acquired at 1 hr, 3 hrs, 6 hrs, 9 hrs and 24 hrs by using the high-resolution gamma camera.This was required for maintaining an efficient down-regulation of galectin-3 expression in stable interfered ARO cells, growing + xenografts was obtained between 6 and 9 hours from injection of the radiotracer, whereas galectin-3 negative tumors were not detected at all  over the implanted tumor and over the counter lateral muscle taken as background. As expected the optimal visualization of the ARO-Gal-399mTc-labeled mAb to galectin-3 (30 µg protein) and 5 groups of 4 animals each were sacrificed at 3 hrs, 6 hrs, 9 hrs, 12 hrs and 24 hrs post-injection, respectively.In a different set of experiments, 20 ARO xenografted mice were injected with 100 µCi of in vivo bio-distribution of radio labeled galectin-3 mAb expressed as percentage of injected dose per gram of tissue (%ID/g), showed a rapid blood clearance of the radiopharmaceutical within 3 hours from injection. Most of the radioactivity was detected in the liver and kidneys indicating a prompt clearance of the tracer via the hepatic and urinary tract . The ry tract table 1.99mTc-labelled mAb increased from 3 to 9 hrs post-injection. At 6 hrs post-injection the target tumors were clearly visible while the whole-body activity had essentially cleared (and ).As expected the tumor uptake of the galectin-3 specific ex-vivo. To this aim a fresh cervical lymph node surgically excised from a patient bearing a papillary thyroid carcinoma metastasis , was used as target for studying the binding efficiency of the Gal-3 specific radiotracer.Although translation of these findings in the clinical setting will require the use of humanized mAbs linked to different radionuclides and more extensive preclinical studies, we attempted to simulate a galectin-3-based imaging of human thyroid cancer 99mTc-mAb to Galectin-3 (0.1 µg protein) and 1% human serum albumin, in presence or absence of 100 µg of unlabelled (cold) anti-galectin-3 mAb (displacement assay).Immediately after surgical removal the lymph node was cut in half through the cancer lesion and was incubated at 37° C for 3 hrs in 50 ml saline solution containing 2 µCi of After extensive washing in saline solution a comparative imaging of the two tissue preparations was performed. Results of this experiment are shown in The mini-gamma camera promptly detected the thyroid cancer metastasis (panel A), whereas a scanty tumor uptake of the radiotracer was visible in the control, due to the displacement effect of the cold mAb in excess (panel B).in vivo by using a galectin-3 specific radio-immunoscintigraphy. The use of galectin-3 radiotracer for detection of thyroid cancer in vivo is supported by a solid molecular rationale Altogether these results suggest the possibility to detect thyroid cancer and other galectin-3 expressing tumors 186Re, 177Lu or 90Y, 64Cu, 67Cu). A galectin-3 ‘radiation targeted therapy’ delivers the radiation dose specifically to the galectin-3 positive tumor with limited exposure of normal tissues. Penetration of beta- rays in living tissues is of several millimeters and the therapeutic effect may be obtained also in cancer cells that do not take up directly the radiopharmaceutical (cross-fire effect). This approach could be useful for both detection and treatment of micro papillary thyroid carcinomas , which are currently undetectable preoperatively.Combining galectin-3 imaging with thyroid FNA-cytology, in fact, may allow to distinguishing preoperatively benign from malignant thyroid proliferations with high efficiency. As a consequence many unnecessary thyroidectomies could be avoided and the clinical use of conventional thyroid scintigraphy with radio iodide, which only provides functional information on specific thyroid conditions, could be restricted to more selected clinical questions. Further studies which use humanized galectin-3 specific mAbs conjugated to different radionuclides will be necessary to confirm and validate these findings. If the proposed diagnostic approach will prove successful a targeted radio-ablation of galectin-3 expressing tumors might also be explored by using galectin-3 specific mAbs conjugated to different radio compounds  strips at different time points. The graph shows retention of technetium ranging between 100% and 85% during the first six hours (around 360 minutes) with a slight decrease from 6 to 24 hours (upper panel). The activity for radiolabel mAb anti galectin-3 has been assessed in a titration experiment. The best mAb/Tc ratio has been calculated by varying the activity of 99mTc added to a stable volume of reduced antibody. The labelling efficiency (LE) was evaluated by ITLC-SG. The optimal mAb/Tc ratio found was 1.200.000/1 corresponding to 30–40 mCi of activity and a labelling efficiency up to 95%. An activity exceeding this value did not increase the LE (lower panel B).(8.52 MB DOC)Click here for additional data file.Figure S2Tumor imaging in vivo by using 99mTc-labelled mAb to Galectin-3 and a high-resolution portable mini gamma camera. A) The high-resolution portable mini gamma camera used in this study. B) Image captured by the high-resolution gamma camera in a mouse bearing the galectin-3 positive thyroid carcinoma xenograft ARO, after 6 hrs from i.v. injection of 100 µCi of 99mTc-labelled mAb to Galectin-3. The tumor confirmed at histology was 1.2 cm in diameter and showed a large central necrotic area corresponding to the lacuna that failed to fix the radiotracer (arrow).(10.39 MB DOC)Click here for additional data file.


Sir,P=0.05 level.I read with interest the article of In our opinion, the role of CD1 family molecules in antitumour immune responses, and in particular of CD1a, should be more debated, since its expression was recently described not only in monocyte-derived dendritic cells, but also in nonmesenchymal cytotypes, that is, epithelial cells (We recently described the immunoexpression of CD1a by metaplastic cells in a large series of Barrett's metaplasia, both gastric- and intestinal-type (Based on all these observations, we strongly recommend further studies on the antitumour role of CD1a and its potential role during immuno-surveillance, not only in invasive cancers but also during the carcinogenetic steps.


Recent reports indicate the existence of breast cancer cells expressing very high levels of the Arylhydrocarbon receptor (AhR), a ubiquitous intracellular receptor best known for mediating toxic action of dioxin and related pollutants. Positive correlation between the degree of AhR overexpression and states of increasing transformation of mammary epithelial cells appears to occur in the absence of any exogenous AhR ligands. These observations have raised many questions such as why and how AhR is overexpressed in breast cancer and its physiological roles in the progression to advanced carcinogenic transformation. To address those questions, we hypothesized that AhR overexpression occurs in cells experiencing deficiencies in normally required estrogen receptor (ER) signaling, and the basic role of AhR in such cases is to guide the affected cells to develop orchestrated cellular changes aimed at substituting the normal functions of ER. At the same time, the AhR serves as the mediator of the cell survival program in the absence of ER signaling.We subjected two lines of Michigan Cancer Foundation (MCF) mammary epithelial cells to 3 different types ER interacting agents for a number of passages and followed the changes in the expression of AhR mRNA. The resulting sublines were analyzed for phenotypical changes and unique molecular characteristics.MCF10AT1 cells continuously exposed to 17-beta-estradiol (E2) developed sub-lines that show AhR overexpression with the characteristic phenotype of increased proliferation, and distinct resistance to apoptosis. When these chemically selected cell lines were treated with a specific AhR antagonist, 3-methoxy-4-nitroflavone (MNF), both of the above abnormal cellular characteristics disappeared, indicating the pivotal role of AhR in expressing those cellular phenotypes. The most prominent molecular characteristics of these AhR overexpressing MCF cells were found to be overexpression of ErbB2 and COX-2. Furthermore, we could demonstrate that suppression of AhR functions through anti-AhR siRNA or MNF causes the recovery of ERalpha functions.One of the main causes for AhR overexpression in these MCF breast cancer cells appears to be the loss of ERalpha functions. This phenomenon is likely to be based on the mutually antagonistic relationship between ER and AhR. The arylhydrocarbon receptor (AhR), a ubiquitous basic Helix-Loop-Helix (bHLH) receptor expressed in various tissues in vertebrate species, is best known for its role in mediating the toxic actions of dioxin. On the molecular level it is known that: (a) dioxin binds to AhR, which exists in cytosol as a complex with a number of chaperone proteins, (b) the resulting dioxin-bound AhR migrates into nucleus where it forms a dimer with ARNT, another bHLH protein, (c) it is this dimer, which binds to the dioxin response element (DRE) on the promoters of the dioxin target genes, and (d) thereby causes induction of a number of detoxification enzymes. Tremendous efforts have been made in the past 25 years in elucidating the intricate molecular mechanisms through which this receptor accomplishes its tasks of inducing a number of detoxification enzymes and related proteins, particularly in the liver upon biding of dioxin and related environmental pollutants -3.in vivo in rats ATP (4000 Ci/mmol) was purchased from ICN . TCDD (>99.9% purity) was originally obtained from Dow Chemicals Co. 17-β-Estradiol (E2), and 4-OH-Tamoxifen (Tam) were obtained from Sigma-Aldrich Chemical Co. . β-hexachlorocyclohexane (β-HCH) was purchased from Chem Service , Parthenolide, Resveratrol, and AG879 were obtained from Calibiochem . 