In suchin vitro  and in v[in vivo , and dis[in vivo  and migh[in vivo  and stor[in vivo . Their a[in vivo .Here we study neuronal avalanches in an open-loop system of cortex and striatum. The striatum is the main input structure of the basal ganglia and plays an important role in motor and cognitive functions. Understanding how the striatum responds to cortical inputs has crucial importance for clarifying the overall functions of the basal ganglia. The projection neurons of the striatum have a high threshold for activation and receive excitatory input from different regions of the cerebral cortex. Although the striatum contains several distinct cell types, 90-95% are GABAergic medium spiny projection neurons (MSNs). These cells are major targets of cortical inputs, the recurrent connection among them mediate weak feedback inhibition and neighboring MSNs are not likely to share cortical inputs . Fast-spPreliminary experiments indicate that activity bursts in the striatum do not follow a power law with characteristic exponent of -1.5. Here we developed an abstract model of the cortico-striatal network that reproduces statistics observed in experimental data. We discuss which kind of connectivity between cortex and striatum, as well as connectivity and strength of inhibition within striatum can lead to results that are in line with experimental data.


Plaque morphology directly correlates with risk of embolism and the recently developed dual-source computed tomography angiography (DSCTA) may help to detect plaques more precisely. The aim of our study was to evaluate the prevalence and morphology of carotid and cerebrovascular atherosclerotic plaques in patients with symptomatic type 2 diabetes mellitus (DM) by DSCTA.From July 2009 to August 2010, DSCTA was prospectively performed in 125 consecutive patients with symptomatic type 2 DM. We retrospectively analyzed plaque type, distribution, and extensive and obstructive natures were determined for each segment for all patients.st to 5th segments was observed in 67 (58.8%) patients and from the 6th to 10th segments in 35 (30.7%) patients. The most common site of all detected plaques was the cavernous segment. Regarding stenosis, there were significantly more nonobstructive than obstructive stenosis .Atherosclerotic plaques were detected in 114 (91.2%) patients. Relatively more noncalcified (45%) and calcified (39%) plaques and less mixed (16%) plaques were observed (p < 0.001). Noncalcified plaques were found mainly in the intracranial arteries (81.8%), mixed plaques in the intracranial arteries (25.2%) and intracranial internal carotid artery (ICA) (56.1%). Calcified plaques were found mainly in the intracranial ICA (65.9%) and extracranial arteries (28.2%) . Extension of plaques from the 1DSCTA detected a high prevalence of plaques in patients with symptomatic type 2 DM. A relatively high proportion of plaques were noncalcified, as well as with nonobstructive stenosis. The distribution of plaques was extensive, with the cavernous portion of ICA being the most common site. The number of people with diabetes mellitus (DM) in 2010 is estimated to be 285 million, which was approximately 7% of the adult world population . RecurreAlthough digital subtraction angiography (DSA) is the gold standard for assessing degree of stenosis , it cannWithin a 14-month period from July 2009 to August 2010, we retrospectively analyzed 125 consecutive patients with symptomatic type 2 DM who were clinically suspected with stenosis of the carotid and intracranial arteries. Inclusion criteria were patients with type 2 DM having neurologic symptoms. Symptoms were classified as follows: (1) transient symptoms, i.e., transient ischemic attack or amaurosis fugax and (2) prior stroke, i.e., any ischemic event with neurologic symptoms. Exclusion criteria were as follows: history of allergy to iodine-containing contrast medium, renal insufficiency , pregnancy, and lack of laboratory or clinical data. All patients underwent DSCTA of the carotid and cerebrovascular arteries. The following baseline demographics, laboratory findings, and medical history were obtained: age, gender, serum cholesterol level, serum HbA1c level, history of DM, hypertension, daily smoking, and cerebral infarction (CI). DM was diagnosed based on a random plasma glucose level ≥11.1 mmol/L or a fasting plasma glucose level ≥7.0 mmol/L. Hypertension was defined as treatment with antihypertensive agents at the time of admission, systolic blood pressure ≥140 mm Hg, or diastolic blood pressure ≥90 mm Hg. Subjects were classified as smoking if they smoked at least a cigarette per day in the year before the study. CI, including lacunar infarct and territorial infarct, was confirmed by CT or MRI.All examinations were performed with a DSCTA scanner  using a standardized enhanced carotid and cerebrovascular artery imaging protocol. The scanning parameters in the dual-source mode were as follows: 330-ms gantry rotation time, tube A  and tube B  with a collimation of 2 × 64 × 0.6 mm and pitch 0.65. The delay before CT acquisition after injection was determined using bolus tracking software, and a circular region of interest (ROI) for attenuation measurement was placed in the right common carotid artery (CCA). As soon as the signal intensity in ROI reached a threshold of 100 HU, data acquisition was started. A nonionic contrast medium  was administered immediately followed by 40 mL of saline chaser solution was administered through an 18-gauge intravenous antecubital catheter at a flow rate of 6 mL/s with a dual-head power injector . CT data was acquired in a caudocranial direction from the aortic arch to the mid-skull.Image reconstruction was performed at another 3 D image analysis workstation  with the following parameters: a soft convolution kernel, slice thickness of 0.75 mm, and increment of 0.4 mm. The DSCTA reader was permitted to utilize any or all available postprocessing image reconstruction algorithms, including maximal intensity projection, multiplanar reformat, curved planar reformat, or volume rendered technique and dual energy direct bone removal CTA (DE-BR-CTA).All images were evaluated by 2 experienced observers unaware of the clinical history of the patients. In case of disagreement, a joint reading was performed and a consensus decision was reached.In order to improve reproducibility of the results, the analysis was performed on a segmental basis. According to the criteria of North American Symptomatic Carotid Endarterectomy Trial (NASCET) , carotidContinuous variables were expressed as the mean ± standard deviation. Categorical variables were presented as percentages. Comparisons between different types of plaque, degree of stenosis, and plaque location were performed by the nonparametric chi-square test. All statistical analyses were performed using SPSS version 16.0 . A p value < 0.05 was considered statistically significant.Carotid and cerebrovascular DSCTA was performed in 125 patients with symptomatic type 2 DM. A total of 4723 arterial segments were included in this analysis, while 277 vascular segments were not evaluated because of deficiency or diameter <1.5 mm. Baseline characteristics of the 125 patients who were analyzed are summarized in Table st-5th diseased segments, and 35 (30.7%) 6th-10th diseased segments. The most common site of all detected plaques in the patients was the cavernous portion of ICA  followed by C5  and CB .DSCTA revealed 11 (8.8%) patients without CeVD and 114 91.2%) patients with CeVD. A total of 658 segments with plaque were identified, of which 296 (45%) were noncalcified, 107 (16%) were mixed, and 255 (39%) were calcified (p < 0.001). Regarding plaque distribution, noncalcified plaques were mainly located in the intracranial arteries (81.8%), mixed plaques in the intracranial arteries (25.2%) and intracranial ICA (56.1%), and calcified plaques in the intracranial ICA (65.9%) and extracranial arteries (28.2%) , 155 24%), 51 (8%), and 9 (1%) segments, respectively (p < 0.001). In general, 598 (91%) showed nonobstructive CeVD and 60 (9%) showed obstructive CeVD (p < 0.001). There were different grades of stenosis in different plaques. Noncalcified plaques led to 233 (78.7%) mild, 52 (17.6%) moderate, 10 (3.4%) severe, and 1 (0.3%) occlusion. Mixed plaques led to 69 (64.5%) mild, 29 (27.1%) moderate, 7 (6.5%) severe, and 2 (1.9%) occlusions. Calcified plaques resulted in 141 (55.3%) mild, 75 (29.4%) moderate, 33 (12.9%) severe, and 6 (2.4%) occlusions , confirming the findings of previous studies %, confir,25. UsinAs was shown in this study, the noncalcified plaques were primarily located in the intracranial arteries, mixed plaques in the intracranial arteries and intracranial ICA and calcified plaques in the intracranial ICA and extracranial arteries. A similar distribution of calcified plaques has been shown in another study using 16-slice MDCT . In our In our study, the most common site of plaques was the cavernous portion of ICA. Wojak et al.  confirmeOutlining the significance of our study, the prevalence and morphology of carotid and cerebrovascular atherosclerotic plaques in patients with symptomatic type 2 DM by DSCTA has been systematically reported for the first time. The DSCT findings, which depended on a large study sample with cutting-edge technology, accurately reflects characteristics of plaque and stenosis in patients with symptomatic type 2 DM, and can be used to conduct a further treatment plan.There were several limitations to this study. First, this study is a single centre, retrospective study. A study with a larger patient population from various centers is warranted to confirm these data. Second, the visualization of noncalcified plaques by DSCT is limited by plaque size; smaller plaques located predominantly in the smaller arteries may therefore be difficult to identify accurately with the current generation of CT scanners.DSCTA detected a high prevalence of CeVD in patients with symptomatic type 2 DM. A relatively high proportion of plaques was noncalcified and calcified, primarily leading to nonobstructive stenoses. The distributions of plaques were different and extensive, with the most common site being the cavernous segment of ICA.The authors declare that they have no competing interests.All authors participated in the design and coordination of the study, reviewed the analysis and took part in writing the manuscript. They also read and approved the final manuscript.


