IL-15 is already recognized as a promising candidate for tumor immunotherapy. [1,2] The principle findings in this study are that IL-15 is critical in the inhibition of tumorgrowthin this model and that IL-1a expression is targeted by IL-15-dependent anti-tumor activity (FIG. 5). Taken together these findings provide a rationale for testing IL-15, an anti-IL-1a antibody or a combination of both in lymphoma. The combination of IL-15 and cetuximab increased ADCC activity against triple negative breast cancer cell lines and enhanced cetuximab efficacy against HER1-positive head and neck cancer. [44,45] IL-15 has also been shown to enhance rituximab-dependent cytotoxicity against chronic lymphocytic leukemia cells.  Moreover, the combination of IL-15 and anti-CD40 is more efficacious than either therapy alone in mouse models of metastatic colon cancer or metastatic renal cell carcinoma. [47–49] An anti- IL-1a monoclonal antibody (MABp1, Xbiotech) is currently undergoing clinical trials for treatment of advanced leukemia and other cancers, and has shown evidence of anti-tumor activity.  Alone and in combination, IL-15 and anti-IL-1a therapies are worth exploring against hematopoietic malignancies.
FVB/N-Tg(MMTV-PyVT)634Mul/J transgenicmice were obtained from the Jackson Laboratory (Bar Harbor, Maine) . Since the female PyVT transgenicmice were defective in litter delivery and lactation, all breedings were carried out using male PyVT transgenicmice. The male heterozygote PyVT(+/ 2) mice were cross-bred with female adiponectin knockout mice  and back-crossed for at least 12 generations to obtain mice with reduced adiponectin expression in both C57BL/6J and FVB/N backgrounds. The genotype was verified by PCR analysis of their genomic DNA using primers listed in Table 3. In addition, serum adiponectin levels were monitored using an in-house ELISA, with the standard curve generated from known concentrations of recombinant adiponectin. Note that mice with the genotype of PyVT(+/2)/ADN(2/2) (transgenic PyVT with adiponectin null alleles) could not be found in all generations of alive litters, which included over 800 mice. On the other hand, their embryos were found to be dead at the early stage of foetal development. As a consequence, the sizes of litters with abnormal adiponectin expressions (3–5) were consistently smaller when compared to those of control PyVT breeding pairs (8–10). Therefore, the PyVT transgenicmice with adiponectin deficiency were referred to those with PyVT(+/2)/ADN(+/2) genotypes in this study. The circulating levels of adiponectin in PyVT(+/2)/ADN(+/2) FVB/N and C57BL/6J mice range from 3–15 mg/ml and 0.2– 5 mg/ml respectively, whereas PyVT(+/ 2)/ADN(+/+) micein both FVB/N and C57BL/6J background have a much higher adiponectin level of over 20 mg/ml and 10 mg/ml respectively, with the median values increased by 4–5 folds. Tumor development was closely monitored every 2–3 days. Tumor latency was recorded as the age of mice when palpable tumors
The mice were tested in the social recognition test (SR) as described elsewhere (Choleris et al., 2003; Prado et al., 2006) to assess their social recognition memory and novelty reaction. Seven days before the SR test, the animals were kept in individual cages to establish territorial dominance. Six-week-old Swiss male mice were used as intruders. Before the first trial, an empty chamber was placed in the test cage with the subject mouse to allow spontaneous exploration (Figure 1). During an “initial encounter”, an intruder was placed inside a transparent acrylic chamber with several orifices on the walls. The sessions consisted of five trials of 5 min each, separated by 10 min intervals. In the subsequent four trials, the subject mouse was exposed to the same intruder. In the last trial (5th), a new intruder (2nd intruder) was placed in the same acrylic chamber (which was properly cleaned to remove the odor of the previous intruder), and the time spent sniffing was quantified again (Figure 1). The time spent sniffing in the social interactions was scored with a stopwatch by an observer blinded to the phenotype or treatment. The duration of investigation by the host mouse, consisting of sniffing the intruder through the orifices, was summed over the course of the trial and was used as a measure of social recognition. A reduction in the