3xERE-TATA-luciferase cDNA was a kind gift from D. McDonnell  was a kind gift from Dr. Josef Abel Human recombinant EGF, serum and other media supplies were obtained from Gibco BRL . ATP (Amersham Life Sciences) and T4 polynucleotide kinase  according to the standard methods. DNA-protein-binding reactions were carried out in a total volume of 20 μl containing 15 μg of nuclear protein, 40,000 cpm of DNA oligonucleotide, 25 mM Tris buffer, pH 7.5, 50 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 0.5 mM dithiolthreitol, 5% glycerol, and 1 μg of poly (dI-dC). Supershift analysis was performed by adding 2 μg of monoclonal ARNT, polyclonal RelB (Active Motif), or polyclonal AhR  antibodies to the reaction mixtures. Competition experiments were performed in the presence of a 100-fold molar excess of unlabeled DNA fragments. The samples were incubated at room temperature for 20 min. Protein-DNA complexes were resolved on a 5% non-denaturating polyacrylamide gel and visualized by exposure of the dehydrated gels to x-ray films. In all cases each EMSA test was repeated more than two times to ascertain the reproducibility of the overall pattern of nuclear protein binding to the labeled oligonucleotides.Nuclear extracts were isolated from MCF10AT1 cells as described by to Dennler et al . In brie®, West Pico  as recommended by the manufacturer. For quantitative analysis, respective bands were quantified using a ChemiImager™ 4400 .A polyclonal anti-human AhR (SC-5579), a polyclonal antibody against human ACTIN (SC-1616), a horseradish peroxidase conjugated secondary antibody, and pre-stained standard markers (SC-2361) were obtained from Santa Cruz Biotechnology . Whole cell lysates (30 μg) were separated on a 10% SDS-polyacrylamide gel and blotted onto a PVDF membrane . The antigen-antibody complexes were visualized using the chemiluminescence substrate SuperSignal4/well). After 24 hrs, cells were washed once with PBS, and 1.6 ml of fresh serum/phenol red free growth medium was added before transfection complexes were applied drop by drop to the cells. Cells were transiently transfected for 16 hrs by using 10 μl per well Effectene  with 0.5 μg per well of respective luciferase reporter constructs of the 3 xERE-TATA promoter according to the manufacturer's instructions. The serum and phenol-red free media was then refreshed. After an additional 24 h incubation period, the cells were washed twice with PBS and lysed with 300 μl of passive lysis buffer. Luciferase activities were measured with the Luciferase Reporter Assay System  using a luminometer . Relative light units were normalized to protein concentration, using a Bradford dye assay (Bio-Rad). Experiments were repeated three times and three wells of cells were analyzed per experiment.For transient transfection experiments, cells were plated in 6-well culture plates  for 100 seconds and incubated at 37°C for 4 hrs. All media except for 200 μl was removed from each plate. Afterwards, 2 μl CaCl2(20 mM), 2 μl annexin V-fluorescein isothiocyanate  and 2 μl of propidium iodide (PI) solution (50 μg/ml) were added directly to each plate. The cells were gently mixed and incubated for 10 minutes at room temperature in the dark. Apoptotic cells were counted directly by using the fluorescence microscope . In each experiment, ten representative fields were counted for apoptosis assay. Both annexin V-positive and annexin V-PI-double-positive cells were considered to be apoptotic.Cells were seeded at 1 × 10t test at the significant level of P < 0.05.All quantitative experiments were repeated a minimum of three times and results are expressed as means ± standard deviations. Data were evaluated statistically by one-way ANOVA followed by Student's 2, 10 nM 4-hydroxy-tamoxifen, or 1 μM β-hexachlorohexane (β-HCH), an estrogenic pesticide, throughout a number of passages under our standard culture conditions using "selection medium" which was phenol red-free and contained heat-inactivated serum (2.5%) [2 associated tyrosine kinase, and 3-methoxy-4-nitroflavone (MNF), a specific antagonist to the Ah receptor (AhR) on those cells  or by β-HCH  of AhR overexpression in mammary epithelial cells, we began this project by exposing MCF10AT1 cells to three estrogen receptor interacting chemicals, 1 nM Em 2.5%) . After 2.5% . Aft2. Fortunately we have already conducted a similar experiments on MCF-7 cells which had been selected by the same set of agents utilizing essentially identical procedures for 35 generations [2-selected lines of MCF10AT1 and MCF-7  and β-HCH. A parallel assay on CYP1A1 expression, which is being used here as a marker for the functional activation of AhR, indicated that, while E2-selected ones (i.e. P35E and P20E for MCF-7 and MCF10AT1) still showed the highest expression among all sub-lines within each MCF cell group, β-HCH selected cells (as compared to "mock" selected control cells) exhibit also relatively higher expressions of the ratio of CYP1A1/AhR than Tam-selected ones. This set of data indicated that the level of induction of AhR is not exactly identical to that of CYP1A1, implying that the functional activation of AhR may be governed by a different set of cellular conditions or factors than its mRNA induction. Nevertheless, in both cases, E2-selected cell lines showed clearly the highest levels of AhR and CYP1A1 mRNA expressions among all selected lines. The observation that E2-selection in both MCF10AT1 and MCF-7 cell lines resulted in AhR overexpression and an increased proliferation indicates that estrogen signaling suppression might play a dominant role in these processes. In addition, these observations indicate some growth factor signaling such as that mediated by ErbB2 or their downstream signaling cascades are also likely to play important roles as well. As for the status of the estrogen receptor (ER), these two E2-selected cell lines show some different characteristics  was found to show a drastically low mRNA level of ERα, but E2-selected MCF10AT1 line (i.e. P20E) did not show such a decrease in the mRNA expression of ERα as compared to the matched "mock"-selected control cell line (designated as P20C) (not shown). However, P20E MCF10AT1 cells showed no detectable level of expression of either progesterone receptor (PR) as in the case of P35E . Of great interest to this study was the differential upregulation of AhR and ErbB2 in cell lines selected by E2, 4-OH-tamoxifen and β-HCH respectively. The differences among them might represent different routes through which MCF10AT1 cells could be affected by these chemicals to develop transformation. An interim conclusion we have reached from this follow-up study are: (a) E2 selection resulted in the greatest up-regulation of AhR mRNA expression, (b) β-HCH, on the other hand, greatly increases mostly ErbB2 mRNA expression, and (c) 4-OH-tamoxifen produces an intermediate type of cells showing modest increases in the expression of both of these markers. These findings support our notion that AhR overexpression is a likely result of alteration of the status of the estrogen receptor as a result of exposure to excess E2 or to 4-OH-tamoxifen.In the case of ErbB20E and P20C cells. The results summarized in Table 20E cells both in the presence and the absence of exogenous heregulin (HRG). In addition, the anti-inflammatory agent parthenolide was also quite effective in suppressing proliferation. Resveratrol, which has both AhR antagonist [20E cells. To confirm the role of AhR on cell proliferation further, we have tested the effectiveness of two siRNA preparations -irradiation on cells maintained under a modestly serum-deprived culture conditions (i.e. 24 hrs serum deprivation)   Figure . This UVeviously , but mos) Figure . Each of20E and P20C cells both in the presence and the absence of MNF to gain insight to the differential contribution of AhR in expressing each of these marker expressions between these two cell lines. The results  expression was higher under fresh serum conditions than in serum starved conditions. On the other hand, the effects of serum or MNF on PI3K were not significant. This set of experiments established that the contributions of AhR to the expression of those selected markers are greatly affected by the condition of serum in culture medium. It appears at the same time, there are at least 2 different types of types of markers: (a) those expression is higher under fresh serum conditions (e.g. TGF-α and HRG), and (b) those favor serum starved conditions (CYP1A1 and TGFβ1). The AhR dependency of those mRNA expressions, as judged by the blocking action of MNF, is mostly confined to P20E compared to P20C (except in the case of CYP1A1 and AhR under serum starved conditions), and is more noticeable under the condition where the given mRNA expression is high  than the conditions promoting low expression.At this stage we have decided to investigate the contributions of the overexpressed AhR on the expression of several diagnostic markers under both fresh serum (3 hrs after the medium change with fresh serum) and serum starved (72 hrs after the last medium change with serum). For this purpose we conducted additional qRT-PCR studies on the expression of selected mRNA markers in Pts Table  showed t20E cells under fresh serum conditions only. The results summarized in Table 20E cells were NOX-2 (NADPH oxidase 2), and NFkB. Two anti-inflammatory agents, NS389 (a COX-2 inhibitor) and quercetin  were used to assess the extent of their participation in causing the cellular state of inflammation. The result showed that, as expected, these two agents are reasonably effective in suppressing the expression of NFκB. In addition, they generally reduced the expression of other markers to roughly the similar extents as those achieved by MNF. These findings support the rationale of selecting these mRNAs as the markers for inflammation. They also indicated that the expression of these markers are definitely higher in P20E than in P20C, and that, judging by the suppressive effect of MNF, AhR contributes to their increase expression to certain extent.One of the characteristic functions of AhR is to mediate the action of dioxin to induce cellular inflammatory responses . Prelimi20C and P20E cells under fresh serum (6 hrs after medium change with fresh serum) and serum starved (72 hrs after the last serum change/24 hours since last media change) conditions. In addition, as a positive control, the effect of TCDD (10 nM) was also determined under these conditions to confirm the presence of the AhR:ARNT-DRE complex. The results in Figure 20E cells than those prepared from "mock-selected" P20C cells. This observation indicates that these DRE binding proteins (likely including AhR) are functionally active in terms of their DRE binding even without the presence of the exogenously added ligand under this test condition. Thus, in this system, overexpressed AhR confers P20E cells the additional influence of functionally active AhR over that is found in P20C. Interestingly the effect of TCDD appears to be more pronounce in cells under serum starved conditions, while the extent of protein binding to the DRE sequence in the absence of TCDD is actually higher in serum fresh conditions than starved conditions. This finding suggests that under fresh serum conditions the level of constitutively activated AhR function in P20E, even in the absence of exogenously added ligands, is already very high (i.e. higher than that is found in P20C exposed to 10 nM of TCDD). To test the specificity of AhR functionality, gel-shift (i.e. supershift) assays were performed onP20C and P20E cells under fresh serum conditions  reporter plasmid transfection approach. The result  or that against ARNT (a required dimerization partner for the AhR target gene activation) clearly caused up-regulation of the luciferase expression, indicating that AhR and its dimer with ARNT work antagonistic to the nuclear transcription