Using G-CSF loss- and gain-of-function approaches, we demonstrated for the first time that: 1) abrogating G-CSF production significantly diminished granulocytic MDSC accumulation and tumor growth; 2) ectopically over-expressing G-CSF in G-CSF-negative tumors significantly augmented granulocytic MDSC accumulation and tumor growth; and 3) treatment of naïve healthy mice with recombinant G-CSF protein elicited granulocytic-like MDSC remarkably similar to those induced under tumor-bearing conditions. Collectively, we demonstrated that tumor-derived G-CSF enhances tumor growth through granulocytic MDSC-dependent mechanisms. These findings provide us with novel insights into MDSC subset development and potentially new biomarkers or targets for cancer therapy.Myeloid-derived suppressor cells (MDSC) are induced under diverse pathologic conditions, including neoplasia, and suppress innate and adaptive immunity. While the mechanisms by which MDSC mediate immunosuppression are well-characterized, details on how they develop remain less understood. This is complicated further by the fact that MDSC comprise multiple myeloid cell types, namely monocytes and granulocytes, reflecting diverse stages of differentiation and the proportion of these subpopulations vary among different neoplastic models. Thus, it is thought that the type and quantities of inflammatory mediators generated during neoplasia dictate the composition of the resultant MDSC response. Although much interest has been devoted to monocytic MDSC biology, a fundamental gap remains in our understanding of the derivation of granulocytic MDSC. In settings of heightened granulocytic MDSC responses, we hypothesized that inappropriate production of G-CSF is a key initiator of granulocytic MDSC accumulation. We observed abundant amounts of G-CSF  Myeloid-derived suppressor cells (MDSC) constitute heterogeneous populations of monocytic and granulocytic-like cells reflecting various stages of differentiation that are well-regarded to play integral roles in immune suppression during diverse pathologic conditions, notably chronic inflammation, infection, trauma, graft-versus-host disease and neoplasia +Gr-1+ cells +Ly6Chigh Ly6G− or CD11b+Gr-1low cells whereas granulocytic MDSC are defined as CD11b+Ly6Clow Ly6G+ cells or CD11b+Gr-1highDespite the fact that much attention has been dedicated to unraveling mechanisms by which MDSC mediate immune suppression, a larger gap remains in our understanding of the mechanisms that initiate their development. Understanding how they appear is also critical to the design of new therapeutic strategies that impede MDSC involvement in order to potentiate anti-pathogen immunity. In the case of neoplasia, it is generally thought that tumor-derived factors (TDF) govern diverse facets of MDSC biology, including their mobilization, recruitment and activation Although much interest has been dedicated to monocytic MDSC biology +Gr-1+) MDSC response 1) Does tumor-derived G-CSF drive granulocytic MDSC generation in vivo? 2) Is the granulocytic MDSC response pro-tumorigenic in vivo? and 3) Does recombinant G-CSF administration elicit a myeloid response phenotypically, functionally and molecularly similar to that of G-CSF-producing tumors? The latter question not only addresses the mechanistic link between G-CSF and granulocytic MDSC development in vivo, but also the potential implications of aphysiologic G-CSF levels in non-neoplastic disease or clinical settings.Indeed, G-CSF production has also been shown to correlate with aberrant granulocytic or MDSC-like responses in several mouse tumor models +Gr-1+ expression +Gr-1+ MDSC responses are observed under autochthonous  and implantable  tumor settings To test our hypothesis, we made use of two implantable orthotopic mouse models of mammary cancer that are proficient at MDSC generation, termed 4T1 and AT-3. 4T1 is a well-characterized mammary tumor model We first screened tissue culture supernatants of 4T1 cells for cytokines and chemokines commonly associated with myelopoiesis and MDSC biology 3) and extensive tumor burden . Not surprisingly, G-CSF levels increased with increasing tumor burden. In contrast, little-to-no G-CSF was observed in the non-tumor-bearing controls  in 4T1-TB vs. WT comparison and G-CSF vs. WT comparison respectively, and only 22 genes were differentially expressed between 4T1-TB and G-CSF using the same significance criteria  mice more so than those of the non-tumor-bearing control  at a molecular level. To that end, splenic CD11bce as in  were purce as in . Differece as in . Specificriteria . Hierarccriteria . OverallTo test the hypothesis that G-CSF production is causally linked to MDSC generation and/or tumor growth, we sought to inhibit G-CSF in tumor-bearing mice using two different approaches. First, treatment with anti-G-CSF mAb significantly reduced primary tumor growth compared to mice treated with the isotype control Ab , suggestSecond, we made use of a loss-of-function approach, whereby G-CSF expression was down-regulated in AT-3 cells via RNA interference. Individual stable cell lines were generated using different short-hairpin (shRNA) sequences delivered via retroviral-based methods. G-CSF knockdown, relative to the non-silencing (scrambled sequence) control, was strongly observed with all constructs, as measured by RT-PCR . In contin vivo. To that end, syngeneic female B6 mice were implanted orthotopically with AT-3 shRNA 1 cells or the control tumor cells. Consistent with our observations using Ab-based G-CSF blockade . First, we observed significant reductions in splenocyte numbers in mice bearing AT-3 shRNA 1 tumor cells compared to the control (+Gr-1+ cells compared to the control (19.7% to 48.01% respectively)  . Third, fraction . Importafraction  panel C fraction  of granu+Gr-1+ cells were isolated and tested for their ability to inhibit allo-specific T cell proliferation . Mice bearing G-CSF-producing CMS4 cells also had an approximately 7-fold higher percentage of CD11b+Gr-1+ cells compared to those implanted with the control cells . The reduction in tumor growth became more apparent over time, consistent with a time-dependent component for anti-Gr-1 mAb treatment to build up to a biologic effect. In contrast, anti-Gr-l mAb treatment had no effect on the growth of the vector control tumor compared to treatment with the isotype control  loss-of-function using RNA interference; 2) gain-of-function using a tumor model over-expressing G-CSF protein; and 3) direct injection of purified G-CSF protein. Using multiple approaches, our data support the hypothesis that tumor-derived G-CSF facilitates granulocytic MDSC generation which displays both immunosuppressive and pro-tumorigenic activities. Furthermore, G-CSF protein alone was sufficient to generate granulocytic-like MDSC, which strongly recapitulated phenotypic, functional and molecular characteristics observed with tumor-induced granulocytic MDSC. These data imply that high concentrations of G-CSF acting in a systemic and aphysiologic manner can impair normal myeloid cell development or differentiation, leading to the accumulation of granulocytic-like MDSC. Thus, exogenous or endogenous stores of G-CSF may have paradoxical roles during certain pathologic circumstances, including neoplasia.Three systematic Our data do not necessarily preclude the potential role of host-derived G-CSF. However, the loss- and gain-of-function studies directly implicate a prominent role of tumor-derived G-CSF in the accumulation of granulocytic MDSC. The possibility that a ‘G-CSF-granulocytic MDSC-tumor axis’ may be important in a broader sense is supported by our data in multiple tumor models or approaches, and the fact that G-CSF production has been identified in several human cancer types +Gr-1high cells from G-CSF-treated mice were collected, admixed with 4T1 cells and implanted orthotopically into naïve recipients. We found that such myeloid-tumor admixtures significantly enhanced tumor growth, demonstrating that granulocytic-like populations induced by G-CSF treatment are pro-tumorigenic within the tumor microenvironment. Moreover, depleting Gr-1+ cells in mice bearing G-CSF-producing, but not non-G-CSF-producing tumors facilitated a significant reduction in tumor growth, supporting a causal link between G-CSF-induced granulocytic MDSC accumulation and tumor progression.In addition to its affects on granulocytic MDSC burden, we observed that tumor-derived G-CSF affects tumor growth rate. Through G-CSF blockade, as well as G-CSF loss- and gain-of-function approaches, we found that altering G-CSF levels had a direct impact on tumor growth. To directly demonstrate that granulocytic MDSC are pro-tumorigenic activity, highly purified CD11bOur data also implicate an important effect of G-CSF on the monocytic MDSC subset. Indeed, modulation of tumor G-CSF levels was accompanied by quantitative changes in the monocytic fraction, albeit, not to the same magnitude. Moreover, our data indicate that the monocytic fraction from tumor-bearing mice expressed appreciable levels of the G-CSF receptor . Althoug1) a novel role of G-CSF in myeloid biology, acting at the level of granulocytic MDSC accumulation; 2) that granulocytic MDSC can facilitate tumor growth, indicating a previously unrecognized causal link between this MDSC subset and neoplastic progression; 3) that recombinant G-CSF administration alone to healthy mice can promote granulocytic MDSC development, which may help to explain in part the basis of certain immune-associated adverse events in healthy individuals that receive G-CSF injections to mobilize progenitor cells for use in hematopoietic transplant settings 4) that experimental approaches that target the initiating TDF  leads to tumor growth inhibition under conditions of host immunosurveillance. Current MDSC-based pharmacologic approaches include vitamin D3, retinoic acid (ATRA), KIT-specific antibody, VEGF blockade and gemcitabine (reviewed in ref. 1). Thus, therapeutic targeting of TDF that initiate granulocytic MDSC development may offer additional ways to abrogate MDSC-mediated mechanisms of tumor growth, thereby enhancing immunotherapy efficacy.Taken collectively, we demonstrated: middle Tantigen controlled through the MMTV-LTR promoter b) background and kindly provided by S. Gendler . MTAG mice develop multifocal mammary carcinomas.All experiments were conducted and approved under our Institutional Animal Care and Use Committee (IACUC) at Roswell Park Cancer Institute under protocol ID numbers 1108 M and 1117 M and in accordance with institutional regulations, NIH and Public Health Service policies. Female C57BL/6 (B6) or BALB/c (6 – 8 wks of age) mice were obtained from the NCI-Frederick Cancer Research Animal Facility . The MMTV-PyMT/B6 transgenic mouse, termed MTAG, expresses the polyomavirus In vitro assays were performed in a RPMI-based medium.The 4T1 mammary tumor cell line  was derived from BALB/c mice 4 4T1 cells. Female B6 mice were injected similarly with 5×105 AT-3 cells, including those cell lines used in the knockdown studies. For the G-CSF over-expression tumor studies, syngeneic BALB/c mice were injected subcutaneously into the flank with 4×105 CMS4 cells. In the admix experiments, 5×104 4T1 tumor cells were mixed with MACS-purified, MHC-matched CD11b+Gr-1+ cells