ESCs and RST613 single cell suspensions formed embryoid bodies (EBs) by forced aggregation into AggreWell 400 inserts (Stem Cell Technologies, Vancouver, CA) . After 24 hours of culture in the wells, aggregates were transferred into Petri dishes containing 10 ml of serum-free N2B27 media, which consisted of DMEM/ F12 (50/50) medium (Gibco, Grand Island, NY) supplemented with N2 (Gibco), 25 m g/L bovine serum albumin (BSA), 100 U/ mL penicillin, 100 m g/mL streptomycin, and 0.25 m g/mL am- photericin (Mediatech), 2 mM l-glutamine (Mediatech), all com- bined 1:1 with Neurobasal medium (Gibco) supplemented with B27 (Gibco) . Aggregates were maintained on a rotary orbital shaker at 40 rpm. Mesoderm induction was attained by additional supplementation with 10 ng/mL BMP-4 (R&D Systems, Minne- apolis, MN) during days 5–14 of culture. On day 14, EBs were treated with vehicle (0.01% ethanol) or 10 28 M 1a,25(OH) 2 D 3 for
Acute kidney injury (AKI) is characterised by an abrupt decline in renal function, resulting in the inability of the kidneys to perform their basic functions of excretion and maintenance of fluid and electrolyte homeostasis in the organism [8, 9]. Ischaemia–reperfusion injury (IRI) is one of the major causes of AKI. The pathogenesis of renal IRI is complex and not yet completely understood, but the inflammatory response is now accepted as an important pathogenic component, such as endothelial injury and reactive oxygen species (ROS) [10, 11]. Patients with AKI have a higher incidence of complications, such as respiratory failure . Inmice and rats, after ischaemic AKI, lung injury characterised by neutrophil infiltration, pulmonary oedema and increased chemokine and cytokine expression can be observed [13- 15]. AKI can cause increased pulmonary permeability, pulmonary oedema and increased pulmonary neutrophil recruitment, all of which are consistent with acute lung injury.
Since metastasis is the major cause of death from solid tumors in patients with PCa, we evaluated the effect of MIC-1/GDF15 on incidence and extent of metastasis in TRAMP mice. Although TRAMP fmsmic-1 mice live longer and have slower primary tumorgrowth (Fig. 1 A, B, C and D), a significantly higher proportion of these mice demonstrated organs with metastasis. 15 out of 30 (50%) TRAMP fmsmic-1 mice had distant organ metastasis com- pared to 6 out of 30 (20%) of TRAMP mice (Fig. 4A). A significantly higher proportion of TRAMP fmsmic-1 mice had macroscopically detectible metastatic lesions in the liver, kidney and rectum. Whilst the proportion of TRAMP fmsmic-1 mice with surface lung tumors was also increased, this fell short of statistical significance (Fig. 4A). Interestingly rectal metastases developed in 6 out of 30 TRAMP fmsmic-1 but not in any of the TRAMP mice (Fig. 4A). There was no difference in lymph node weight (data not shown), suggesting that lymphatic spread was not increased in TRAMP fmsmic-1 mice. To ensure that the increase in the number
IGF-I or IGF-IR. Indeed, plasmids or oligonucleotides carrying non-methylated CpG sequences are known to trigger innate immune responses and can develop strong toxicity in human cells [36,37]. Here, we used siRNAs rather than antisense RNA vectors or antisense oligonucleotides to analyze the effects of IGF-IR inhibition on immune response triggering. Since specific motifs in unmodified siRNA duplexes may activate cellular sensors of foreign RNA, leading to interferon induction and cell death , the siRNAs used inin vivo experiments were modified at uridine positions of the sense strand with 2 9-O-methyl-uridine. This modification reportedly blocks the immunostimulatory effect of the RNA duplex without significantly attenuating RNAi . However, we noticed a reduction of efficiency with modified siRNA even though 2 9-O-methyl residues were not introduced at positions 9 and 10 in the sense strand; modifications at these positions are known to reduce RISC assembly . Similarly, a detectable reduction in silencing efficacy was found with modified IGF-IR siRNAs, where 29-O-methyl nucleotides were placed in alternating positions on both strands . Moreover, we showed that our modified siRNAs targeting IGF-IR did not inhibit insulin receptor, which possess high homology with IGF-IR, unlike anti- Figure 4. Transfection with siRNAs targeting IGF-IR alters in vivo growth of C4HD cells. (A) Western blot after 48 h transfection of C4HD cells with 29-O-methyl siRNAs. Representative experiment is presented and quantitative analysis of three independent experiments is shown on the right of the blot with means 6 SEM with black bars corresponding to mock transfected cells (NT). ** P,0.01. (B) After 48 h, treated cells were inoculated s.c. into mice. C4HD cells were transfected either with 29-O-methyl ADT siRNA, which targets mIGF-IR (