factors (likely including the ER) binding to ERE as positive regulators.In view of the lack of the difference in the level of expression of ERα protein between these two cell lines, we hypothesized that the actual difference may be in the function of ERα itself. To test this hypothesis, we assessed the levels of estrogen response element (ERE)-mediated gene expression in Pt Figure  clearly 20E) as well as in MCF-7(P35E) cells, E2 was the most effective agent, among tested in terms of eliciting AhR overexpression, and (b) E2 selection resulted in transformed cells with a phenotypic expression of the increased cell proliferation in both cell lines. The above finding suggests that the estrogen receptor (ER) is likely involved in this process of AhR up-regulation at least in these cell lines under our test conditions. In the case of MCF-7 cells we found the mRNA expression of ERα itself in E2-selected cells (P35E) is totally suppressed. On the other hand in the case of E2-selected MCF10AT1 cells (P20E) the extent of down-regulation of expression of ERα mRNA, in comparison to P20C cells, was not relatively modest. Instead we found that the level of mRNA expressions of progesterone receptor (PR) and PS2, both estrogen receptor element (ERE) controlled proteins, were very low, indicating possibly that the function of the ERα is impaired in P20E as a result of its overexpression of AhR. In this regard, the results of ERE-Luciferase (ERE-Luc) assays . In other words, even the remaining AhR still expressed in P20E cells is powerful enough to keep the expression of the ERE-target genes suppressed. An alternate possibility is that there is another factor other than AhR which makes the functional activation of ERE less effective in P20E cells than in P20C. The third possibility, which is even less likely, is that the transfection efficiency of these cells with the ERE-Luc plasmid was compromised due to molecular differences between the two sublines. Nevertheless, the main conclusion derived from these experiments that the functions of ERα and ERE are reduced in P20E cells in comparison to P20C is still firm, clearly supporting our conclusion.Our main strategy for the current study has been first to identify the most likely cause for these MCF breast cancer cells to overexpress AhR by checking the effectiveness of three estrogenic agents in stimulating the expression of AhR for a number of passages, and second to identify phenotypic signs of transformation along the process of selecting those cells by these agents. This approach has yielded two major tangible results: (a) in our main study cell lines, MCF10AT1 , we decided to study MCF10AT1 as our main cellular model in examining this phenomenon of AhR overexpression in more detail. In this regard, the most noticeable finding of the current study has been that AhR overexpression through E2-selection is accompanied with increased cell proliferation in both MCF cells lines. This observation merits special attention, since increased cell proliferation is clearly one of the major phenotypic expressions of advanced transformation of mammary epithelial cells. The most significant pieces of evidence procured for the critical contribution of AhR in helping P20E cells to express the above transformation characteristics were first, the effectiveness of MNF, a specific blocker of AhR and second, that of siRNA against AhR in effectively suppressing cell proliferation of P20E cells. Thus, by this approach we could satisfactorily meet the second objective of this project to unequivocally show, at least in one case, that AhR indeed contributes significantly to the progression of transformation of mammary epithelial cells. To support the above diagnosis of the influence of AhR on progression of cellular transformation, we have further conducted the apoptosis resistance study, which clearly showed that AhR overexpression is intimately associated with the phenotypic expression of apoptosis resistance in P20E cells.The second major objective has been to find the effect of the overexpressed AhR on the increased expression of at least one phenotype indicating the AhR-dependent progression to more advanced state of transformation. It must be noted that MCF-7 Pferation . These cferation . Since m20E and P20C, on the other hand (Table 20E cells is still based on this circumstantial evidence only, and therefore this subject still remains to be resolved in the future.The third topic needing discussion is the relationship between AhR overexpression in these cells and their inflammatory status. The extent of contribution AhR on the expression of these inflammation markers was assessed by the effectiveness of MNF in reducing the mRNA expression of those markers Table . However20E cells are functionally active even without exogenous ligands (e.g. see Figure One final topic needing a brief discussion is how the elevated AhR in MCF cells becomes functionally activated even without addition of exogenous ligands. It must be pointed out that we avoided the use of typical ligands such as TCDD throughout this study (except in Figure 2-selection causes induction of AhR overexpression in two MCF breast cancer cell lines, and that such a phenomenon is accompanied with increased cell proliferation that is significantly dependent on the presence of AhR. The evidence suggests that the basic cause for up-regulation of AhR by E2-selection is related to the E2-induced down-regulation of either expression of ERα as in the case of MCF-7 cells, or the loss of the function of estrogen responsiveness in MCF10AT1 cells.We could clearly establish in the current study that E2: 17-β-estradiol; ERE: Estrogen Response Element; HRG: heregulin; MCF: Michigan Cancer Foundation; MNF: 3-methoxy-4-nitroflavone; qRT-PCR: quantitative-Real-Time – Polymerize Chain Reaction; TCDD: 2,3,7,8-Tetrachlorodibenzo-p-Dioxin.AhR: Arylhydrocarbon Receptor; ARNT: Arylhydrocarbon nuclear transferase; DRE: dioxin response element; EMSA: Electrophoretic Mobility Shift Assay; ER: estrogen receptor; EThe authors declare that they have no competing interests.This study was conceived jointly by both PSW and FM. PSW was responsible for experimental design and completion of all laboratory work unless noted contained in this article. EMSA data Figure  was compThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/9/234/prepub


Liver microsomes of the rat contain a group of hydroxylating enzymes which are coupled to a greater or lesser degree to the electron flow system. In our studies, enzymes believed to be directly associated with the electron flow chain of NADPH, ferricyanide reduction, cytochrome c, cytochrome P-450 and substrate hydroxylation have been observed in livers obtained from normal, tumor-bearing and whole body irradiated rats as well as in Morris hepatoma 7777 and dimethyl-amino-biphenyl induced breast tumors.5 were decreased in the tumor-bearing animal.A significant difference appeared to exist in the activity of NADPH oxidase, NADP-ferricyanide reductase and benzopyrene hydroxylase when normal liver was compared with the liver obtained from a breast-tumor-bearing animal. Both cytochrome P-450 and cytochrome bTissue distribution of benzopyrene hydroxylase in normal, lactating and tumor-bearing Wistar rats has been studied.With the exception of NADPH oxidase, the activities of NADP-cytochrome c reductase, NADPH-ferricyanide reductase, benzopyrene hydroxylase and P-450 were markedly different in liver from Morris hepatoma 7777-bearing Buffalo rat when this was compared with homologous tissue obtained from normal Buffalo rat.Whole-body irradiated animals showed increased P-450 and NADPH oxidase activity in liver as a function of irradiation and there further appeared to be a correlation with decreased ferricyanide reductase activity.


We appreciate the helpful comments by Dr. Rogers but with all due respect there are a number of problematic issues with his remarks on our most recent publication . Firstlyet. al, 2011[et. al, 2012 (n = 349) because it is different project and time setting. Pethlert project was run after Insawang project. Not all participants in Insawang project were recruited into Pethlert project. Though the participants were the same group, some participants did not meet the requirements of Pethlert project such as unavailable during study period, did not return the informed consent, the levels of daily alcoholic drinking were exceeded the criteria of non-alcoholic fatty liver. Therefore, the number of Pethlert project is less than Insawang project.1) The number of participants (n = 315) in Pethlert  al, 2011 is not t2) Commercial MSG is always available and is not expensive for villagers to obtain it. Normally, only one person preparing food for family and that is the key person for MSG consumption in the family. We gave enough information to participants including key persons about the use and the objective of MSG measurement. Participants knew that it doesn’t matter how much the MSG left in the provided box, it will belong to them after 10-day of study. Therefore, it is unlikely that individuals use more MSG than usual because it is free.3) Nowadays, it becomes a nuclear family in Thailand, including the area of study. Some families do not have children below 10 years old; however, some families have one or two children. Families usually prepare a special meal for young children by avoiding chili, MSG, salty or any spicy ingredients. Thus, it is not suitable to divide the MSG consumption in each family by number of all family members which may include infant, toddler, or pre-school children. Therefore, we excluded children under 10 for more reasonable calculation of individuals MSG consumption.4) The association of the median and percentage values of the five criteria of ATP III individually and MSG intake were not observed in our study. This is probably due to the small number in sample size. However, when combined the metabolic disorders as a cluster, the association was found. Every 1-g of MSG intake significantly increased the risk of having metabolic syndrome with the odds ratio of 1.14. In other word, the estimated risk of having metabolic syndrome increased 1.14 fold for each gram of daily MSG consumption. If a person consumed MSG 5 g/day the estimated risk of having metabolic syndrome would be increased 5.7 fold. Therefore, it shall not classify as very week association.5) It is barely to find non-MSG users in the area of study. Moreover, it is hard to identify a person with non-MSG user because if he/she eats out or buys food from retailer or restaurant; generally he/she cannot avoid MSG exposure. Therefore, non-MSG users were not included in our study.Finally, we note that none of the investigators in our paper has any industrial or personal disclosure.We submit that MSG poses a host risk in susceptible individuals and that further data is needed to define those at risk.