at a 2∶1 ratio, respectively, prior to orthotopic implantation. Spleens, serum and tumor tissue were recovered, as appropriate. For the G-CSF blockade studies, B6 mice were injected orthotopically, and on day 7 when tumors became palpable, mice were injected intraperitoneally with 10 µg of either neutralizing anti-G-CSF mAb or rat IgG isotype (R&D Systems) for 8-consecutive days, as described in vivo depletion experiments, BALB/c mice were injected ip with anti-Gr-1 Ab  or control Ab . Antibodies were administered at 200 µg/mouse once tumors became palpable, and continued twice weekly for the duration of the experiment, similarly as described 3 or for other health considerations. Tumor growth was measured 2 – 3 times/week in two dimensions and tumor volume was calculated using the formula (width2×length)/2. For recombinant G-CSF in vivo studies, mice were injected subcutaneously with recombinant G-CSF  for 5 consecutive days and serum/tissues collected, similarly as described Female BALB/c mice were injected orthotopically in an abdominal mammary gland with 5×10+Gr-1+ cells were purified from the indicated groups using similarly established protocols + magnetic beads from Miltenyi Biotec  on an AutoMACs system. The percentage of CD11b+ cells was routinely >95%. Of these cells, >90% co-expressed CD11b and Gr-1 markers in both control and tumor-bearing mice, as previously reported Splenic CD11bMyeloid preparations were pre-incubated with anti-CD16/32 mAb  to block Fc receptor binding, followed by incubation with directly conjugated primary mAb, as described 4 cells/well. Isolated CD11b+Gr-1+ cells were added to the plate at multiple CD11b+Gr-1+ cell:T cell ratios. Plates were incubated for 48 hr at 37°C, after which 3H-thymidine (1 µCi/well) was added for an additional 24 hr. Proliferation determined by measuring 3H-thymidine uptake , one-way allogeneic H-2b anti-H-2d or H-2d anti-H-2b cultures was established. Responders (1×105 unfractionated splenocytes/well from naïve mice) were cultured with irradiated (20 Gy) stimulators  in 96-well, U-bottomed plates. MHC-matched CD11b+Gr-1+ splenocytes were then added to the allo-assay at one or more cell densities. Plates were incubated for 96 hr at 37°C, after which 3H-thymidine was added for an additional 24 hr, and cells harvested as above.Flat-bottomed, 96-well plates were coated with anti-CD3 mAb (1 µg/well) (BD Biosciences). Total splenic T cells were purified from naïve mice using the AutoMACS system, and plated at 5×10+ Gr-1high myeloid cells from WT control, 4T1 tumor-bearing and recombinant G-CSF treated mice, we first incubated total splenocytes with CD11b  and Gr-1 (BD Biosciences) antibodies as indicated previously. 4T1 tumor-bearing mice had tumor burdens of >1000 mm3, whereas mice treated with recombinant G-CSF were given a dose regimen as previously indicated. The cells were sorted on a FACSAria cell sorter (BD Biosciences) and analyzed using FACSDiva software (BD Biosciences). The FACS resulted in >98% recovery of the CD11b+ Gr-1high population. Total RNA was extracted from 5×106 sorted cells using Qiagen's miRNeasy kit, which was quantified and subsequently used for microarray analysis.To efficiently isolate CD11b5′-AAAAAACACCATGGCTCAACTTTCTGCCC-3′; reverse:  expression plasmid , amplified, and purified under endotoxin-free conditons for use in transfection. CMS4 cells were then transfected with the expression vector or empty vector as a control using Lipofectamine (Invitrogen) per manufacturer's instructions. Cells were stably propagated under G418 (800 µg/ml) selection.RNA interference methods were used to silence G-CSF expression in AT-3 tumor cells. Stably, transduced cell lines were generated by the shRNA core facility at Roswell Park. Briefly, the retroviral vector, pSM2, was engineered to contain several independent sequences targeting murine G-CSF expression , 2 (68539), 3 (64790) or non-silencing/scrambled control) . AT-3 tumor cells were transduced with the individual G-CSF shRNA-containing retroviral supernatants. Cells were stably propagated under puromycin selection (2 µg/ml). For the G-CSF over-expression studies, murine G-CSF cDNA was PCR-cloned from AT-3 cells using the following primers: forward: 6 cells/ml) were collected after incubation for 24 hr at 37°C. Serum was also collected, and all samples were stored at −80°C until assayed. Comprehensive analysis of cytokines was initially performed using Quansys multipex ELISA services . Subsequently, G-CSF levels were quantified using G-CSF-specific ELISA .Cell-free supernatants from tumor cell lines  forward: 5′-AGTCACTGCTACTGCACCCAG-3′ and reverse: 5′-CTCTCCTCAGTTCCTCGCTCC-3′; VEGF-A forward: 5′-CTGTGCAGGCTGCTGTAACG-3′ and reverse: 5′-GTTCCCGAAACCCTGAGGAG-3′; VEGF-C forward: 5′-TGTGGGGAAGGAGTTTGGAGC-3′ and reverse: 5′-CGGCAGGAAGTGTGATTGGC-3′; COX-2 forward: 5′-ACAACATCCCCCTCCTGCG-3′ and reverse: 5′-GCTCCTTATTTCCCTTCACACCC-3′, GAPDH forward: 5′-CATCACCATCTTCCAGGAGCG-3′ and reverse: 5′-ACGGACACATTGGGGGTAGG -3′. PCR products were separated on a 1% agarose gel and the images captured with the Chemidoc Imaging System .Total RNA was isolated using RNeasy Mini kits  according to manufacturer's instructions. cDNA was synthesized using the ThermoScript RT-PCR system (Invitrogen). The cDNA was then used as the template for PCR amplification of the indicated murine genes in a PTC-200 thermal cycler  under the following standard conditions: 94°C for 2 min, 30 cycles  and 72°C for 10 min. The following mouse primer sets were used: G-CSF forward: +Gr-1high cells from the different experimental groups was quantified by a ND-1000 spectrophotometer  and evaluated for degradation using a 2100 Bioanalyzer . Samples requirements were as follows: RIN >7, an OD 260∶280 of 1.9-2.0, and an OD 260/230 >1.8. The MouseWG-6 whole-genome gene expression array and direct hybridization assay was used for expression profiling . Initially, we converted 250–500 ng total RNA to cDNA, followed by in vitro transcription to generate biotin-labeled cRNA . 750 ng of the labeled probes were then mixed with hybridization reagents and hybridized overnight at 58°C to the MouseWG-6 v2 BeadChips. Following washing and staining with Cy3-streptavidin conjugate, the BeadChips were imaged using the Illumina iScan Reader to measure fluorescence intensity of each probe, where intensity corresponds to the quantity of the respective mRNA in the original sample. The expression profiles have been deposited in NCBI's Gene Expression Omnibus (GEO) with GSE accession number GSE32209. All data is MIAME compliant.Total RNA from highly purified splenic CD11blumi module in the R-based Bioconductor Package was used to transform the expression intensity into log2 scale Limma program in the R-based Bioconductor package to calculate the level of gene differential expression for each comparison P<0.01. Results are reported from data collected on two biologic replicates of each group.BeadChip data files were then analyzed with Illumina's GenomeStudio gene expression module and R-based Bioconductor package to determine gene expression signal levels t-test or exact Wilcoxon Rank Sum tests, where appropriate. P-values less than 0.05 were considered significant.For comparisons between control and experimental groups, data were recorded as mean ± SEM of the indicated number of mice or experiments. Statistical analysis was determined using a two-sided unpaired Figure S1High levels of G-CSF produced by DA-3 and EMT-6 mammary carcinomas. Cell-free supernatants from DA-3 or EMT-6 mammary carcinoma cell lines were collected and analyzed by ELISA for G-CSF secretion (pg/ml/106 cells/24 hr). DA-3 cells produced high levels of G-CSF ; EMT-6 cells also produced an appreciable level of G-CSF . Data expressed as the mean ± SEM of triplicate determinations.(TIF)Click here for additional data file.Figure S2+ Gr-1+ myeloid cells.Recombinant G-CSF administration results in a selective increase in CD11b (A) BALB/c mice were treated with recombinant mouse G-CSF protein (10 µg/day for 8 consecutive days). Two days following the last injection, spleens were collected. Splenocytes from untreated or G-CSF-treated mice were analyzed by flow cytometry for the indicated cell surface marker. CD11b+Gr-1+ cells were strongly increased following G-CSF treatment, which was accompanied by a corresponding reduction in other major lymphoid populations. Data represent the mean percentage positive cells ± SEM (n = 5 mice/group). (B) Small aliquots of the indicated CD11b+Ly6ChighLy6G- and CD11b+Ly6Clow Ly6G+ cells in (TIF)Click here for additional data file.Figure S3Impact of G-CSF knockdown on tumor growth in vitro, serum G-CSF levels and MDSC frequences in vivo. (A) Control and shRNA 1 AT-3 cells were measured for potential differences in proliferation in vitro by the MTS assay, a variation of the MTT assay. Tumor cell lines were incubated in flat-bottomed, 96-well plate for 24 hr at multiple cell densities. After incubation time, the MTS solution was added and the extent of proliferation was determined by measuring OD at 490 nm. (B) Stability of G-CSF knockdown in vivo was determined by measuring systemic G-CSF levels from both groups of mice with comparable tumor volumes (800 – 1000 mm3). Data expressed as the mean ± SEM . (C) Splenocytes were isolated from mice bearing control and shRNA 1 AT-3 cells at comparable tumor volumes, and analyzed for the percentages of granulocytic and monocytic subsets. G-CSF knockdown in AT-3 cells led to a significant decline in granulocytic MDSC. Data expressed as the mean positive staining ± SEM .(TIF)Click here for additional data file.Figure S4Impact of G-CSF over-expression on tumor growth in vitro, serum G-CSF levels and MDSC frequences in vivo. (A) CMS4-vector control (CMS4-VC) and G-CSF-producing CMS4 cells (CMS4-GCSF) were measured for potential differences in proliferation in vitro as in B) Each cell line was also analyzed for G-CSF receptor expression by flow cytometry. One of 3 representative experiments is shown. (C) Stability of G-CSF over-expression in vivo was determined by measuring systemic G-CSF levels from the two groups of mice with equivalent tumor volumes (∼1000 mm3). Data expressed as the mean ± SEM . (D) Photograph of representative spleens from CMS4-VC or CMS4-GCSF tumor bearing mice (∼1000 mm3). (E) Splenocytes in D were isolated from mice bearing CMS4-VC or CMS4-G tumors, and analyzed by flow cytometry for the percentages of granulocytic and monocytic subsets, based on differential staining with anti-CD11b, Ly6C and Ly6G mAb. G-CSF over-expressing CMS4 tumors showed a significant rise in granulocytic MDSC, with a corresponding drop in monocytic MDSC relative to the control. Data expressed as the mean positive staining ± SEM .(TIF)Click here for additional data file.Figure S5G-CSF receptor expression on monocytic and granulocytic MDSC. G-CSF receptor expression was analyzed on splenic MDSC subsets from the indicated CMS4 tumor-bearing mice. Splenocytes were stained for CD11b and Gr-1 expression and then further gated based on differential Gr-1 levels (Gr-1high or Gr-1low) to phenotypically distinguish granulocytic from monocytic MDSC subsets, respectively. The gated cells were then analyzed for G-CSF receptor expression relative to isotype control staining.(TIF)Click here for additional data file.