Many infectious diseases which affect pregnant women can be prevented through vaccination. But vaccination in pregnant individual with live attenuated vaccine strains is always fraught with the theoretical possibility of transmission of infection to the fetus. Transmission has been shown for certain live attenuated virus like the triple vaccine for measles, mumps and rubella (MMR) and vaccinia virus . The diseases for which vaccination is recommended during pregnancy by CDC’s (Centers for Disease Control and Prevention) Advisory Committee for Immunization Practices (ACIP) includes hepatitis, influenza  (viral), pertussis, tetanus, and diphtheria (bacterial). For all the above diseases, the vaccines used are subunit, killed or toxoid vaccines which do not cause transmission of disease to the fetus and induces a Th2 mediated immune response. The two approved Salmonella vaccines for human are Ty21A (live attenuated) and Vi polysaccharide vaccine. The Vi antigen of Salmonella is encoded by highly unstable and mobile DNA island called Salmonella pathogenicity Island 7 (SPI-7) and the mutation has been shown in the viaB locus [22,23] The isolation of Vi negative clinical isolates of S. Typhi from human patients indicates that the Vi vaccine will not protect the individuals against infection caused by Vi-negative Salmonella strains [24,25,26]. The live attenuated vaccinia virus should be given at least three months before the onset of pregnancy  to rule out the possibility of its transmission to the fetus. In our current vaccination regime, we have used 1 dose followed by two booster doses of our live attenuated vaccine strain DpmrG-HM-D (here after DV-STM-07); prior to mating. We found that DV-STM-07 is completely safe in terms of transmission to fetus and is effective in preventing abortion when challenged with WT during pregnancy. In our previous study, we reported that live attenuated vaccine DV- STM-07 is a very potent candidate as a vaccine and was able to reduce the bacterial load of virulent Salmonella from the organs of the vaccinated mice. Very low dose of vaccine is required to protect the mice from lethal dose of virulent Salmonella . In our current study, we have tried to further elucidate the mechanism by which our vaccine strain achieves protection and at the same time prevents abortion. We have shown in this study that the natural Th2 bias of pregnancy is not altered by the vaccine strain whereas the wild type Salmonella induces a Th1 mediated immune response with increased Th1 cytokines (IFNc, TNFa) leading to abortion [28,29]. We have also shown that the protection is through a strong Th2 response. We demonstrated that the vaccine strain is able to confer immunity to the next generation. Pups of vaccinated
appropriate markers can be used for the unequivocal identification of type II NKT cells, these cells have been less well studied than iNKT cells, and knowledge of their function is largely inferred from comparisons of phenotypes in Jalpha18 2/2 and CD1d1 2/2 mice. The majority of data from solid tumor models suggests that type II NKT cells suppress the anti-tumor response [2,12–14]. The mechanism for this suppressive effect is generally thought to be mediated by inducing a Th2 cytokine bias through secretion of cytokines such as IL-4 and IL-13. However it is also apparent that other unknown mechanisms, distinct from altering in the Th1/ Th2 cytokine balance, might also be involved [2,35]. In contrast to their suppressive functions in solid tumors, our data suggest that type II NKT cells are activating in the anti-lymphoma immune response. Development of better techniques for type II NKT cell identification and function would greatly aid in understanding their function the cancer microenvironment.