Our aim was to determine whether individuals with asthma and elevated circulating eosinophils express signs of different regulatory immune mechanisms compared with asthmatics with low blood eosinophils and non-asthmatic control subjects. In addition, determine the relationship between eosinophilia and circulating Th cell subsets.The immune process driving eosinophilic and non-eosinophilic asthma is likely driven by different subsets of T helper (Th) cells. Recently, in vitro stimulation with anti-CD3/anti-CD28 there was a significant increase of single expressing GATA-3+ and co-expressing T-bet+GATA-3+ cells in the EOS high asthmatics in comparison with control subjects. In addition, T-bet−GATA-3+RORγt+FOXP3+ were decreased in comparison to the EOS low asthmatics. Finally, in a group of control subjects we found that the majority of proliferating Th cells (CD4+CD25+Ki67+) expressed three or four transcription factors.Participants were selected from a random epidemiological cohort, the West Sweden Asthma Study. Immunophenotypes of fresh peripheral blood cells obtained from stable asthmatics, with and without elevated eosinophilic inflammation (EOS high and EOS low respectively) and control subjects, were determined by flow cytometry. No differences in the number of Th1 (T-bet), Th2 (GATA-3), Th17 (RORγt) or Treg (FOXP3) cells were observed between the groups when analysing each subset separately. However, in all groups, each of the Th subsets showed expression of additional canonical transcription factors T-bet, GATA-3, RORγt and FOXP3. Furthermore, by in vivo.The ability of human Th cells to express several regulatory transcription factors suggests that these cells may display plasticity  Asthma is a heterogeneous disease; patients express different phenotypes and have differences in severity, natural history, and response to treatment in vitro studies to suggest that Th cell subsets are not irreversibly differentiated, but can exhibit plasticity by changing transcription factor expression or by expressing multiple transcription factors In addition to the well characterised T regulatory (Tregs) cells and T helper (Th) cells, such as Th1 and Th2, new effector cells, including Th17, Th9 and Th22 have been identified in the last few years Our hypothesis was that patients with a specific asthma endotype could be distinguished by the immunological profile of their circulating Th cells. Therefore, we compared the absolute numbers of circulating Th cells expressing the transcription factors, T-bet, GATA-3, RORγt and FOXP3, in two groups of asthmatics. All participants, both asthmatics and healthy subjects, were selected from an epidemiologically based asthma cohort study, the West Sweden Asthma Study. The asthmatics displayed similar clinical disease profiles, but with distinct differences in the number of circulating eosinophils, and were compared to healthy subjects.1 reversibility greater than 12%. The skin prick test (SPT) was performed using a standard panel of 11 inhalant allergens composed of birch, mugwort, timothy, horse, dog, cat, cockroach, Cladosporium, Alternaria, Dermatophagoides farinae and D. pteronyssinus . The SPT test was considered positive with a wheal and flare reaction ≥3mm for at least one allergen. Asthmatics were considered to have high numbers of eosinophils if blood eosinophils were ≥0.3×109/L and low eosinophils if values were ≤0.2×109/L. Control subjects did not report asthma symptoms, were non-reactive to methacholine or non-reversible, were SPT negative and had a low number of blood eosinophils. All participants were non-smokers.Study participants were selected from questionnaire respondents in the West Sweden Asthma Study (aged 16–75 years) who attended a detailed clinical examination at the Krefting Research Centre, Gothenburg, Sweden and for whom clinical data was available (Visit 1) i.e. NSAIDs). The time period separating Visit 1 and Visit 2 ranged between 3 months to 2 years.The study population consisted of 11 asthmatics with high blood eosinophils (EOS high group), 12 asthmatics with low blood eosinophils (EOS low group) and 9 non-asthmatic healthy controls (control group). Participants were recruited in two separate blocks either during winter time (December to February) or during spring time (April to early June). During a clinical visit (Visit 2) blood samples, nasal lavage (NAL) and induced sputum were collected in addition to spirometry and fractional exhaled nitric oxide (FeNO) measurements were taken. During four weeks prior to Visit 2, none of the subjects had received any vaccination, changed their asthma medication, had any worsening of asthma symptoms, reported any symptoms of infection/cold, had any surgery, had any antibiotics, had any new medication, or had any anti-inflammatory medication .Peripheral whole blood cells were stained for flow cytometry within 1 hour of sampling. The total cell count was obtained using Trucount™ tubes  together with antibodies (Ab) detecting CD45/CD3/CD4 or CD45/CD3/CD4/CD14/CD19 according to manufacturer’s protocol. Additionally, the following Ab panels were used: a) CD4/CD25/T-bet/GATA-3/RORγt/FOXP3 with or without CD3, b) CD45RO/CD45RA/CD3/T cell receptor (TCR)αβ/TCRγδ/CD4/CD8, c) CD45/CD56/CD16/CD19/CD14, d) CD4/CD25/Ki-67/T-bet/GATA-3/RORγt and e) CD4/CD25/Ki-67/T-bet/RORγt/FOXP3. All monoclonal antibodies were purchased from BD Biosciences, eBioscience or Invitrogen  were isolated by density centrifugation using Ficoll-Paque™  and cultured in serum-free AIM-V® + AlbuMAX® medium  alone or in plates pre-coated with anti-CD3/anti-CD28 antibodies  for 48 hours, before flow cytometry staining with antibody panel A (see above). Cell viability was analysed using 7AAD exclusion in the forward scatter-side scatter (FSC-SSC) gate. Cell viability was >96% (range 96.3–99.1%) in cells harvested from medium alone and >88% (range 88.4–95.8%) in cells harvested from anti-CD3/anti-CD28 coated wells.i.e. controls containing all markers except the one of interest were used to set gates  and data were analyzed with FlowJo software . Gating of transcription factors and surface markers were determined using control samples by the Fluorescence Minus One (FMO) approach et gates .Cells from induced sputum and NAL were stained with May-Grünwald and Giemsa  and counted using a Zeiss Axioplan microscope  and stained with the following combinations: a) T-bet/GATA-3, b) FOXP3/GATA-3, c) T-bet/RORγt and d) FOXP3/RORγt. The detection signal was enhanced using fluorochrome-conjugated secondary antibodies . CytospiData were tested for adherence to a normal distribution with the Kolmogorov-Smirnov test . If a normal distribution of sample means could be assumed, unpaired test or paired t-test was used; otherwise the Kruskal-Wallis test followed by the Mann-Whitney U test were applied. Values in tables and graphs are mean±SEM, except in scatter plot graphs where median values are given. Correlations were performed using Pearson correlation test. Values of p<0.05 were considered statistically significant.Clinical characteristics and standard hospital laboratory measurements were similar between the study groups, except for a higher number of eosinophils in the blood in the EOS high group compared with the EOS low and control groups p<0.001; . No diff+CD25+ cells and the expression of the transcription factors T-bet, GATA-3, RORγt and FOXP3 was analysed in the groups. The total number of CD4+CD25+ T cells did not differ between the groups , T-bet/RORγt (ex vivo), FOXP3/GATA-3 (ex vivo) and FOXP3/RORγt (ex vivo).To confirm the flow cytometry data of co-expression of transcription factors in CD4t/GATA-3 , ex vivoet/RORγt , ex vivo3/GATA-3 , ex vivoP3/RORγt , ex vivoin vitro stimulation of the cells could change the co-expression pattern of transcription factors, PBMCs taken from all groups during winter time were cultured with a combination of anti-CD3 and anti-CD28 antibodies. After 48 hours, cells were harvested and stained for co-expression of T-bet, GATA-3, RORγt and FOXP3. The number of cells expressing multiple and single transcription factors was expressed as a percentage of the total CD4+ T cell count and the effect of stimulation compared with medium alone. A sub analysis showed that most Th subsets increased after stimulation compared to medium alone but not all , T-bet/RORγt (in vitro), and FOXP3/GATA-3 (in vitro) and FOXP3/RORγt (in vitro) in CD4+ cells both from stimulated cells as well as in medium alone treated cells. However, in the medium alone treated samples, co-expressing cells were fewer overall than in the stimulated samples.To determine if  not all . Single um alone . Three sow group . Non-spe+CD25+ cells in Panel D and 7.44% of CD4+CD25+ cells in Panel E comprise of Ki-67+ cells  between any cell subset and any of the clinical parameters was single expressing GATA-3+ cells versus FEV1 reversibility. In addition, two more GATA-3 subsets, T-bet+GATA-3+ and T-bet+GATA-3+FOXP3+, showed correlation to FEV1 reversibility -17+ cells expressing RORγt still showed suppressive function in vitro+IL-17+IL-13+IL-4+ cells co-expressed RORγt and GATA-3 after local or systemic immunization with inflammatory dendritic cells Sub-analysis of the T-bet, GATA-3, FOXP3, and RORγt cell populations, enabled up to 15 different populations to be defined. These populations expressed single transcription factors or up to four transcription factors, arguing for the presence of Th cell plasticity in humans as has been earlier proposed from animal models or +CD25high cells that expressed FOXP3 and did not address the co-expression with others transcription factors The only subset that was different after sub-analysis was single FOXP3 cells, which was increased only in EOS high asthmatics compared to controls. Increased Tregs in asthmatics has previously been shown in vitro, a substantial increase in the number of Th cells expressing multiple transcription factors was observed. Moreover, only a small percentage of stimulated cells were found to express a single transcription factor. This strongly argues that circulating Th cells, expressing a master regulatory transcription factor such as GATA-3 also have the capacity to express additional transcription factors upon further stimulation. The question remains unanswered, of whether Th cells that co-express multiple transcription factors are undergoing lineage commitment, or whether an already committed cell can express multiple master transcription factors  showing CD4+CD25+ cells gated for T-bet (Th1), GATA-3 (Th2), RORγt (Th17) and FOXP3 (Treg). One individual from each group is presented in the top three rows. In the final row, the fluorescence minus one (FMO) control used for analyses is shown.(TIF)Click here for additional data file.Figure S2Technical controls for confocal microscopy. Z sectioning is not performed on the following samples. Micrographs of ex vivo samples: 1) Only DAPI staining; 2) DAPI/mIgG1isotype control (IC)/Goat anti-mouse IgG-AF555; 3) DAPI/ratIgG2a IC/Goat anti-rat IgG-AF488; 4) DAPI/ratIgG2b IC/Goat anti-rat IgG-AF488; 5) DAPI/No primary antibody/Goat anti-mouse IgG-AF555/Goat anti-rat IgG-AF488; 6) DAPI/mouse anti-human FOXP3/Goat anti-mouse IgG-AF555; 7) DAPI/mouse anti-human T-bet/Goat anti-mouse IgG-AF555; 8) DAPI/Rat anti-human GATA-3/Goat anti-rat IgG-AF488; 9) DAPI/Rat anti-human RORγt/Goat anti-rat IgG-AF488; Micrographs of in vitro samples: 10) DAPI/mIgG1IC/Goat anti-mouse IgG-AF555 ; 11) DAPI/mIgG1IC/Goat anti-mouse IgG-AF555 (stimulated); 12) DAPI/ratIgG2a IC/Goat anti-rat IgG-AF488 (medium); 13) DAPI/ratIgG2a IC/Goat anti-rat IgG-AF488 (stim.); 14) DAPI/ratIgG2b IC/Goat anti-rat IgG-AF488 (medium); 15) DAPI/ratIgG2b IC/Goat anti-rat IgG-AF488 (stim.); 16) DAPI/No primary antibody/Goat anti-mouse IgG-AF555/Goat anti-rat IgG-AF488 (medium); 17) DAPI/No primary antibody/Goat anti-mouse IgG-AF555/Goat anti-rat IgG-AF488 (stim.); 18) DAPI/mouse anti-human FOXP3/Goat anti-mouse IgG-AF555 (medium); 19) DAPI/mouse anti-human FOXP3/Goat anti-mouse IgG-AF555 (stim.); 20) DAPI/mouse anti-human T-bet/Goat anti-mouse IgG-AF555 (medium); 21) DAPI/mouse anti-human T-bet/Goat anti-mouse IgG-AF555 (stim.); 22) DAPI/Rat anti-human GATA-3/Goat anti-rat IgG-AF488 (medium); 23) DAPI/Rat anti-human GATA-3/Goat anti-rat IgG-AF488 (stim.); 24) DAPI/Rat anti-human RORγt/Goat anti-rat IgG-AF488 (medium); 25) DAPI/Rat anti-human RORγt/Goat anti-rat IgG-AF488 (stim.).(TIF)Click here for additional data file.Table S1Experimental laboratory data.(DOCX)Click here for additional data file.Table S2Fold change expression of in vitro transcription factors.(DOCX)Click here for additional data file.Table S3Antibodies used in the study.(DOC)Click here for additional data file.Methods S1Sputum induction and processing.(DOC)Click here for additional data file.