The use of expanded criteria donor kidneys (ECD) had been associated with worse outcomes. Whole gene expression of pre-implantation allograft biopsies from deceased donor kidneys (DDKs) was evaluated to compare the effect of pulsatile pump preservation (PPP) vs. cold storage preservation (CSP) on standard and ECD kidneys.99 pre-implantation DDK biopsies were studied using gene expression with GeneChips. Kidneys transplant recipients were followed post transplantation for 35.8 months (range = 24–62). The PPP group included 60 biopsies (cold ischemia time (CIT)  = 1,367+/−509 minutes) and the CSP group included 39 biopsies  (P<0.001). Donor age  and the percentage of ECD kidneys  were significantly different between groups. A two-sample t-test was performed, and probe sets having a P<0.001 were considered significant. Probe set level linear models were fit using cold ischemia time and CSP/PPP as independent variables to determine significant probe sets (P<0.001) between groups after adjusting for cold ischemia time. Thus, 43 significant genes were identified (P<0.001). Over-expression of genes associated with inflammation  was observed in the CSP group. Cell-to-cell signaling and interaction, and antigen presentation were the most important pathways with genes significantly over-expressed in CSP kidneys. When the analysis was restricted to ECD kidneys, genes involved in inflammation were also differentially up-regulated in ECD kidneys undergoing CSP. However, graft survival at the end of the study was similar between groups (P = 0.2). Moreover, the incidence of delayed graft function was not significant between groups.Inflammation was the most important up-regulated pattern associated with pre-implantation biopsies undergoing CSP even when the PPP group has a larger number of ECD kidneys. No significant difference was observed in delayed graft function incidence and graft function post-transplantation. These findings support the use of PPP in ECD donor kidneys. Kidney transplantation (KT) represents the treatment of choice for patients with end-stage renal disease and provides survival benefit when compared with long term dialysis therapy, in terms of quality of life and life expectancy The increasing disparity between organ supply and demand challenges the transplantation community to maximize efforts and optimize the use of organs from all consented donors. Recent increases in graft availability from deceased donors have been a result of expansion of the donor acceptance criteria, including increasing use of older donors, donation-after-cardiac-death (DCD), and deceased donors with other characteristics that might be associated with increased risk of graft dysfunction ex-vivo biopsies and machine preservation technology However, ECD kidneys had been often associated with worse short and long term renal function, and the limited nephron reserve might result in a relative risk of graft loss when compared with kidneys from standard criteria donors (SCD) Furthermore, it is well-established that post-transplantation occurrences such as DGF and ACR are risk factors for poorer intermediate-term graft survival, increasing the burden of patients returning to dialysis therapy From the time a patient is identified as a potential donor it is critical to maintain adequate organ perfusion and avoid hypoxemia. Currently, once a deceased donor kidney is recovered, there are two main methods of preservation including cold hypothermic storage preservation (CSP) and pulsatile perfusion preservation (PPP) More importantly, recent reports support the use of PPP demonstrating clinical benefit despite of the fact that the patho-physiological mechanisms involved in the “improved” graft function are still unclear. Assessment of the transcriptome in the donor organ itself is an appealing strategy to determine organ quality and predict subsequent graft performance, as molecular pathways may provide a comprehensive measurement of the individual graft's response to acute injury factors. In the current study, the transcriptome of 99 deceased donor pre-implantation biopsies of deceased donor kidney grafts preserved with CSP and PPP was evaluated.The study was conducted at Virginia Commonwealth University and at the University of Virginia after Institutional Review Board (IRB) approval was obtained at both institutions . Patients received a deceased donor kidney (DDK) transplant between January 2006 and January 2010. They were aware of the collection of biopsies and they signed a consent form (that includes description of risk associated with biopsies). They had the right to agree/deny the use of a pre-transplant biopsy. All organ donors (or their next of kin) consented for their samples to be used in research . Additionally, because the present research results are part of an overall study that includes the use of protocol biopsies and more than “minimal risks to the individual”, a Data and Safety Monitoring Board has been established for the study evaluation. Complications (expected and unexpected) are reported to the Institutional Review Boards  and to the study's sponsor as part of the yearly progress report. No living donors, HIV positive or re-transplantation patients were included in the study. Allograft biopsies from kidneys preserved using both cold preservation (CSP) and pump perfusion preservation (PPP) were included in the study. All the transplant recipients underwent the same immunosuppressant protocol that included a calcineurin inhibitor based plus mycophenolate mofetil and prednisone. Acute cellular rejection episodes were always treated with steroids boluses (three days followed by prednisone taper).later (Ambion) immediately after collection. Biopsies were collected at pre-implantation time . Estimated GFR (eGFR) was calculated using the abbreviated Modification of Diet in Renal Disease (MDRD) formula Kidney allograft tissue was obtained through an 18 gauge biopsy needle and all samples were placed in RNAThe sample preparation protocol follows the Affymetrix GeneChip® Expression Analysis Manual . Briefly, total RNA was reverse-transcribed using T7-polydT primer and converted into double-stranded cDNA , with templates being used for an in vitro transcription reaction to yield biotin-labeled antisense cRNA. The labeled cRNA was chemically fragmented and made into the hybridization cocktail according to the Affymetrix GeneChip protocol, which was then hybridized to HG-U133A 2.0 GeneChips. The array image was generated by the high-resolution GeneChip® Scanner 3000 by Affymetrix®.GeneChip HG-U133Av2 arrays were hybridized for 99 pre-implantation samples. Quality control parameters that included scaling factor, average background, percent of probe sets called present, and the 3′∶5′ ratio for GAPDH and B-actin, were checked. The robust multiarray average method was used to obtain probe set expression summaries. Prior to performing statistical analyses, control probe sets and probe sets called absent across all 99 arrays were excluded, leaving 17,654 probe sets.P<0.001 were considered significant. Because there was a significant difference between the CSP and PPP groups with respect to cold ischemia time (CIT) (P = 0.0003), probe set level linear models were fit whereby expression was modeled using CIT and CSP/PPP as independent variables, to identify probe sets that were significantly different between the two groups after adjusting for CIT.To identify probe sets differentially expressed between the pump (n = 60) and no pump (n = 39) groups, for each probe set a two-sample t-test was performed and probe sets having a P<0.001 were considered significant.First, the dataset was restricted to patients receiving a graft with a CIT<1200 minutes (20 hours) and for each probe set a two-sample t-test was performed. Second, the dataset was restricted to graft with a CIT>1200 minutes and again, for each probe set a two-sample t-test was performed. For each analysis, probe sets having a vs. 7K1b, 49K1 vs. 50K1, and 82-K vs. 83-K. For each pair of GeneChips, the probe level data were read into the R programming environment and the Affymetrix MAS5 detection call algorithm was applied. Probe sets that were absent in both GeneChips were removed from subsequent analysis. Thereafter, the S-Score algorithm S-Score package in R P-values were used to estimate the false discovery rate using the q-value method q-value was less than 0.05. The log2 transformation was applied to probe set expression summaries obtained using the Affymetrix MAS5 algorithm for producing scatterplots and calculating fold changes.Three pairs of Affymetrix HG-U133Av2 GeneChips were considered: 6K1 http://www.ingenuity.com).Gene ontology and gene interaction analyses were executed using Ingenuity Pathways Analysis tools 9.0  for CD86, CD209, and EGFR2 mRNAs in the same RNAs samples that were subjected to microarray study. Total RNA was subjected to reverse transcription using TaqMan® Reverse Transcription Reagents  according to the manufacturer's protocol. QPCR reactions then were carried out using TaqMan® Gene Expression Assays (Applied Biosystems). Data was analyzed according to the comparative cycle threshold method and was normalized with a housekeeping gene ). Pearson's correlation coefficient (r) was calculated to examine the relation between microarray and QPCR results. Descriptive statistics were reported for demographic and clinical variables, including proportions for categorical variables and mean ± standard deviation for continuous variables.later (Ambion) and sent to the molecular laboratory. An additional sample from each kidney was sent for pathological evaluation.A total of 99 pre-implantation kidney graft biopsies from 92 deceased donors were studied. Seven donors provided paired kidneys. Patients were followed post transplantation for 35.8 months (range = 24–62). All biopsies were performed by the same surgical team, and consisted of a