Our results also suggest a role for innate immunity inIL-18- induced tumor suppression. In RAG1 2/2 mice, which fail to develop adaptive immunity, the presence of IL-18 resulted in significantly smaller tumors (Fig. 4 A). Furthermore, in both RAG1 2/2 and immunologically intact mice, IL-18 correlated with greater tumor infiltration of macrophages (Fig. 3 E & F, Figure S3 A & B) and neutrophils (Fig. 3 G & H, Figure S3 C & D). An involvement of macrophages in the anti-tumour effect of IL-18 is further supported by the finding that intratumoral injection of rIL-18 into subcutaneous tumours elevated serum levels of MCP- 1 (Fig. 6 E). Macrophages express the IL-18R, and can be activated by and secrete IFN-c , and may be central to the development of adaptive immunity induced by IL-18. Kito et al. demonstrated that macrophages activated through stimulation with IL-12 and IL-18, produce IFN-c which induces nitric oxide secretion that is cytotoxic for tumors . IL-18 is chemoat- tractive for neutrophils , induces the expression of IL-8 which Figure 6. Intratumoral injection of rIL-18 inhibits growth of subcutaneous tumors. C57BL/6 mice were injected subcutaneously with 2610 6 RM1(BM)/B4H7-luc cells. IL-18 (1.5 ug) or vehicle (Ctrl) was injected intratumorally in15 ml volume, or 100 ml when injected i.p., for 5 consecutive once tumors reached 6 mm 66 mm in size. A) post-mortem tumor weights from two independent experiments which showed similar results. Statistical analysis was performed by non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparison’s test. B) Growth rates of subcutaneous tumors injected intratumorally with IL-18 or vehicle (Ctrl) are shown from one representative experiment. Staining for F4/80 (macrophages) in tumors treated with IL-18 (C) or vehicle (D) is shown. E) Concentration of cytokines inmice serum from Fig. 6 A is shown. Graphs show median concentration and range. Statistical analysis was performed by non-parametric Mann-Whitney test.
The ablation of a conditional Ets2 loxP allele  was studied both in mammary stromal fibroblasts and in mammary tumor epithelial cells in the MMTV-PyMT (PyMT) mammary cancer model . For this purpose, Fibroblast Specific Protein 1 promoter-Cre (Fsp-Cre) was used for specific deletion intumor fibroblasts, while MMTV-Cre was used to target epithelial cells [15,29]. Initial experiments indicated that deletion of both alleles in homozygous Ets2 loxP/loxP mice was inefficient with several different Cre-driver lines [14,15], including Fsp-Cre (data not shown). In order to facilitate the complete ablation of Ets2 within the fibroblast and epithelial compartments, mice were generated carrying the Cre transgene along with one conditional Ets2 loxP allele and a conventional Ets2 knockout allele targeting the DNA- binding domain, Ets2 db . We evaluated tumor development in both PyMT;Fsp-Cre;Ets2 db/loxP and PyMT;MMTV-Cre;Ets2 db/loxP experimental groups and in control PyMT;Ets2 db/loxP mice. No difference was observed intumor initiation; however tumor progression was significantly impeded by the ablation of Ets2 specifically in mammary fibroblasts. The average weight of tumors 30 days after tumor initiation in PyMT;Fsp-Cre;Ets2 db/loxP mice was 1.5-fold lower than in either control PyMT;Ets2 db/loxP or PyMT;MMTV-Cre;Ets2 db/loxP mice (P,0.01, Fig. 1A). Additionally, H&E staining of inguinal mammary glands from control PyMT;Ets2 db/loxP mice showed solid tumors throughout the mammary gland, whereas tumors had not invaded the entire fat pad of PyMT;Fsp-Cre;Ets2 db/loxP mammary glands (Fig. 1B, yellow outline indicates area with less tumor invasion).