The recurrence rate of periocular nodular basal cell carcinoma (PNBCC) following treatment with imiquimod (IMQ) has not yet been established. Previous studies did not include histological follow-up. The aim of this analysis was to evaluate the efficacy of topical immunotherapy with 5% IMQ cream for the treatment of PNBCC.Study design: A prospective, non-randomized, and uncontrolled longitudinal case series study. No participants were blinded. Punch biopsy confirmed PNBCC patients were included at the Ophthalmology Clinic of São Paulo University Medicine School Hospital (from 2008 to 2012). Patients were treated with 5% IMQ cream once a day, 5 days per week, for 8–16 weeks. Standard lesion photographic documentation was done during the study. Three months after treatment ended, an image-guided biopsy was performed. Patients were followed at 6-month intervals and annually for control biopsies. Main outcome measures were clinical and histological clearance rates. Data were analysed by frequency distribution for qualitative group characteristics and central tendency measures for quantitative data.Twenty-four patients met the inclusion criteria, 19 of whom remained until the end of treatment. The histological clearance rate was 89.5% and 84.2%, respectively, at 3 and 39.5 months. The 3-year histological clearance rate was 81.8% (9/11) for lesions >10 mm, and 100% (8/8) for lesions <10 mm. Three patients did not tolerate the side effects of the medication and left the study. Two patients were excluded for treatment interruption related to comorbidities.Our results indicated that 5% IMQ cream was a useful alternative treatment for NBBCC, especially for lesions <10 mm. IMQ also showed a significant neoadjuvant effect on lesions >10 mm.NCT 00803907.ClinicalTrial.gov Registration Dec 3, 2008: # The incidence of basal cell carcinoma (BCC), the most common human neoplasm, has increased significantly worldwide over the past few years ,2. More BCC is the main cause of required reconstructive surgery in the periocular region. For facial BCC, Mohs surgery is considered the method with the best chance of cure, with 5-year recurrence rates of up to 6.5% [For cases in which surgery is not possible , topical immunotherapy may be an alternative. Imiquimod (IMQ), an immune modulator, acts by stimulating innate and adaptive immunity and by inducing apoptosis in tumour cells . The ideIMQ use, as an alternative treatment for periocular BCC, was suggested using results from a small case series with an almost 100% reported cure rate -11. HoweThe purpose of the present study was therefore to evaluate clinically and histologically the effects of 5% topical IMQ cream on PNBCC by evaluating for residual tumour rate and recurrence.This was an interventional prospective, non-randomized, and uncontrolled longitudinal case series study, conducted between 2008 and 2012. The study followed the tenets of the Declaration of Helsinki, and was approved by the University of São Paulo Medical School Hospital Institutional Review Board Ethics Committee. All participants gave their informed consent. The ClinicalTrial.gov number was NCT00803907.Patients with periocular biopsy diagnosed as NBCC were included in this study. We limited eyelid margin lesions up to 20 mm, and medial canthus lesions up to 30 mm (largest diameter), with no infiltrating deeper tissues on palpation. All lesions had not had prior treatment. Recurrent BCC lesions and patients with clinical signs of orbit invasion were excluded. Uncooperative patients with no caregiver assistance to correctly apply the medication were also excluded.http://www.nss.gov.au/nss/home.nsf/). All patients were submitted to IMQ cream treatment. No participants were blinded.Main outcome measures were clinical and histological clearance rates. To detect a histological tumour clearance rate of 50% at 3 months, with a 5% significance level and 95% confidence, a sample size of 22 patients was necessary, given an anticipated dropout rate of 10%  was applied once each day at bedtime. To ensure patient safety, we instructed patients and caregivers (or relatives) to use lubricating gel in the conjunctival sac before IMQ cream application. We stressed the correct medication application, using a swab, and taking care to keep the border of the eyelid away from the eye. The cream remained in contact with the tumour for 8 to 10 hours. In the morning, the periocular area was washed with neutral liquid soap. In cases of accidental contact with the ocular surface and conjunctiva, the patient was instructed to wash abundantly with saline solution and apply lubricating eye gel.Artificial tears were prescribed and provided free of charge to the patients, for application every 6 hours during the day. IMQ was used once each day, 5 days a week, for a minimum of 8 weeks and a maximum of 16 weeks. Within this period, treatment was discontinued once the lesion became undetectable by slit lamp examination and palpation.During the treatment period, patients were followed biweekly and information was collected through questionnaires, slit lamp examinations, visual acuity testing, photography, and measurement of lesions, using Image J software (version 1.42) .Baseline measurements were obtained from photographs taken after the initial biopsy so that the amount of tissue removed would not influence the results. The final measurements were based on photographs taken 3 months (12 weeks) after treatment end.We used 2-mm trephine for all biopsies. An image-guided biopsy of the region was performed 3 months (12 weeks) after treatment. Patients with clinical or histological findings of residual lesions were referred for surgical excision and reconstruction. Patients were followed at 6-month intervals, with annual control biopsies.Data were analysed by frequency distribution for qualitative group characteristics and central tendency measures for quantitative data.Throughout the study, 24 patients met the inclusion criteria, 19 of whom remained until the end of treatment. Patients were recruited until December 2012 when the required sample size was obtained. Three patients did not tolerate the side effects of the medication and left the study. One patient suffered an ischemic cerebrovascular accident, and one patient died during the treatment period. Both cases were associated with previous diseases and high surgical risk  interrupted treatment for 2 weeks because of intense local inflammation associated with systemic symptoms. This patient was also the only patient who experienced tumour recurrence, as confirmed by biopsy 2 years after treatment , followed by keratitis (84%), foreign body sensation (79%), lacrimation (58%), low visual acuity (53%), and ectropion (37%). Treatment consisted of frequent lubrication with eye drops. All patients presented with some degree of skin reaction such as hyperaemia, crusting, ulceration, and bleeding during treatment. No medication was prescribed except for cold compresses. No side effects were permanent and all were resolved after the end of treatment.The current investigation confirmed several previous studies reporting favourable results with IMQ ,13 and sPrevious PNBCC studies reported treatments lasting 6 weeks, at seven applications per week and with 71% efficacy, and 50–59% efficacy with three applications ,16. HoweIn the present study, 57.9% (11/19) of the patients had lesions >10 mm. Interestingly, the 3-year histological clearance rate was 81.8% (9/11) for lesions >10 mm, and 100% (8/8) for lesions <10 mm. Eigentler et al. obtained the best results in smaller lesions , and observed that the larger the tumour, the less efficacious the medication and the longer the required treatment [Thus, a treatment duration of 6 weeks was reported to be a negative prognostic factor in the treatment of periocular lesions larger than 10 mm . This fiOnly two of our patients had partial tumour clearance. Both had lesions larger than 10 mm, and in both cases the medication had a neoadjuvant effect, significantly reducing tumour size and thereby facilitating surgery  measured 13.5 mm. Adding a 2-mm safety margin on each side, an area equivalent to half the eyelid (17.5 mm), would have required reconstruction of the eyelid. After treatment with 5% IMQ cream, the diameter was reduced to 4.2 mm. Including the safety margin, the area to be reconstructed (8.2 mm) was less than one third the size of the eyelid. In this patient the safety margins were free and the lesion was submitted to pentagonal excision, cantholysis, and closure.Lesion 8  was reduced from 27.2 mm to 7.6 mm. Thus, instead of performing a complex surgery with glabellar flap rotation and advancement, the patient was submitted to simple excision and direct closure of the tumour-free surgical margins.The patient with lesion 12 interrupted the treatment for 2 weeks because of intense local inflammation mimicking preseptal cellulitis, but systemic symptoms were also observed, especially diarrhoea. The interruption may have influenced the evolution because this was the only patient with recurrence of BCC  .Despite longer treatment, our patients had only minor ocular symptoms  and no permanent eye damage was observed. Cannon et al., in a retrospective study, described conjunctivitis and eye burning sensation as the most common symptoms during treatment of periocular lesions, despite the fact that only three in a sample of 47 patients had periocular BCC, and IMQ was administered three times per week for 4–6 weeks . All symThe main limitation of the present study was the lack of a comparative group . Regarding generalization to other cases, our cases consisted of primary PNBCC lesions without signs of deep infiltration or orbit invasion, therefore it was a limited group of patients.Although surgical excision remains the gold standard for PNBCC and is associated with the highest cure rates , in the The recurrence rate of periocular nodular basal cell carcinoma (BCC) following treatment with IMQ was low. The 3-year histological clearance rate was 100% (8/8) for lesions <10 mm and 81.8% (9/11) for lesions >10 mm. Importantly, IMQ had significant neoadjuvant effects on PNBCC lesions >10 mm.