single 18 gauge needle biopsy taken from the cortex (2–15 mm deep) kidney upper pole, and right before implantation was started (at back-table and post-ischemia time). Samples were submerged immediately in RNATable S1). Demographic and clinical information for the enrolled study patients were separated according to the preservation method used (PPP vs. CSP) as shown in P<0.001). Clinically, the two patient groups were similar regarding recipient age, recipient and donor gender, last donor creatinine, warm ischemia time, incidence of acute rejection during the first year post-kidney transplant.No significant differences were observed in the histology of donor biopsies (pre-implantation) between groups (P = 0.009). The number of extended criteria donor (ECD) kidneys vs. 12.8%, P = 0.012). The donor age difference between groups relates with the higher number of ECD donor kidney in the PPP group.Donor age was significantly different between groups (P = 0.2)). Moreover, the incidence of DGF was not statistically significant between PPP vs. CSP groups (28.3% (17 out of 60) vs. 28.2% (11 out of 39) (P = 0.18), respectively.Of interest, graft survival at the end of the study was similar between groups (88.5% in PPP group and 94.3% in CSP group (vs. CSP (n = 39) groups using a two-sample t-test and P<0.001. Core analysis was performed to interpret the data set in the context of biological processes, pathways and molecular networks. The associated functions to the top scored network (score 40) that related with the differentially expressed genes included antigen presentation and cell-to-cell signaling and interaction (Forty-three probe sets were significantly different when comparing the PPP (n = 59) eraction Figure 1From the analysis of the genes differentially expressed between groups, genes involved in immune response  were significantly over-expressed in CSP kidneys. Moreover, the specific analysis of the over-expressed genes showed association with inflammation , cell movement of dendritic cells (CD209), activation of naïve T lymphocytes , pro-inflammatory response of macrophages (INPP5D), and binding and accumulation of macrophage .P = 9.4E-05 to 4.8E-02) and antigen presentation (P = 1.2E-04 to 4.0E-02) were the more important functions of genes significantly over-expressed in CSP kidney samples. Seventeen probe sets were significantly different when adjusting the analysis for CIT using probe set level linear models . Six probe sets were significant when adjusting the analysis for CIT using probe set level linear modelsWhen the dataset was restricted to patients with a CIT<1200 minutes, there were 24 patients in PPP and 22 patients in CSP. There were 5 significant probe sets when comparing PPP P<0.001) (P = 0.08). Importantly, all the over expressed genes identified from this analysis, belong to the same network (score 23)  Table 3 ECD kidneys represented an important sub-group in our study population (27.7%) and in line with the reported use of these grafts in the USA.vs. CSP) were compared, 102 probe sets were statistically differentially expressed (P<0.01). From the analysis of these genes, we observed that genes involved in inflammation were down-expressed in the ECD kidneys preserved with PPP as it is shown in the vs. 20%, P = 0.27 (one tailed Fisher Exact Probability Test). Moreover, there was not statistical significantly difference in graft function between ECD kidneys undergoing PPP or CSP at 24 months post-kidney transplantation.Characteristics of the ECD kidneys by preservation group are shown in the In the present study, seven donors provided paired kidneys (14 kidneys). We also evaluated how the gene expression patterns differ among paired kidneys. From the time a patient is identified as a potential organ donor it is critical to maintain adequate organ perfusion and avoid hypoxemia. In the 1970s, most of the donated kidneys were preserved by pulsatile perfusion preservation However, PPP has resurged in the last decade based upon the belief that this preservation method leads to a reduced rate of graft ischemia, DGF and eventually, improved long term graft function. At the same time, comparative studies between PPP and CSP started to show benefit of using PPP et al. P = 0.01) as well as a reduced serum creatinine level at 2 weeks post-kidney transplant and reduced risk of graft failure. Also, graft survival at 1 year was better in kidneys preserved by PPP (P = 0.04). As reported by the editorial in the same journal A report by Moers Up-to- date, the published reports about the advantages and/or disadvantages of the use PPP over CSP It is now well described that molecular profiling detects changes not seen by histological evaluation and clinical markers. However, the identification of predictive gene sets and the application to an individualized patient management needs the integration of clinical and pathology-based variables, as well as objective reference markers of graft function, post-transplant complications, and long-term outcomes At present, PPP is widely used in most areas of USA in renal transplantation, allowing a greater use of ECD kidneys and increasing the number of organs available for transplantation. In addition, perfusion parameters, such as flow rate and resistance are factors frequently used to assess and estimate the downstream functionality of renal grafts vs. PPP in a prospective study.To the best of our knowledge, this is the first report showing gene expression profiles of kidney donor biopsies in association with clinical/demographic donor and recipient characteristics to compare CSP vs. PPP) were evaluated. As expected, kidneys preserved by PPP had a tendency to have longer CIT; the analysis was therefore controlled for CIT. Our study included consecutive cadaveric donor kidney transplants that were performed following clinical/surgical team decisions based on organ quality despite the preservation method. The present study represents a realistic situation about the decisions and clinical practice that most regions in the USA challenge daily.Gene expression profile data of 99 consecutive deceased donor kidneys preserved with different methods (CSP Table S1). There were not statistical significant differences among groups. We did not identify relationships between glomerulosclerosis, tubular atrophy, and/or interstitial fibrosis and graft function with gene expression profiles. Moreover, these results are in concordance with the publication from Edwards et al.In the present cohort of patients, histological evaluation at pre-implantation time was performed for all the donor kidneys . In the last decade, the proportion of deceased donors older than 50 years of age has increased from 21% to 30% in USA An imbalance in metabolic supply and demand within the ischemic organ results in profound tissue hypoxia and microvascular dysfunction. Subsequent reperfusion further enhances the activation of innate and adaptive immune responses and cell death programs. Recent advances in understanding the molecular and immunological consequences of ischemia and reperfusion may lead to innovative therapeutic strategies for treating patients with ischemia and reperfusion-associated tissue inflammation and organ dysfunction Our analysis demonstrated that the expression of CD209 gene was up-regulated in CSP kidneys. This gene encodes a transmembrane receptor and is often referred to as DC-SIGN because of its expression on the surface of dendritic cells and macrophages. Dendritic cells and macrophages play an important role in the innate and adaptive immune response of acute ischemia-reperfusion injury (IRI). In the kidney they reside in the interstitial extracellular compartment and are poised to interact with substances transported from the tubule lumen into peritubular capillaries, endogenous molecules released from parenchymal cells, exogenous invading organisms, or with resident or infiltrating immune cells including lymphocytes, nature killer T (NKT) cells, epithelial cells and fibroblasts. Dendritic cells and macrophages are key initiators, potentiators and effectors of innate immunity in kidney IRI and induce injury either through inflammatory signals to other effector cells or directly through the release of soluble mediators The expression of CD86 was also up-regulated in CSP kidneys. This protein is expressed by antigen-presenting cells, and it is the ligand for two proteins at the cell surface of T cells, CD28 antigen and cytotoxic T-lymphocyte-associated protein 4. Binding of this protein with CD28 antigen is a costimulatory signal for activation of the T-cell. Targeting the activation or effector function of lymphocytes is a potentially effective approach to treat or modify the immune response When the groups were sub-stratified according with CIT, kidneys with longer CIT (when no significance was observed in CIT between groups) presented an inflammatory signature when preserved with CSP. This finding demonstrates the effect of PPP in decreasing inflammation even on those kidneys with longer CIT.From the evaluation of paired kidneys undergoing the same preservation method, we observed that two sets of paired ECD kidneys  but also in the functional validation of the results. All the studied biopsies were from kidney grafts that were transplanted in recipients with end stage kidney disease and followed for at least 24 months post-transplantation. The present findings, based in the study of an important number of samples and patient characterization, support the importance of the use of PPP in ECD donor kidneys.Biological modulation of ischemic acute kidney injury aims to reduce the incidence of delayed graft function and to safely increase the number of kidney transplantations using organs that have suffered prolonged warm and cold ischemia Table S1Histological evaluation of pre-implantation biopsies classified by sub-groups.(DOC)Click here for additional data file.