Since general hyperactivity does not appear to be the explanation of the current data, decreased anxiety inIL-1RI 2/2 mice appears the more likely account. The finding that the disruption of IL-1RI expression is associated with a decrease in anxiety is consistent with one previous study inIL-1RI 2/2 mice . Since it is well described that acutely elevated IL-1b induces anxiety [21,22] and that the anhedonic and decreased neurogen- esis effects of chronic unpredictable stress are mediated by IL-1 , it seems reasonable to argue for a role for endogenous IL-1 in inducing anxiety in normal animals and that decreased respon- siveness to endogenous IL-1 drives decreased anxiety. Since increased activity in a novel open field likely reflects the triumph of exploratory drive over anxiety in an unfamiliar environment, it might be that the increased activity in the open field and the increased time in the open arms of the elevated plus maze are both expressions of decreased anxiety and that this is brought about by decreased endogenous IL-1 action in brain anxiogenic centres. It is, of course, impossible to pinpoint the locus of reduced anxiety in the brain of a global knockout animal. However, there is considerable evidence to suggest an important role for the hippocampus, and particularly the ventral subregion of the hippocampus, during ethological, unconditioned tests of anxiety like the elevated plus maze and the open field [24,25,26,27,28,29,30]. It has been argued that during these unconditioned tests, anxiety is generated by the approach/ avoidance conflict experienced by the animals [27,31]. For example, on the elevated plus maze the mouse is faced with the choice of either approaching and exploring the open arms, or avoiding them and staying safe in the enclosed sections of the maze. An important component of the anxiety response in normal animals is the behavioural inhibition of motor activity and, in particular, inhibition of the approach response towards the open arms [31,32]. In the present study IL-1RI 2/2 mice were less able to inhibit the approach response during these approach/avoidance conflict tests. It is notable that the IL-1RI 2/2 mice were also less able to inhibit their approach responses to the decoy beacon during the spatial discrimination version of the MWM task. Although we should be cautious about over-interpreting what is only a mildly significant effect in the beacon watermaze task, it nevertheless may help to identify a key psychological process (behavioural inhibition of approach responses) which could be important, not only in interpreting certain tests of cognition but also during anxiety, and which could also be a key target for modulation in sickness behaviour.
Malaria remains a major source of morbidity and mortality throughout the tropical regions of the world causing up to 1 million deaths every year, mainly in children. Although infection with malaria parasites is common, only 1 to 2% of infections lead to severe life-threatening disease charac- terized by a range of clinical features including coma, severe anemia, respiratory distress, metabolic acidosis, or multiorgan failure. Animal models of infection are indis- pensable tools to better understand the dynamic host- parasite interactions that lead to the onset of different infection outcomes. We now show that DBA/2 mice infected with P. berghei ANKA constitute a rodent model for malaria-associated acute lung injury (ALI). Up to 60% of these infected mice develop respiratory problems includ- ing dyspnea, airway obstruction and hypoxemia and die soon after. The most common pathological findings were pleural effusion, pulmonary hemorrhage and edema, features common to human malaria patients that show life-threatening respiratory distress. Malaria-associated ALI in this model correlates with high levels of circulating vascular permeability factor, VEGF, and its blockage by different means leads to protection from ALI. The existence of such a model of disease will certainly contribute to a better understanding of malaria-associated pathology and possibly to the design of novel intervention strategies.
The human lung adenocarcinoma cell lines A549, SPCA-1and H1975 were purchased from the National Cancer Institute (Bethesda, MD, USA). The human lung squamous cell line SK- MES-1 and the human bronchial epithelial cell line BEAS2B were obtained from American Type Culture Collection (Manassas, VA, USA). Human umbilical vein endothelial cells (HUVECs) were purchased from American Type Culture Collection (Manassas, VA, USA). The BEAS2B, A549, SPCA-1 and H1975 cells were cultured in Roswell’s Park Memorial Institute (RPMI) 1640 medium (Hyclone, Logan, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Gaithersburg, USA). The SK-MES-1 cells were grown in MEM (Gibco, Carlsbad, CA, USA) supplemented with 20% FBS. HUVECs were cultured in endothelial cell growth medium M199 supplemented with 15% FBS, 1 mg/ml low serum growth supplements and 2 mM glutamine. All cells were incubated in 5% CO 2 at 37uC. RBP2
fibrogenesis after chronic toxic injury or bile duct ligation . These findings are in line with clinical studies showing an association between the CCR5D32 polymorphism and the degree of the HCV-related hepatitis . Furthermore, analysis of fibrogenesis in CCR1 and CCR5 chimeric mice revealed a divergent function of these chemokine receptors in the liver. Interestingly, CCR1 mediates its pro-fibrogenic effects predomi- nantly through hematopoietic cells, whereas CCR5 mediates liver fibrosis mainly through resident liver cells, especially through hepatic stellate cells . Their shared other ligand CCL5 is known to be involved in this process, as CCL5 2/2 mice also showed lower degree of experimental liver fibrosis associated with reduced stellate cell activation and immune cell infiltration . Accordingly, systemic administration of Met-CCL5 or (44)AANA(47)-CCL5, a mutated CCL5 protein, led to a strong attenuation of experimental liver fibrosis in vivo [10,29]. These findings, together with the knowledge that CCL3 is also a prominent ligand of the receptors CCR1 and CCR5, has brought us to further investigate the functional role of CCL3 during experimental liver damage.