Myogenic differentiation, which occurs during muscle development, is a highly ordered process that can be regulated by E2F transcription factors. Available data show that E2F3b, but not E2F3a, is upregulated and required for myogenic differentiation. However, the regulation of E2F3b expression in myogenic differentiation is not well understood. To investigate whether E2Fb expression is controlled by miRNAs, we used bioinformatics to combine the database of microRNAs downregulated during myogenesis and those predicted to target E2F3. This identified miR-17 and miR-20a as miRNAs potentially involved in E2F3 regulation. We found that miR-17-92 controls the expression of E2F3b in C2C12 cells during myogenic differentiation. Moreover, we confirmed that miR-20a regulates the expression of E2F3b proteins in vivo using a muscle regeneration model. Skeletal muscle differentiation occurs during muscle growth and regeneration ,2. This MicroRNAs (miRNAs) are 21~23-nucleotide, noncoding RNA molecules involved in post-transcriptional regulation through targeting mRNAs to inhibit its expression . In the To validate the antibody for the detection of E2F3a and E2F3b, we transducted pLVX-E2F3a and pLVX-E2F3b, respectively, into C2C12 cells and measured their protein levels by western blotting. As shown in To confirm a role for E2F3 in myoblast differentiation, we constructed the sh-E2F3 lentiviral vector and verified its efficiency by real-time PCR  and westIncreasing evidence suggests that microRNAs (miRNAs) play a critical role in regulating myogenesis. Because E2F3b is upregulated during C2C12 cell myogenesis, miRNAs directly targeting E2F3b would be expected to be downregulated. We therefore analysed the expression profiles of 720 miRNAs in myogenesis. To investigate E2F3b and miR-20a in vivo, we constructed a muscle regeneration model by injecting cardiotoxin (CTX) into the tibialis anterior muscles of mice to mimic myogenesis in vivo . HematoxWe further injected adenovirus vectors expressing miR-20a (Ad-miR-20a) or control (Ad-miR-Ctrl) into the tibialis anterior muscles of mice 10 days before CTX-injury. Our study shows that E2F3b is indispensable in myogenic differentiation, and suggests for the first time that E2F3b is regulated by miR-17 and miR-20a during C2C12 cell myogenesis. More importantly, we report that E2F3b and miR-20a levels are negatively correlated in a muscle regeneration model and that miR-20a overexpression inhibits the expression of E2F3b in vivo and delays muscle differentiation.We revealed that miR-17-92 regulates E2F3b during myogenesis, and thus promotes muscle differentiation in vivo. However, other miRNAs can also target E2F3. Our screening of miRNAs that target E2F3 and are downregulated in myogenic differentiation also identified miR-15a, miR-490, miR-128, miR-106a, and let-7. Previous studies have shown that miR-128 targets E2F3 and inhibits cell proliferation in glioma . AdditioIn this study, we were unable to construct separate shRNA vectors for E2F3a and E2F3b because of the limited difference in base sequences between E2F3a and E2F3b, although some researchers have previously used E2F3a- and E2F3b-specific knockouts to explore their functions . In our E2F3 reversibly regulates the expression of miR-17-92. The existence of an autoregulatory feedback loop between E2F factors and miRNAs from the miR-17-92 cluster has been proposed . Woods e2 at 37 °C. HEK293A and 293T cells were cultured in DMEM with 10% FBS.C2C12, HEK293A, and HEK293T cells were purchased from the American Type Culture Collection . C2C12 cells were maintained in Dulbecco’s modified Eagle medium  supplemented with 10% fetal bovine serum  until they reached 80–90% confluence. At this point, myogenic differentiation was induced by changing the culture medium to DMEM with 2% horse serum  in a humidified incubator with 5% COAll the protocols were approved by the Animal Ethical and Welfare Committee of Shenzhen University . The male C57BL/6 mice  were used to generate a mouse regeneration model by injecting cardiotoxin (CTX), as described previously . AdenoviTotal RNA was extracted from cultured C2C12 cells or tibialis anterior muscles using RNAiso Reagent  according to the manufacturer’s instructions, and quantified using the NanoDrop2000c Spectrophotometer . miRNAqRT-PCR was performed using the S-Poly(T) Plus method with SNORNA234 as the mouse miRNA control  ,18. SYBREcoRI/XhoI restriction sites. Empty vector was used as overexpression control. Plasmid vectors containing shRNA targeting E2F3 (sh-E2F3) or a nonspecific control (sh-Ctrl) were constructed based on pLVX-hU6. shRNA sequences containing BsmBI restriction site were synthesized using primers listed in To construct overexpression plasmids, the open reading frame of mouse E2F3a was synthesized by Viewsolid Biotech  and E2F3b was amplified from C2C12 myoblast cDNA using the primer listed in EcoRI/XhoI restriction sites of the miRGlo vector .To construct the E2F3 3′UTR luciferase reporter plasmid, the E2F3 3′UTR was amplified from mouse genomic DNA using the primers listed in Lentiviral vectors overexpressing miR-17, miR-20a, and miR-17-92 and adenoviral vectors (Ad-miR-20a and Ad-miR-NC) were constructed as described previously .The transfection of plasmids into C2C12 cells and E2F3 3′UTR luciferase assays were performed as previously described . PlasmidTotal protein isolation was performed as previously described . We collCellular immunostaining was carried out using previously reported protocols with slight modifications . Brieflyt-test with STATGRAPHICS (Centurion XVI.I) software . A p-value of <0.05 was considered statistically significant.All results are expressed as the mean of at least three triplicates for each treatment. Pairwise comparisons were performed using a two-tailed Student’s In conclusion, this study focused on the association between E2F3b and myogenic differentiation, and showed that the miR-17-92/E2f3b axis has an important regulatory role in C2C12 myogenesis and the muscle regeneration model. Our results contribute to the understanding of E2F3b function in myogenesis, and the fundamental mechanism of expression regulation by miR-17-92 in myogenic differentiation.