Brachypodium, and oats, all in the grass subfamily Pooideae, the change in phyllotaxis does not occur; primary inflorescence branches are produced distichously. It is unknown whether the distichous inflorescence originated at the base of Pooideae, or whether it appeared several times independently. In this study, we show that Brachyelytrum, the genus sister to all other Pooideae has spiral phyllotaxis in the inflorescence, but that in the remaining 3000+ species of Pooideae, the phyllotaxis is two-ranked. These two-ranked inflorescences are not perfectly symmetrical, and have a clear “front” and “back;” this developmental axis has never been described in the literature and it is unclear what establishes its polarity. Strictly distichous inflorescences appear somewhat later in the evolution of the subfamily. Two-ranked inflorescences also appear in a few grass outgroups and sporadically elsewhere in the family, but unlike in Pooideae do not generally correlate with a major radiation of species. After production of branches, the inflorescence meristem may be converted to a spikelet meristem or may simply abort; this developmental decision appears to be independent of the branching pattern.The shoot apical meristem of grasses produces the primary branches of the inflorescence, controlling inflorescence architecture and hence seed production. Whereas leaves are produced in a distichous pattern, with the primordia separated from each other by an angle of 180°, inflorescence branches are produced in a spiral in most species. The morphology and developmental genetics of the shift in phyllotaxis have been studied extensively in maize and rice. However, in wheat,  Arabidopsis thaliana) and a couple of hugely important crops . Much less work has been done to extend these data to wild species.Inflorescence development controls plant reproduction and hence, fitness. The number of branches produced, and the pattern and timing of their production, dictate the number of flowers, the number of vascular bundles entering the inflorescence  begins when the shoot apical meristem converts from its vegetative state, producing leaves on its flanks, to an inflorescence meristem. Bracts form as in many other flowering plants, but their growth is suppressed Evans, ; the matIn grasses, as in many other flowering plants, the phyllotaxis of lateral structures in the inflorescence may continue the same phyllotactic pattern as the leaves, or it may change. In all grasses and their close relatives in the “core” Poales [the clade consisting of Anarthriaceae, Centrolepidaceae, Restionaceae, Flagellariaceae, Joinvilleaceae, Ecdeiocoleaceae, and Poaceae  pasture grasses in subfamily Pooideae, and some of the C4 grasses in subfamily Panicoideae.The literature on inflorescence development in grasses hints at a phylogenetic correlation with inflorescence phyllotaxis, but sampling is uneven Table . AlthougEcdeiocolea monostachya produces large bracts on its flanks, with floral meristems forming in the axils of the bracts. The inflorescence meristem thus produces floral meristems directly . The fate of the inflorescence meristem in Centrolepis is unknown, but if it ultimately becomes a flower, this must occur late enough in development that it has not been observed in developmental studies. Sokoloff et al.  that terminate in flowers; while initiation of these branches is not documented, figures of slightly later stages suggest that they are arranged in a spiral  and Zizania aquatica (wildrice)  together include about 60% of grass species; within this large clade most data come from subfamily Panicoideae, tribe Paniceae. The inflorescence meristems of most species produce branches in a spiral, forming multiple orthostichies or parastichies depending on the shape of the axis Table . In someBonnett, , but theBonnett, . We haveChionochloa macra, in which the primary branch primordia are distichous  at this stage, using the OTOTO or OTO method of Murphy . The material was then transferred to 70% ethanol for storage. In a few cases, fixed material was rehydrated and infused with osmium tetroxide .Phaenosperma globosa Figures . The inflorescence of P. globosa is two-ranked and approximately distichous initially. Higher order branching occurs predominantly on one side. We do not have data on late development to determine the fate of the inflorescence meristem.Nassella spp. Figure . Data are presented for Nassella filiculmis, but observations for N. manicata (= Stipa formicarum) and N. tenuissima were similar, and we infer that the results are general for the genus. The primary branches of the Nassella inflorescence are two-ranked and apparently distichous, separated by a branch angle very close to 180°  Figure . Although the inflorescence meristem produces lateral structures in a distichous pattern in very early development . The terminal spikelet differentiates rapidly, well before the few primary branches  that the common ancestor of Nardus plus the remainder of Pooideae had spiral phyllotaxis; under this model, two-ranked phyllotaxis originated after the divergence of Nardeae. All other origins appear the same with ML and parsimony optimization.Mapping phyllotaxis on to the phylogeny of grasses shows that the two-ranked inflorescence is synapomorphic for the Pooideae excluding m Figure . Two-ranm Figure , ML indiNardus, but is lost in Diarrhena. However, in all Pooideae outside Triticeae and Poeae, higher order branching occurs only on one side of the inflorescence, suggesting an underlying bias to the inflorescence that creates a “front” and a “back.”Within the Pooideae with two-ranked inflorescence branching, distichy appears after the divergence of The inflorescence meristem of most grasses terminates in a spikelet Table . HoweverNardus and Diarrhena the inflorescence is not strictly distichous, but is biased to one side, a pattern called “biased distichous” by Ikeda et al. . Even in the spiral inflorescences of Brachyelytrum erectum, branches on one side are separated by an angle of almost 180°, whereas angles on the other side are appreciably lower. This one-sided pattern is consistent enough that it appears to represent some sort of biophysical constraint or a regulated, genetically established developmental mechanism. Cresswell et al.  of the inflorescence apex. In The relevance of our data to grasses with leafy inflorescences is not clear. Most bamboos and some Andropogoneae have complex flowering shoots that are extensively branched and bracteate, as though the entire structure is neither fully vegetative nor fully floral. Bracts are generally suppressed below the spikelets and below certain inflorescence branches; however, other branches in the inflorescence are subtended by bracts and bear prophylls, hinting at a different pattern of regulation entirely. While it is common for the terminal inflorescence branches in these groups to be two-ranked , it is not universal; thus the correlation between inflorescence branching and bract or spathe development remains unclear.Most descriptions of inflorescence morphology in the grasses attempt to force the variation into a few standard terms borrowed from dicots. Thus, inflorescences in which the spikelets are sessile on the inflorescence axis are called “spikes,” those in which the spikelets are pedicellate are “racemes,” and any inflorescence in which there are higher orders of branching is called a “panicle.” Under this set of definitions, most grasses have some sort of panicle. However, many authors have noted the problems with this approach. One problem is simply that the spikelet itself is a part of an inflorescence and thus is not strictly equivalent to a flower. Because of this, Endress  describeA second problem with using the term “panicle” in the grasses is the sheer diversity of forms. These are diagrammed by Vegetti and Anton . While rThe data presented here add another dimension to the architectural complexity already documented. “Panicles” in Pooideae originate from primary branches that initiate in a distichous fashion, whereas those in Ehrhartoideae and Panicoideae originate from primary branches initiated in a spiral. Thus, the primary phyllotaxis of the inflorescence is different, even though subsequent branching appears to be morphologically similar.Hyparrhenia), the condition is uncertain. In taxa in which the inflorescence meristem produces a single branch and then aborts , the branch then develops in its normal two-ranked pattern. It is easy to imagine that if the inflorescence meristem aborts early, the branch meristem would be wrongly interpreted as the inflorescence meristem. This may be particularly common in the PACMAD clade.Phyllotaxis of the inflorescence meristem cannot be determined in all cases, because the term has meaning only if the meristem produces at least two lateral structures. Therefore, in taxa in which there is only one primary branch  always produce two ranks of branch or spikelet primordia; these may or may not be separated by angles of 180°. In addition, the inflorescence meristem itself often shifts from producing branch primordia to producing spikelet primordia; in this case, the spikelet primordia are produced in two ranks. [The lone exception is the staminate inflorescence (tassel) of the domesticated Brachypodium and Nassella. Alternatively, the meristem may continue producing lateral structures but these may become smaller and smaller, as though the number of meristematic cells becomes increasingly limited; this appears to be the case in several of the grass outgroups but is less obvious in the grasses themselves. More commonly in the grasses, the inflorescence meristem simply terminates blindly, as described for Oryza.Phyllotaxis of the primary branches is apparently independent of the fate of the inflorescence meristem Table . The infThe presence or absence of a terminal spikelet is variable between tribes and genera of grasses. Although the majority of species in the family exhibit terminal spikelets, virtually all major clades have several members in which the terminal spikelet is lacking Butzin, . ReinheiThe functional significance of primary branch phyllotaxis is unknown. However, the fact that it can be stable among groups of species, including the several thousand species of subfamily Pooideae, suggests that it is preserved either by selection or developmental constraint. Because inflorescence architecture controls the timing and position of pollen presentation, the timing of seed maturation, the extent of seed provisioning, and the extent of seed dormancy, it is likely to be under selection Simpson, . Seed seArabidopsis. Mutations in FON-like genes lead to greatly enlarged inflorescence meristems; while they affect phyllotaxis they also disrupt many aspects of normal inflorescence patterning. ABPHYL1, a two-component response regulator, also regulates meristem size  of rice convert the normally spiral phyllotaxis of the inflorescence to two-ranked  of maize also affects phyllotaxis by shortening plastochron length so that the meristem produces lateral organs in a spiral  in the maize ear (Bommert et al., Apo1 has recently been shown to be the gene underlying the QTL Primary Branch Number in rice; higher levels of Apo1 transcript lead to more primary branches and lower levels lead to fewer (Terao et al., Despite the highly disruptive effects of knock-out mutations in the genes described above, slight modulations in their expression can change branching patterns without affecting other aspects of meristem function. For example, minor alterations in Brachyelytrum, and that the two-ranked phyllotaxis gives rise to distichous phyllotaxis somewhat later in the evolution of the subfamily. Analogous changes occur in other clades as well but appear more sporadic in evolutionary time. We hypothesize that the change in phyllotaxis may be caused by a change in meristem size or aspect ratio. Finally, we hypothesize that modulation of APO1 levels, perhaps relative to LFY levels, could create the observed phenotypic variation, although variation in the CLV pathway could also be involved.In summary, we have shown that two-ranked phyllotaxis is apparently synapomorphic for the grass subfamily Pooideae, excluding The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Survival of Colorectal cancer (CRC) patients is considerably stage-dependent; therefore, early diagnosis is a pivotal factor in decreasing mortality and morbidity associated with this cancer. GM-CSF and IL-7 are reported to increase in different cancers and we aimed to investigate the pre-treatment serum levels of GM-CSF and IL-7 in Iranian patients with colorectal cancer. 