Pancreatic cancer may have a distinct miRNA expression pattern that may differentiate it from normal pancreas and chronic pancreatitis. miRNA expression patterns may be able to distinguish between long- and short-term survivors, but these findings need to be validated in other study populations.

microRNAs are a highly conserved class of noncoding RNAs with important regulatory functions in proliferation, apoptosis, development, and differentiation. To discover novel regulatory pathways during megakaryocytic differentiation, we performed microRNA expression profiling of in vitro-differentiated megakaryocytes derived from CD34 ؉ hematopoietic progenitors. The main finding was down-regulation of miR-10a, miR-126, miR-106, miR-10b, miR-17 and miR-20. Hypothetically, the down-regulation of microRNAs unblocks target genes involved in differentiation. We confirmed in vitro and in vivo that miR-130a targets the transcription factor MAFB, which is involved in the activation of the GPIIB promoter, a key protein for platelet physiology. In addition, we found that miR-10a expression in differentiated megakaryocytes is inverse to that of HOXA1, and we showed that HOXA1 is a direct target of miR-10a. Finally, we compared the microRNA expression of megakaryoblastic leukemic cell lines with that of in vitro differentiated megakaryocytes and CD34 ؉ progenitors. This analysis revealed up-regulation of miR-101, miR-126, miR-99a, miR-135, and miR-20. Our data delineate the expression of microRNAs during megakaryocytopoiesis and suggest a regulatory role of microRNAs in this process by targeting megakaryocytic transcription factors.leukemia ͉ hematopoiesis

Activated monocytes produce proinflammatory cytokines (monokines) such as interleukin (IL)-12, IL-15, and IL-18 for induction of interferon-gamma (IFN-gamma) by natural killer (NK) cells. NK cells provide the antiinflammatory cytokine transforming growth factor (TGF)-beta, an autocrine/negative regulator of IFN-gamma. The ability of one signaling pathway to prevail over the other is likely important in controlling IFN-gamma for the purposes of infection and autoimmunity, but the molecular mechanism(s) of how this counterregulation occurs is unknown. Here we show that in isolated human NK cells, proinflammatory monokines antagonize antiinflammatory TGF-beta signaling by downregulating the expression of the TGF-beta type II receptor, and its signaling intermediates SMAD2 and SMAD3. In contrast, TGF-beta utilizes SMAD2, SMAD3, and SMAD4 to suppress IFN-gamma and T-BET, a positive regulator of IFN-gamma. Indeed, activated NK cells from Smad3(-/-) mice produce more IFN-gamma in vivo than NK cells from wild-type mice. Collectively, our data suggest that pro- and antiinflammatory cytokine signaling reciprocally antagonize each other in an effort to prevail in the regulation of NK cell IFN-gamma production.

Next‐generation sequencing has aided characterization of genomic variation. While whole‐genome sequencing may capture all possible mutations, whole‐exome sequencing remains cost‐effective and captures most phenotype‐altering mutations. Initial strategies for exome enrichment utilized a hybridization‐based capture approach. Recently, amplicon‐based methods were designed to simplify preparation and utilize smaller DNA inputs. We evaluated two hybridization capture‐based and two amplicon‐based whole‐exome sequencing approaches, utilizing both Illumina and Ion Torrent sequencers, comparing on‐target alignment, uniformity, and variant calling. While the amplicon methods had higher on‐target rates, the hybridization capture‐based approaches demonstrated better uniformity. All methods identified many of the same single‐nucleotide variants, but each amplicon‐based method missed variants detected by the other three methods and reported additional variants discordant with all three other technologies. Many of these potential false positives or negatives appear to result from limited coverage, low variant frequency, vicinity to read starts/ends, or the need for platform‐specific variant calling algorithms. All methods demonstrated effective copy‐number variant calling when evaluated against a single‐nucleotide polymorphism array. This study illustrates some differences between whole‐exome sequencing approaches, highlights the need for selecting appropriate variant calling based on capture method, and will aid laboratories in selecting their preferred approach.