The study design is illustrated in Figure 1. Twenty 6 weeks old female NMRI (Naval Medical Research Institute) nude mice were acquired from Taconic Europe, (Lille Skensved, Denmark). After 1 week of acclimatization, the mice were inoculated with H727 cells (5–7610 6 cells in 100 uL medium mixed with 100 uL Matrixgel TM Basement Membrane Matrix; BD Sciences, San Jose´, CA, USA). The inoculates were injected subcutaneously on the left and right flank during anesthesia with 1:1 V/V Hypnorm R (Janssen Pharmaceutica NV, Beerse, Belgium)/Dormicum R (Roche, Basel, Switzerland) i.p. Tumors were allowed to grow for 2 weeks. Half of the mice were FLT-PET scanned and the other half were FDG-PET scanned. At day 0 ‘‘baseline’’ examinations with FDG or FLT PET-scans as well as CT-scans were performed. Thereafter we randomized the mice to treatment with everolimus (n = 10) or vehicle (n = 10). The mice were treated daily i.p with 0.250 ml solution, either vehicle consisting of 18 ml 5% glucose and 2 ml DMSO, or 10 mg of everolimus (Sigma- Aldrich, Copenhagen, Denmark) diluted in 18 ml 5% glucose and 2 ml DMSO. Daily dose of everolimus was 5 mg/kg . The mice were sacrificed on day 10. CT scans for measurements of tumor size were performed at baseline and at day 1, 3, 7 and 10. FDG and FLT PET imaging were performed at baseline, and at day 1, 3 and 10. Twenty tumors were inoculated for each PET- scan group. At the time of PET imaging, sixteen tumors in the FLT group and 20 tumors in the FDG group were suitable for measurements.
anti-angiogenesis [12,14–16,35,36], direct suppression of tumorgrowth , and inhibition of cyclooxygenase-2 expression , depending on the characteristics of individual tumors. In the present study, we further explored the molecular mechanism whereby IL-27 induces direct suppression of tumorgrowth of human melanomas. We elucidated that IL-27 augments the expression of TRAIL and TLR3 together with RIG-I and MDA5 in human melanomas, and that IL-27 and a TLR3 agonist, poly(I:C), cooperatively enhance TRAIL expression and inhibit their tumorgrowth partly in a TRAIL-dependent manner as illustrated in Fig. 6. The cooperative effect could be ascribed to the enhanced expression of TLR3, but not RIG-I or MDA5, by IL-27 (Fig. 3A, B, D-G and Fig. S2A-D), which is consistent with facts that RIG-I and MDA5 sense transfected poly(I:C), but not naked poly(I:C), in non-phagocytic cells [38,39]. Since IL-27 augmented only marginal apoptosis even in collaboration with poly(I:C) (Fig. 2C and D), IL-27 is considered to mediate cytostatic activity rather than cytotoxic activity as reported before . Furthermore, the combination of IL-27 and poly(I:C) significantly suppressed in vivo tumor progression in the human melanoma xenograft model using immunodeficient NOD/SCID mice (Fig. 5). When a higher dose (100 m g) of poly(I:C) was injected, tumor progression was more markedly inhibited regardless of the combination with IL-27 (data not shown) as reported previously [32,40]. However, we noticed that 3 out of 5 mice died even without any tumor after the experiment was completed. In contrast, intriguingly, no mice died with treatment based on combined IL-27 and poly(I:C) (data not shown). Although further studies are necessary, the combination treatment may be beneficial in reducing the potential toxicity arising from a higher dose of poly(I:C) .