127 patients  entered this study before receiving chemotherapy or radiotherapy. A control group of 50 healthy age/sex matched individuals  were included in the study. The serum levels of GM-CSF and IL-7 were measured using commercial enzyme linked immunosorbent assays. A significantly higher level of GM-CSF was found in the sera of patients with colorectal cancer compared to healthy age/sex matched controls (P=0.013). However, there was no significant difference between the levels of IL-7 in sera of patients and controls. We observed a significant elevation in the level of GM-CSF in poorly differentiated tumors (P=0.024). Also a significant correlation between lymphatic invasion and the level of GM-CSF in sera of CRC patients was detected (P=0.01). We found an increase of the level of IL-7 in four patients presenting moderate stages of tumor concomitant with a decrease of the level of GM-CSF. It can be concluded that the increase of the level of GM-CSF is accompanied by CRC progression in Iranian patients. Potential therapeutic effect of IL-7 in this disease, however, needs further investigations. Annually one million new cases of colorectal cancer (CRC) are diagnosed and half a million death occurs due to this cancer . It reprThe immune system plays a dual role in the defence and development of many tumors. Interestingly, tumor cells may exploit or produce effectors of the immune system for their own advantage. Colony Stimulating Factors (CSFs) are among the cytokines that are produced by cancer cells as well as the immune system in the course of tumorigenesis . CytokinGranulocyte Macrophage-Colony Stimulating Factor (GM-CSF) is a cytokine that acts as a growth factor for white blood cells, and stimulates bone marrow to produce granulocytes and macrophages . GM-CSF On the other hand, CRC cells with microinstability (MSI) gene can generate abnormal peptides that stimulate the secretion of cytokines (including GM-CSF), the infiltration of lymphocytes and stimulate the immune system against tumor, which can lead to a better prognosis for the patients . An incrT-cells and downregulates TGF-beta in macro-phages, thereby accelerating anti-tumor immune responses  . The expesponses .-Experimental immunodeficient tumor-bearing mice have provided the evidence of the anti-tumor properties of recombinant human IL-7 (rhIL-7) on a human colon tumor . In addi18Increased GM-CSF mRNA, protein and its receptor have been found in many colon cancer cell lines, as well as in surgical specimens -22. GM-CIn the present study we aimed to determine the pre-treatment serum levels of GM-CSF and IL-7 in Iranian patients with CRC and investigate their possible correlation with clinicopathological chara-cteristics of the patients. The potential diagnostic use of circulating GM-CSF and IL-7 and further clinical applications in Iranian patients with colorectal cancer were explored as preliminary data.PatientsThis study was approved by the Ethics Committee of Shiraz University of Medical Science (SUMS). The patients were informed about the aim of this study as well as safety and security measures before their consents were obtained. The cases were selected among CRC patients who were referred for surgery to hospitals related to SUMS between November 2009 and September 2010. 127 cases  aged between 13-83 years entered the study. None of the patients had been treated by chemotherapy or radiotherapy before sample collection. The clinicopathological charac-teristics of the patient group is shown in The control group was selected from 50 healthy adults aged between 21-83 years  who had no acute or chronic diseases such as autoimmune disease, diabetes mellitus, thyroid disease, recent common cold, hypertension, hyper-lipidemia, ischemic heart disease, cerebrovascular, renal, skin and pulmonary disorders as well as infectious diseases and were not receiving any medications.The group of 127 CRC patients  and 50 healthy controls  were matched based on age and gender . The meaSamplesFour ml blood was collected from peripheral veins of patients on the day before surgery. The samples were brought to Shiraz Institute for Cancer Research (ICR) immediately. Samples were centrifuged and sera were preserved at -20˚C till used. On the day of operation, the tissue biopsies were assessed by collaborative pathologist. The pathologist confirmed colon cancer and evaluated invasion of tumors to perineural, perivascular, perilymphatic and local area lymph nodes. Cancer was staged according to tumor-node-metastasis (TNM) by the American Joint Committee on Cancer Classification and stage grouping.ELISA assaysThe plasma level of GM-CSF was measured by a commercial enzyme linked immunosorbent assay (ELISA)  according to the manufacturer's instructions. The sensitivity of this assay was 0.6 pg/ml and the range of detection was between 7.8-500 pg/ml. The serum level of IL-7 was measured using a commercial ELISA assay  according to the manufacturer's instructions. The sensitivity of this assay was less than 3 pg/ml and the range of detection was between 6.25-200 pg/ml.Statistical analysisStudent's t-test was used for the analysis of age and gender distribution between the case and the control groups. One-way ANOVA or t-test was used for the comparisons between the two groups using SPSS software . When the data points were less than 30 in categories, the normality of data was checked and parametric or non parametric  analyses were performed. Statis-tically significant differences were defined as comparisons resulting in p<0.05.A significantly higher level of GM-CSF (12.49±27.28 pg/ml) was found in the sera of patients with colorectal cancer compared with healthy age/sex matched controls (2.21 ± 15.68 pg/ml), (P= 0.013). Due to the high variance of data the analysis was performed by non-parametric comparison of the means (Mann-Whitney) and the results were confirmed (P=0.000). However, there was no significant difference between the level of IL-7 in sera of patients (0.16 ± 1.38 pg/ml) and healthy controls (0.06 ± 0.16 pg/ml) which was also confirmed by the non-parametric statistical evaluation (P=0.088). A high percentage of CRC patients  had some level of GM-CSF in their sera while only one out of 50 (2%) healthy controls were found positive for GM-CSF (Roc curve cut off point= 5.04 pg/ml). Conversely, a higher number of healthy controls  in the serum level of GM-CSF in patients with different histological grades of colorectal cancer. The level of GM-CSF showed an increasing trend with the loss of differentiation in the tumor. Conversely, the highest level of IL-7 was detected in patients with well differentiated tumors and no IL-7 was detected in patients with poorly differentiated tumors .A significant difference in the level of GM-CSF in sera of CRC patients with or without lymphatic invasion was also observed (P= 0.01). Accordingly, the level of GM-CSF was much higher in sera of patients with lymphatic invasion compared to those without lymphatic invasion . No diffWe also compared the level of the two cytokines between CRC patients presenting low stages (Stages 1 and 2), medium stages (Stages 3a and 3b) and high stages (stages 3c and 4) tumors. We found a significant increase in the level of IL-7 in moderate stage tumors (P<0.05). This increase was concomitant with a decrease in the level of GM-CSF in sera of the patients with medium stage tumors .The mean serum levels of GM-CSF did not show any difference between male (9.49 ± 8.80 pg/ml) and female (15.81 ± 38.36 pg/ml) CRC patients. There was also no significant difference between the level of IL-7 in male (0.04 ± 0.28 pg/ml) and female (0.29 ± 1.98 pg/ml) patients.There was no significant correlation between tumor type, tumor side, tumor size, perineural invasion, vascular invasion and lymph node involvement with either of GM-CSF or IL-7 cytokines levels.In this study we observed a significantly higher level of GM-CSF in Iranian patients with CRC compared to their age/sex matched controls. Previous studies have shown that the increased level of local cytokines in the site of tumor stimulates the immune system against tumor and CSFs have the most important role in this event . On the The exact explanation for the variable results on the production of GM-CSF in CRC and the mechanisms behind its dual (or multiple) role against tumor cells is not known. However, it has been suggested that a slight elevation in the serum GM-CSF levels may reflect a protective response in cancer patients, while higher levels of autologous GM-CSF may stimulate the metastatic properties of cancer cells . AccordiA previous report showed that the level of IL-8 but not GM-CSF was significantly different between the well differentiated adenocarcinomas and other types of colorectal tumors , howeverIn our study, IL-7 was only elevated in 4 CRC patients and did not differ between patients and controls. Despite the low number of IL-7 positive CRC patients, an inverse pattern of GM-CSF and IL-7 increase was observed in the four IL-7 positive cases. Increase of IL-7 was accompanied by a decrease of the GM-CSF in those patients. The low number of IL-7 positive cases hampers to draw any conclusion but it may be suggestive of a beneficial role of IL-7 in CRC. It can also be a part of efforts of the immune system to respond to the tumor. There is limited information on the level of IL-7 in colorectal cancer and the results of the available studies are contradictory. In one study, IL-7 was only detectable in higher stages of the tumor. In our hands, IL-7 was only produced by tumors in stage 3a where the production of GM-CSF decreased. On the contrary, there are other reports showing that IL-7 is increased in colorectal cancer and it can be used as a diagnostic or prognostic factor in this cancer - 20. MorThe mean concentrations of both IL-7 and GM-CSF are reported to be high in females with colorectal cancer but did not reach the significant level . MroczkoCurrently it is difficult to interpret the discrepancy of the IL-7 data obtained in our study and some previous studies but as mentioned by others, what actually operate in vivo are the cytokine cascades and cytokine networks . In addiWe did not find any correlation between tumor type, tumor side, tumor size, perineural invasion, vascular invasion, and lymph node involvement with either GM-CSF or IL-7 cytokines. Similarly, another study reported no correlation between the serum levels of GM-CSF and the clinicopathological findings in colorectalcancer .In summary, due to a high percentage of patients with elevated serum level of GM-CSF, we suggest that GM-CSF can be used as a tumor marker in Iranian colorectal cancer patients, however, the specificity and sensitivity of the test should be defined in future studies. We also suggest that the role of IL-7 in colorectal cancer needs more investigation and may provide a new target in the immunotherapy of colorectal cancer.