Spiroplasma-induced embryonic male lethality in Drosophila melanogaster. Transcriptomic analysis reveals that many genes related to DNA damage and apoptosis are up-regulated specifically in infected male embryos. Detailed genetic and cytological analyses demonstrate that male-killing Spiroplasma causes DNA damage on the male X chromosome interacting with the male-specific lethal (MSL) complex. The damaged male X chromosome exhibits a chromatin bridge during mitosis, and bridge breakage triggers sex-specific abnormal apoptosis via p53-dependent pathways. Notably, the MSL complex is not only necessary but also sufficient for this cytotoxic process. These results highlight symbiont's sophisticated strategy to target host's sex chromosome and recruit host's molecular cascades toward massive apoptosis in a sex-specific manner.Some symbiotic bacteria are capable of interfering with host reproduction in selfish ways. How such bacteria can manipulate host's sex-related mechanisms is of fundamental interest encompassing cell, developmental and evolutionary biology. Here, we uncover the molecular and cellular mechanisms underlying  Symbiotic bacteria are able to interfere with host reproduction in ways that are detrimental to the host organism. Here the authors show that Spiroplasma induces DNA damage on the male X chromosome in Drosophila, causing sex-specific apoptosis. The process, mechanism and origin of sex determination have been focal topics in genetics, cell biology, developmental biology and evolutionary biology12Drosophila melanogaster, a female-specific developmental switch gene, Sex lethal (Sxl), counts autosome/sex chromosome ratio in an early developmental stage to establish the choice between male and female alternative developmental pathways at the cellular level. Downstream of Sxl, a cascade of regulatory genes branches into several major pathways, which respectively control sexual differentiation of the soma and neural cells, development of the germ line, and dosage compensation for equalizing X chromosomal transcript levels between males with XY chromosomes and females with XX chromosomesroX1 and roX2), which concentrates on the single male X chromosome and up-regulates its transcriptional level approximately twofoldIn the fruit fly Drosophila species, are commonly associated with symbiotic bacteria6Wolbachia, Spiroplasma, Cardinium and Arsenophonus cause striking reproductive phenotypes such as cytoplasmic incompatibility, male-killing, parthenogenesis and feminization, whereby these symbionts drive their own infection to spread into their host populations in selfish ways8Diverse insects and other animals, including Spiroplasma of Drosophila species, msl mutant hosts fail to express male-killing252627Ostrinia moths, male-killing Wolbachia was reported to suppress host's masculinizing gene expression, thereby disturbing dosage compensation in male embryosHow these microbes interfere with host's reproduction and development is of fundamental interest, but the mechanisms have been poorly understood9111213141517181921Drosophila in combination with sophisticated cytological, molecular and genomic techniques, we demonstrate a number of previously unrecognized molecular and cellular aspects of Spiroplasma-induced male-killing, which provide an integrative understanding of mechanisms underlying the symbiont's reproductive manipulation at the molecular, chromosomal, cellular and organismal levels.In this study, by making use of ample genetic tools and resources available for Spiroplasma-infected and uninfected Drosophila embryos of both sexes at stage 10–11 when infection-associated male-specific abnormal apoptosis startsSxl gene, Sxl-Pe-EGFP, which expresses GFP only in females  and upd2 are ligands in the JAK-STAT  pathway, which are involved in host's survival upon intestinal-bacterial infection3031GstE5, GstE9, Drsl5 and proPO45), which may reflect the general effects of Spiroplasma infection on host's physiology. Strikingly, no genes constituting the Toll and Imd (Immune deficiency) pathways were assigned to this cluster, which is in accordance with previous observations that Spiroplasma infection does not induce host's innate immune responses by evading host's recognition, presumably due to the absence of cell wall3334Sxl-Pe-EGFP transgene worked well  revealed that p53 was strongly activated in infected male embryos , two types of signals were detected: strong signals covering the whole nucleus  with compromised MSL complex function in comparison with a maternal mutant  with the functional MSL complex. When these fly strains were infected with Spiroplasma, DNA damage and abnormal apoptosis in male embryos were attenuated under the msl3-deficient maternal–zygotic mutant genotype  suppresses toxicity of this transgene due to reduced amounts of ectopic MSL complexmsl1L60/+ heterozygotes , cells are arrested at G2 phase during the rest of embryogenesis, thereby resulting in embryos with fewer and larger cellsstg do not undergo cell division after the formation of the MSL complex, which is required for Spiroplasma-induced DNA damage. Therefore, using stg mutant embryos, we can genetically dissect whether bridge breakage in the male X chromosome has a causative role for the chromosome-specific DNA damage induced by Spiroplasma.In an attempt to gain further insight into the relationship between DNA damage, chromosomal breakage and abnormal apoptosis, we genetically blocked cell division during embryogenesis. String (Stg), a CDC25 homologue of Spiroplasma-infected male embryos mutant for stg, abnormal apoptosis was significantly suppressed in comparison with control embryos  may underlie Spiroplasma-induced neural defects cannot be excluded.Abnormal apoptosis in restored , in comp embryos , but the embryos , which whenotype . These rSpiroplasma-induced male-killing during Drosophila's embryogenesis, which include: (i) a large number of genes related to DNA damage and apoptosis are up-regulated specifically in Spiroplasma-infected male embryos  Sp complex ; (iii) t complex ; (iv) thpoptosis ; (v) bripoptosis ; and  Spiroplasma is enriched extracellularly in Drosophila hostsSpiroplasma-infected male embryos (this study) and (iii) mosaic and gynandromorph analyses reveal specific killing of male cells even when male cells and female cells coexist in the same embryos26Spiroplasma-produced factors, so-called effectors or toxins, may be involved in the process. Some bacterial toxins, such as colibactin of Escherichia coli, typhoid toxin of Salmonella typhi and cytolethal distending toxins of various Gram-negative bacteria, are known to cause DNA crosslinking and induce double-strand breaks in eukaryotic cells, though probably not specific to sex chromosomes59Spiroplasma genome encodes specific prophagesSpiroplasma-infected Drosophila hosts62Hamiltonella defensa produces a phage-encoded toxin, thereby protecting its aphid host against parasitoid waspsSpiroplasma-associated Drosophila species65Spiroplasma may act on the host cells to induce some eukaryotic factors that interact with and damage the MSL-bound X chromosome. Future studies should focus on these possibilities.It remains unknown how Spiroplasma-induced male-killing ; a male-killing Wolbachia suppresses host's masculinizing gene expression and thereby disturbs dosage compensation of the male Z chromosome in Ostrinia mothsWolbachia induces defective chromatin remodelling and subsequent abnormal mitotic spindle formation in the male embryos of Drosophila bifasciataArsenophonus inhibits formation of maternal centrosomes required for early male development in Nasonia vitripennisOstrinia's Wolbachia is of particular interest in comparison with the Drosophila's Spiroplasma in that: (i) the entirely different symbiotic bacteria, Wolbachia (α-Proteobacteria) and Spiroplasma (Mollicutes), cause similar male-killing phenotypes in the entirely different insect hosts, Drosophila  and Ostrinia ; (ii) both symbiotic bacteria interact with host's dosage compensation mechanisms for inducing male-killing; (iii) however, while Wolbachia disturbs the dosage compensation of the male Z chromosome in Ostrinia, Spiroplasma scarcely affects the dosage compensation of the male X chromosome in Drosophila; and (iv) Wolbachia's male-killing in Ostrinia is due to dosage compensation defects, whereas Spiroplasma's male-killing in Drosophila is caused by bridge breakage of the male X chromosome and subsequent p53-mediated massive apoptosis.In theory, any male-specific essential molecular, cellular and/or structural aspects of host organisms can potentially be exploited by symbiotic microorganisms to induce male-killingSpiroplasma-induced male-killing, which encompass molecular, chromosomal, cellular and organismal levels. By accumulating such in-depth knowledge for different host–symbiont systems, we will be able to gain insights into the diversity and commonality of symbiont's strategies for interfering with host's sex-related cellular mechanisms, which should lead to a promising avenue for broadening the frontier of cell biology towards the realm of evolutionary biology and ecology.In this study, we provide an integrative picture as to what mechanisms underlie Drosophila Stock Center (Indiana University), the Drosophila Genetic Resource Center (Kyoto Institute of Technology) and several Drosophila researchers. RNA-seq libraries of Spiroplasma-infected and uninfected embryos were constructed by TruSeq RNA Sample Preparation Kit (Illumina) and sequenced by HiSeq 2000/2500 (Illumina). Short reads were aligned to the reference genome sequence of D. melanogaster provided by University of California, Santa Cruz   for at least three libraries were subjected to identification of differentially expressed genes (Fly stocks used in this study were obtained from the Bloomington lease 5) . Of all ed genes . Immunofhttp://www.ddbj.nig.ac.jp) Sequence Read Archive with the accession numbers PRJDB4469/DRA004268/SAMD00044983-SAMD00044986 (Nucleotide sequence data that support the findings of this study have been deposited in the DNA Data Bank of Japan (DDBJ: 00044986 . All othAccession codes: Nucleotide sequence data have been deposited in the DNA Data Bank of Japan (DDBJ: http://www.ddbj.nig.ac.jp) Sequence Read Archive with the accession numbers PRJDB4469/DRA004268/SAMD00044983-SAMD00044986 .Supplementary Figures 1-5, Supplementary Methods and Supplementary ReferencesCluster analysis and gene annotation of selected differentially expressed genes. Gene sets identified in the cluster analysis  are summarized with their Flybase IDs and gene annotations (sheet 1). In sheets 2-7, red color highlights remarkably enriched GO terms .Summary of RNA-seq datasets and mapping results. The number of total reads obtained from each RNA-seq library. Mapped reads, aligned pairs to the D. melanogaster reference genome sequence  and their percentages are also summarized with their accession numbers in the DNA Data Bank of Japan (DDBJ).Transcriptional changes in Spiroplasma-infected and uninfected embryos. Summary of edgeR differential expression analysis. In sheet 1, gene names and normalized read count  are indicated. Results of significance tests are also shown . In sheet 2, gene names and their locus (chromosome) are indicated with log2-transformed fold-change (logFC), average of log2-transformed counts per million reads (logCPM), P-value, and false discovery rate (FDR).