Inbred B6SJL SOD1 G93A mice (The Jackson Laboratory, Bar Harbor, ME, USA) were used in this study because they provide a suitable ALS disease model. These mice carry a G93A mutation (substitution of Glycine to Alanine at residue 93) in the human gene superoxide dismutase 1 (SOD1). Hemizygous mutants, obtained by crossing a mutant male with a wild-type (WT) female, were used for all of the experiments. The offspring were identified by PCR amplification of DNA extracted from tail tissue as described in The Jackson Laboratory protocol for genotyping hSOD1 transgenicmice (http://jaxmice.jax.org/pub-cgi/proto- cols.sh?objtype = protocol,protocolid = 523). The animals were housed in the Unidad Mixta de Investigacio´n of the University of Zaragoza, in accordance with international guidelines for the use of laboratory animals. Food and water were available ad libitum. Routine microbiological monitoring did not reveal evidence of infection with common murine pathogens. All of the experimental procedures were approved by the ethics committees of our institutions and followed the international guidelines for the use of laboratory animals, particularly the guidelines for the preclinical in vivo evaluation of pharmacologically active drugs for ALS/MND.
Figure 1. Characterization of TFE3 expression and TFE3 rearrangement in the initial tumor and in TF cells. A) Immunohistochemical staining for TFE3 of the initial tumor. Labeling with anti-TFE3 antibodies was also performed on cells from passages 14 and 16 (P14 and P16 TFE3 cells) embedded in paraffin. TFE3 labeling was also performed on ccRCC cells cultured under the same conditions as TF cells. ccRCC cells served as a negative control. Note the cytoplasmic background instead of only nuclear labeling. B) Image a: An uncultured cell suspension from the renal cell tumor hybridized with a dual-color break-apart FISH probe framing TFE3. A rearrangement of TFE3 in the upper nucleus (tumor cell) is observed with BAC probes CTD-2534B7 (red signal; 39 side of TFE3) and CTD-3009K20 (green signal; 59 side of TFE3). The red and green signals are clearly separated as a result of the pericentric inversion of chromosome X (thin arrows). Two rearranged signals are observed because the abnormal chromosome X is duplicated intumor cells. In contrast, red and green signals are closely juxtaposed in the two normal nuclei that contain one X chromosome, respectively (thick arrows). Image b: A normal partial metaphase cell hybridized with a dual color break-apart BAC FISH probe framing TFE3. BAC probes CTD-2534B7 (red signal; 39 side of TFE3) and CTD-3009K20 (green signal; 59 side of TFE3) are closely juxtaposed at Xp11.23 on the short arm of the X chromosome. Image c: A partial abnormal tumor metaphase cell (cell line, passage 9) hybridized with a dual color break-apart FISH probe framing TFE3. As a result of the X chromosome pericentric inversion, BAC probe CTD-3009K20 (green signal; 59 side of TFE3) is translocated from its normal location at Xp11.23 to NONO locus at Xq13.1 on the long arm of the X chromosome. BAC probes CTD-2534B7 (red signal; 39 side of TFE3) remains at the original TFE3 locus at Xp11.23. Image d: A partial abnormal tumor metaphase cell (cell line, passage 9) hybridized with a dual-color break-apart FISH probe framing NONO. As a result of the X chromosome pericentric inversion, BAC probe RP11-753F2 (green signal; 59 side of NONO) is translocated from its normal location at Xq13.1 to TFE3 locus at Xp11.23 on the short arm of the X chromosome. BAC probes RP11-624G23 (red signal; 39 side of NONO) remains at the original NONO locus at Xq13.1. C) Western blot analysis of the presence of TFE3 in cells from the ‘‘TFE3’’ tumor, in ccRCC 786-O cells and ccRCC cells obtained from an independent tumor. 786-O and ccRCC cells served as negative controls. ERK served as a loading control.