IL-6-/- mice with KrasG12D mutant mice, which develop lung tumors after activation of mutant KrasG12D, to investigate whether IL-6 inhibition contributes to tumor progression and survival time in vivo. KrasG12D; IL-6-/- mice exhibited increased tumorigenesis, but slower tumor growth and longer survival, than KrasG12D mice. Further, in order to investigate whether IL-6 deletion contributes to suppression of lung cancer metastasis, we generated KrasG12D; p53flox/flox; IL-6-/- mice, which developed lung cancer with a trend for reduced metastases and longer survival than KrasG12D; p53flox/flox mice. Tumors from KrasG12D; IL-6-/- mice showed increased expression of TNFα and decreased expression of CCL-19, CCL-20 and phosphorylated STAT3 (pSTAT3) than KrasG12D mice; however, these changes were not present between tumors from KrasG12D; p53flox/flox; IL-6-/- and KrasG12D; p53flox/flox mice. Upregulation of pSTAT3 and phosphorylated AKT (pAKT) were observed in KrasG12D tumors with p53 deletion. Taken together, these results indicate that IL-6 deletion accelerates tumorigenesis but delays tumor progression and prolongs survival time in a Kras-driven mouse model of lung cancer. However, these effects can be attenuated by p53 deletion.Interleukin-6 (IL-6) is involved in lung cancer tumorigenesis, tumor progression, metastasis, and drug resistance. Previous studies show that blockade of IL-6 signaling can inhibit tumor growth and increase drug sensitivity in mouse models. Clinical trials in non-small cell lung cancer (NSCLC) reveal that IL-6 targeted therapy relieves NSCLC-related anemia and cachexia, although other clinical effects require further study. We crossed  Accumulating evidence indicates that inflammation contributes to tumorigenesis, tumor progression, and metastasis ,2. Oncogin vivo [IL-6 knockdown, genetic ablation, or treatment with a neutralizing IL-6 antibody inhibits tumor growth in vivo [IL-6 expression can be detected in lung tumors  and in 5in vivo ,26,32. T in vivo ,33. Conv in vivo ,35, whil in vivo . An IL-6 monoclonal antibody therapy would be predicted to inhibit the inflammatory microenvironment in lung cancer. One such therapy, ALD518, has undergone preclinical and Phase I and II clinical trials. It appears to be well tolerated and ameliorates NSCLC-related anemia and cachexia , althougin vivo, we crossed IL-6-/- mice with mutant KrasG12D mice because IL-6 is a downstream effector of oncogenic Ras to promote tumorigenesis[p53 are found in at least 50% of NSCLC cases [KrasG12D activation accompanied by p53 deletion can cause lung tumor metastasis [KrasG12D; p53flox/flox; IL-6-/- mice..To assess the contribution of IL-6 signaling inhibition on tumor progression and survival time rigenesis. NSCLC irigenesis. InactivLC cases , and Kratastasis . To studIL-6-/- mice were purchased from The Jackson Laboratory and maintained in sterile housing [Lox–Stop–Lox KrasG12D (hereafter referred to as KrasG12D) mice [p53flox/flox mice [KrasG12D and KrasG12D; lL-6-/- mice were inoculated with 5 × 106 PFU of adenoviral Cre (adeno-Cre) by intranasal inhalation to activate oncogenic KrasG12D in the lungs. KrasG12D; p53flox/flox and KrasG12D; p53flox/flox;IL-6-/- mice were inoculated with 5 × 105 PFU of adeno-Cre. All experimental procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Institutional Animal Care and Use Committee at Dana-Farber Cancer Institute (permit number 04-094). All surgeries were performed under Avertin anesthesia to minimize suffering. After euthanasia, organs, including heart, liver, spleen, kidney, stomach, intestine, spine, brain, breast, skin, and testis or ovary, were undergone gross inspection for metastases. Lung tumors adhered to the pleura were considered parietal pleural metastases. Suspected metastases were harvested and confirmed by histological features. housing . Conditi2D) mice  and p53lox mice  were desAfter euthanasia, the lungs were removed and fixed in 10% neutral buffered formalin overnight before embedding in paraffin. Five-micrometer sections of mouse lung tissues were cut. Some sections were stained with H&E. For immunohistochemistry, heat treatment with citrate solution  in a decloaking chamber  unmasked antigens for phosphorylated ERK (pERK), BrdU, Ki67, Endomucin and Caspase-3 staining. Whole lung tissue sections were incubated overnight at 4°C with primary antibodies: pERK  at 1:100; BrdU  at 1:200; Ki67  at 1:200; Endomucin  at 1:100; cleaved Caspase-3  at 1:300. Digest-All 2B Trypsin (Invitrogen) was used to retrieve the antigen for MAC2  staining at 1:5000. At 400X magnification, all MAC2-positive macrophages in tumors were counted within 3 microscope fields with the most MAC2-positive macrophages after review of the whole lung section. Three mice per genotype were analyzed.At 20 weeks post-infection, mice were injected intraperitoneally with 10 μL of 10 mM BrdU in PBS per gram of body weight and euthanized after 2 hours. Whole lungs were harvested and processed as described above. At 400X magnification, all BrdU-positive tumor cell nuclei were counted within 3 microscope fields with the most BrdU-positive nuclei after review of the whole lung section. Four mice per genotype were analyzed. Same method was used to calculate Ki67-labeled tumor cells on sections from mice 28 weeks post-infection with adeno-Cre.KrasG12D and KrasG12D; lL-6-/- mice 32 weeks post-infection and from KrasG12D; p53flox/flox and KrasG12D; p53flox/flox; IL-6-/- mice 15 weeks post-infection for Western blot analysis. Tumors were lysed with a homogenizer in RIPA buffer  containing complete mini protease inhibitors (Roche) and phosphatase inhibitors . Nuclear and Cytoplasmic Extraction Kit  was used to extract cytoplasmic (C) and nuclear (N) fractions from tumors. Lysates (20 μg per lane) were separated on 10% polyacrylamide gels, transferred to PVDF filters, and incubated overnight at 4°C with antibodies to β-actin , pERK , total-ERK , pAKT , total-AKT , pSTAT3 , STAT3 , p65 , PARP , GAPDH , or β-catenin . Western blots were exposed to X-ray films or scanned with an ImageQuant LAS 4000mini . Lung tumors were harvested from KrasG12D and KrasG12D; lL-6-/- mice 32 weeks post-infection and KrasG12D; p53flox/flox and KrasG12D; p53flox/flox; IL-6-/- mice around 16 weeks post-infection for analysis. 2μg total RNA was reverse transcribed to cDNA using SuperRT cDNA synthesis kit . Real-time PCR was performed using the BioRad iQ5 Realtime PCR system and StepOnePlus Realtime PCR system (ABI) with Realtime PCR Master Mix containing SYBR Green  and unique primers  (n = 3), both KrasG12D and KrasG12D; IL-6-/- mice had early lung lesions. At 4 weeks post-infection, KrasG12D; IL-6-/- mice had more early lung lesions than KrasG12D mice  . Mice we12D mice . These lKrasG12D; IL-6-/- mice were modestly smaller and less dense than those in KrasG12D mice    despite nfection . BrdU stnfection , and pERnfection ; no statKrasG12D; p53flox/flox; IL-6-/- mice and 19 KrasG12D; p53flox/flox mice were analyzed for metastases around 16 weeks post-infection with adeno-Cre  and 10 of 19 KrasG12D; p53flox/flox mice (52.6%), although this difference was not significant (P=0.19). Metastatic lesions to the parietal pleura, thymus  . Oncogenr groups . KrasG12D; IL-6-/- tumors , a C-C motif chemokine, is overexpressed in pancreatic carcinoma cells and stimulates growth of tumor cells . CCL-19 nfection , but incnfection . These dp53 deletion more dramatically affected KrasG12D-induced lung cancer than IL-6 deletion. To a large extent, p53 deletion attenuated the effects of IL-6 deletion on delayed tumor growth and prolonged survival. p53 deletion enhanced pSTAT3 expression   . These rIL-6 deficiency promotes lung tumorigenesis, but suppresses tumor progression and elongates survival in vivo. However, these effects can be attenuated by p53 deletion.The involvement of inflammation in tumorigenesis, progression, and metastasis is widely accepted; however, whether IL-6-targeted therapies will prolong the survival time of lung cancer patients remains uncertain. Our results indicate anti-IL-6 therapies may have some success in clinical trials. For example, when NSCLC has not metastasized, IL-6 inhibition may prolong survival but increase the risk of further tumorigenesis; if metastasized, IL-6 inhibition may only moderately impact metastasis but may lengthen survival time. Further studies are needed to elucidate these possibilities. In summary, our results provide evidence that Figure S1Kras allelic recombination.PCR analysis of  A 500 bp PCR product represents the floxed, unrecombined KrasG12D allele; a 622 bp fragment represents the wildtype Kras allele; and a 650 bp fragment represents a recombined KrasG12D allele after removal of floxed stop cassette by adeno-Cre. K, KI, KP, and KPI mice were treated with adeno-Cre and the 650 bp recombined band revealed. Abbreviations: WT= wildtype lungs. Floxed=floxed KrasG12D, without adeno-Cre treatment. K=KrasG12D. KI=KrasG12D; IL-6-/-. KP=KrasG12D; p53flox/flox. KPI=G12D; p53flox/floxKras;IL-6-/-.(TIF)Click here for additional data file.Figure S2IL-6 deletion attenuates tumor proliferation determined by Ki67 staining.(A and B) Representative images of Ki67-stained lung tissue sections from (A) K and (B) KI mice 28 weeks post-infection with adeno-Cre. (C) Quantification of Ki67-positive tumor cells in lung tissue sections of K and KI mice (n=3). **P<0.01. Scale bar indicates 50 μm. Abbreviations: K=KrasG12D. KI=KrasG12D; IL-6-/-. (TIF)Click here for additional data file.Figure S3p53 allelic recombination.PCR analysis of  A 212 bp PCR product represents the floxed, unrecombined p53 allele; a 168 bp fragment represents the recombined allele after inoculation with adeno-Cre; and a 130 bp fragment represents the wildtype p53 allele. K, KI, KP, and KPI mice were treated with adeno-Cre. The 168 bp recombined band was showed in KP and KPI mice and 212 bp fragment remained due to tumor stromal cells. Abbreviations: WT=wildtype lungs. Floxed=floxed p53, without adeno-Cre treatment. K=KrasG12D. KI=KrasG12D; IL-6-/-. KP=KrasG12D; p53flox/flox. KPI=G12D; p53flox/floxKras;IL-6-/-.(TIFF)Click here for additional data file.Figure S4KP and KPI mice develop metastatic lesions.(A and B) Representative images of metastatic lesions to the (A) pleura, (B) thymus in KPI mice 15 weeks post-infection with adeno-Cre. Dotted lines in the images indicate metastatic tumor edges. Asterisks indicate center of metastatic tumors. (C) Representative image of heart metastases in KP mice 14 weeks post-infection with adeno-Cre. Metastatic lesions in the heart are left of the dotted line. Scale bar indicates 200 μm (A) or 100 μm (B and C). Abbreviations: KP=KrasG12D; p53flox/flox. KPI=G12D; p53flox/floxKras;IL-6-/-.(TIF)Click here for additional data file.Figure S5Real-time PCR screen of changes in inflammatory cytokines levels.. P<0.05 vs. K tumors. # P<0.05 vs. KP tumors. Abbreviations: K=KrasG12D. KI=KrasG12D; IL-6-/-. KP=KrasG12D; p53flox/flox. KPI=G12D; p53flox/floxKras;IL-6-/-.Three tumors from each genotype were analyzed by real-time PCR without replicate for expression of the indicated cytokine. Gene expression was normalized to β-actin mRNA. *(TIF)Click here for additional data file.Figure S6Nuclear localization of p65 and β-catenin are unchanged.Tumors from each mouse genotype were lysed to obtain cytoplasmic (C) and nuclear (N) fractions. Lysates were analyzed for the presence of nuclear p65 and β-catenin by Western blot. Fraction purity was determined by GAPDH (cytoplasmic) and PARP (nuclear) blots. Abbreviations: K=KrasG12D. KI=KrasG12D; IL-6-/-. KP=KrasG12D; p53flox/flox. KPI=G12D; p53flox/floxKras;IL-6-/-.(TIFF)Click here for additional data file.Table S1Primers for real-time PCR analysis of gene expression.(DOCX)Click here for additional data file.