The questionnaire was answered by 47 paediatric oncologists from 15 countries. iCHG progressing during first line therapy with carboplatin-vincristine would be considered for treatment with alternative chemotherapy by 17 (36%) and with surgery plus chemotherapy by 27 respondents (58%). Components suggested for second line were vinblastine (62%), cisplatin (34%) and cyclophosphamide (26%). For third line therapy bevacizumab (BVZ) was considered as suitable by respondents in 53% (often with irinotecan 40%) and vinblastine by 34% respectively. Experience with BVZ in CHG is shown by 53% of respondents regarding at least 95 patients (median treated 1–5 patients per respondent at any age) with a median BVZ administration over 12 months. Effectiveness was reported varying between stable disease and regression while complications were rarely stated . BVZ would be available to 85% of respondents as therapeutic option for iCHG patients. Multiple anti-neoplastic drug regimens are applied for progressive iCHG, partly considered in combination with surgery if safely feasible. BVZ is commonly used at a satisfactory level in third line, mainly combined with irinotecan.Treatment of infant hypothalamic chiasmatic glioma (iCHG) is challenging, about 30% of the children progress during chemotherapy. Despite subsequent treatments the 5 year overall-survival rate is only 70%. This study investigates treatment strategies currently applied for progressive iCHG. A web-based questionnaire was sent out to the members of the SIOPE Brain Tumour Group asking for current second and third line strategies at progression The online version of this article (doi:10.1007/s11060-017-2630-6) contains supplementary material, which is available to authorized users. Low-grade gliomas (LGG) are the most common brain tumours in childhood, accounting for approximately 35% during the first year of life and up to 50% in older children . The chiThe present survey was conducted following intense discussions among the SIOPE LGG working group on possible treatment strategies for infants with progressive CHG. The survey therefore aimed at evaluating treatment strategies currently adopted for progressive iCHG, i.e. progressive tumour in children diagnosed during the first year of life. Following primary reports on the efficacy of bevacizumab (BVZ) in patients with LGG –23, and http://www.thesistools.de . Per definition, tumours were considered as infant CHG if diagnosed during the first year of life. Treatment at progression or at relapse after the end of intial therapy (independent of the age at relapse) was addressed in the present survey. The survey took around 15 min to fill in and consisted of questions regarding treatment strategies, drugs applied for systemic therapy and experience with bevacizumab in CHG patients. Additional questions were asked on characteristics of the participating medical specialist: profession, country of origin, years of clinical experience, yearly amount of children treated with a brain tumour and the subgroups of LGG, CHG as well as iCHG specifically. As the survey aimed at assessing general treatment strategies, it did not collect data on patient characteristics .A web-based questionnaire on experiences and preferences on relapse treatment for iCHG (during or following first line treatment with carboplatin/vincristine) was created at The invitation to participate in the survey with a directly accessible link was sent to specialists in the field of paediatric neuro-oncology consisting of the 220 members of the multidisciplinary SIOPE Brain Tumour Group via email in February 2014 and repeated after 3 weeks.The questionnaire was answered by 47 paediatric oncologists from 15 countries . Two neuro-surgeons were omitted from the analysis as they indicated not to be involved in the decision on systemic treatment approaches. The professional experience of the respondents was reflected by the fact that 85% worked in a clinic with > 20 new brain tumour patients per year. Their years of experience in neuro-oncology were as follows: <5 years n = 5, 5–10 years n = 8, 10–20 years n = 18, > 20 years n = 15 and missing n = 1. 81% treated 1–6 patients with newly diagnosed CHG yearly, 19% seven or more patients per year. The reported rate of iCHG was low, during a 5 year period 57% of respondents cared for 1–3, 23% for 4–6, while 20% for more than six patients with iCHG.Considering the high rate of patients progressing after chemotherapy, the survey asked on the opinion of prolonging first line chemotherapy (see Table during first line therapy with carboplatin - vincristine would be treated with different chemotherapy by 17 (36%) and with surgery plus chemotherapy by 27 respondents (58%). Vinblastine was selected as single chemotherapeutic agent in 62% followed by cisplatin (34%) and cyclophosphamide (26%) in multi-agent combinations.Therapeutic strategies adopted and systemic therapies administered at progression in patients with iCHG are summarised in Table after the end of combined carboplatin—vincristine chemotherapy 54% of respondents would favour vinblastine monotherapy while about 44% would choose a platinum derivative, 35% vincristine and 20% cyclophosphamide, mostly in combination therapy. In addition to systemic therapy 38% of respondents would consider a neurosurgical option (if safely feasible) in combination with further chemotherapy.In case of progression In third line respondents would use BVZ in 53%, in 19/25 (76%) combined with irinotecan. Vinblastine is considered by 34%.The actual experiences in application of BVZ are listed in Table Radiotherapy was not considered an option in second and third line therapy  and exhibits a poor overall survival of 70% –6. Due tAccording to the current recommendations of the SIOPE LGG group, carboplatin and vincristine are mostly administered over a period of 18 months in first line therapy, but a third of responding experts would consider prolongation of first line treatment to ameliorate PFS.The present survey underlines that systemic therapies are considered the mainstay of relapse therapy in iCHG. Even if neurosurgery is (re-)considered, subsequent systemic therapy approaches are deemed necessary to sustain a possible tumour reduction achieved by neurosurgery. Accordingly none of the respondents would select a stand-alone neurosurgical approach at first tumour progression and 6% at second progression.Radiotherapy was not considered an option in second and third line therapy by respondents of this survey. The vast majority would even only consider radiotherapy after failure of multiple lines of systemic therapy. This reasoning will probably be based on the possible devastating side effects of cranial radiotherapy in small children , 11. AlsThe survey revealed that a wide variety of anti-neoplastic agents are applied in subsequent therapy lines.At first progression vinblastine is chosen most often as 2nd line treatment, 62% for progression during carboplatin-vincristine and 54% for progression after the end first line treatment. The excellent tolerability and efficacy of vinblastine in LGG has been documented earlier and its efficacy was also reported in young children with iCHG and those with diencephalic syndrome, either alone or in combination with carboplatin , 24, 25.A wide variety of drugs is used in case of progression during and/or after first line chemotherapy. We could not define specific subgroups of professionals for instance by nationality and/or years of experience, who provided specific alternative chemotherapy regimens. Only the application of cisplatin in combination with etoposide was reported by Italian neuro-oncologists as published by Massimino et al. , 28 AlthThe antiangiogenic agent BVZ, a monoclonal antibody targeting vascular endothelial growth factor is approved for oncologic treatment in adults and was consecutively applied in a variety of paediatric CNS malignancies. In childhood LGG several studies demonstrated benefit of BVZ, specifically in patients with CHG. Packer et al. were the first to report clinical and radiologic responses in ten cases with multiple relapsed LGG with a combination of two-weekly BVZ and irinotecan . Kalra eIndeed application of BVZ for progressive iCHG seems to be a hopeful strategy as shown in the experiences of 53% of our respondents reflecting experience in at least 95 patients. The previously published results of BVZ in LGG incited us to investigate the current practice of BVZ application in progressive CHG in the clinical practice of paediatric neuro-oncology. The results of the survey suggest that while vinblastine is widely applied in second line, BVZ was indicated as most prevalent choice in third line therapy with a majority of clinical responses despite multiple progressions of their LGG.BVZ is not approved for the use in children with LGG and expensive compared to conventional chemotherapy. Considering its effectiveness in LGG , 21 and Following the increasing understanding of molecular pathways involved in LGG pathogenesis , a novelSince a majority of patients with LGG become long term survivors, more insight is needed in the late outcome by gathering data in a common registry . This isOur survey provides insights in the opinions of a broad group of experienced paediatric neuro-oncologists and reflects clinical practice from many different countries in the choices made in treatment of progressive iCHG. The information regarding BVZ in clinical practice refers to at least 95 patients, extending the information on the use of BVZ, so far limited to reports published on 25 CHG patients treated with BVZ. Due to the nature of an online survey there was the possibility that multiple respondents from one treatment centre may have answered the questionnaire. Indeed, two IP-addresses were used by two respondents each. All four could be identified as independent respondents; one of them was neurosurgeon and left out from analysis (see above). Only 47/220 persons invited via email have responded to the survey, maybe distorting the results. As the mailing list of SIOPE also included neurologists, radiotherapists, neurosurgeons, neuro-pathologists, psychologists, endocrinologists, statisticians, basic scientists and ophthalmologists who were not addressed by the present survey, the percentage of paediatric neuro-oncologists responding is much higher than suggested by this number.The efficacy of other targeted drugs, such as specification of MEK inhibitors were not implicated in our survey yet. The survey allowed however to name alternative drugs and the application of a MEK inhibitor was indicated only twice by the respondents. The group of MEK inhibitors deserves specific attention in a possible follow-up survey in the future.Although clinical response to BVZ is reported by the majority of respondents our survey did not extrapolate on the responses in vision and the differences between children with or without neurofibromatosis type 1.Multiple different drug regimens are applied for progressive iCHG. Also in case of a neurosurgical approach subsequent anti-neoplastic treatment is widely applied. The application of targeted drugs emerges as novel treatment strategy. While vinblastine is the most common drug used in second line treatment, BVZ is often used in third line, mostly in combination with irinotecan. Prospective data on the efficacy of BVZ in LGG are needed and should include the infant population. Whereas the present findings cannot replace the lack of prospective trials for this small group of patients with progressive iCHG, they clearly reflect current strategies applied by an international community of paediatric neuro-oncologists. Still, treatment decisions have to be made on a patient per patient basis by an experienced interdisciplinary paediatric neuro-oncology team as depicted by the wide variety of possible therapeutic strategies. We advocate an international registry for children with (i)CHG to prospectively gather information on the multiple progressions and subsequent treatment strategies.Below is the link to the electronic supplementary material.Supplementary material 1 (PDF 1278 KB)