Activation of fibroblast growth factor receptor (FGFR) signaling through mutations, amplifications, or fusions involving FGFR1, 2, 3, or 4 are seen in multiple tumors including lung, bladder, and cholangiocarcinoma. Currently, several clinical trials are evaluating the role of novel FGFR inhibitors in solid tumors. As we move forward with FGFR inhibitors clinically, we anticipate emergence of resistance with treatment. Consequently, we sought to study the mechanism(s) of acquired resistance to FGFR inhibitors using annotated cancer cell lines. We identified cancer cell lines that have activating mutations in FGFR1, 2, or 3, and treated them chronically with the selective FGFR inhibitor, BGJ398. We observed resistance to chronic BGJ398 exposure in DMS114 (small cell lung cancer, FGFR1 amplification), and RT112 (urothelial carcinoma, FGFR3 fusion/amplification) cell lines based on viability assays. Reverse phase protein array (RPPA) analysis showed increased phosphorylation of Akt (T308 and S473) and its downstream target GSK3 (S9 and S21) in both the resistant cell lines when compared to matching controls. Results of RPPA were confirmed using immunoblots. Consequently, the addition of an Akt inhibitor (GSK2141795) or siRNA was able to restore sensitivity to BGJ398 in resistant cell lines. These data suggest a role for Akt pathway in mediating acquired resistance to FGFR inhibition.

Targeted, capture-based DNA sequencing is a cost-effective method to focus sequencing on a coding region or other customized region of the genome. There are multiple targeted sequencing methods available, but none has been systematically investigated and compared. We evaluated four commercially available custom-targeted DNA technologies for next-generation sequencing with respect to on-target sequencing, uniformity, and ability to detect single-nucleotide variations (SNVs) and copy number variations. The technologies that used sonication for DNA fragmentation displayed impressive uniformity of capture, whereas the others had shorter preparation times, but sacrificed uniformity. One of those technologies, which uses transposase for DNA fragmentation, has a drawback requiring sample pooling, and the last one, which uses restriction enzymes, has a limitation depending on restriction enzyme digest sites. Although all technologies displayed some level of concordance for calling SNVs, the technologies that require restriction enzymes or transposase missed several SNVs largely because of the lack of coverage. All technologies performed well for copy number variation calling when compared to single-nucleotide polymorphism arrays. These results enable laboratories to compare these methods to make informed decisions for their intended applications.

Interleukin-15 (IL-15) is a pleiotropic proinflammatory cytokine with inefficient posttranscriptional processing. We hypothesized that endogenous IL-15 could affect disease progression in the well-described C57Bl/6 (B6) 3 (C57Bl/6 ؋ DBA/2) F1 hybrid ( IntroductionBone marrow transplantation (BMT) is a potentially curative therapy for patients with heritable immunodeficiencies and malignant diseases including leukemia, lymphoma, and myeloma. 1 While the conditioning regimen for BMT leads to direct tumor destruction, donor-derived allogeneic T cells can exert an important graft-versus-tumor (GVT) effect: recipients of allogeneic transplants have a decreased probability of relapse compared with recipients of autologous, syngeneic, or T-cell-depleted bone marrow transplants. 2,3 However, allogeneic BMT also carries a significant risk for graft-versus-host disease (GVHD), an immunologic attack of allogeneic donor T lymphocytes against normal recipient tissues, the magnitude of which depends in large part on the degree of HLA incompatibility between donor and recipient. Even with appropriate prophylaxis, the incidence of acute GVHD ranges from 30% in HLA-identical donor-recipient pairs to 70% for donorrecipient pairs HLA-incompatible at 2 loci. 1,4 Thus, GVHD remains the most significant obstacle to the wider application of BMT for the treatment of malignancy.GVHD is characterized by a cycle of tissue destruction and inflammation initiated by the preparative regimen for transplantation and propagated by alloreactive donor T cells. 5 Transplant recipients are prepared for BMT with a regimen that obliterates their current bone marrow stores and potentially their residual leukemia. However, this regimen is also highly toxic to the gastrointestinal system and allows release of gram-negative bacterial lipopolysaccharide (LPS) across the intestinal mucosa. 6 LPS is a potent stimulator for the production of tumor necrosis factor ␣ (TNF-␣), interleukin-1 (IL-1), and IL-12. 6,7 IL-12 polarizes donorderived T cells toward a proinflammatory Th1/Tc1 phenotype, producing interferon ␥ (IFN-␥) and TNF-␣; 8,9 IFN-␥ and TNF-␣ induce major histocompatibility complex (MHC) expression by host-derived antigen-presenting cells (APCs), which results in efficient presentation of alloantigen to donor-derived T cells. 10,11 These donor-derived Th1/Tc1-polarized T cells in turn produce IL-2, expand, and infiltrate host epithelial tissues. Thus, the inflammation of acute GVHD is influenced in large part by the cytokine cascade within the host after transplantation.The role of IL-15, a T-and natural killer (NK) cell growth factor, in allogeneic GVHD has not been addressed. Originally isolated because of its ability to restore T-cell growth in the presence of IL-2-neutralizing antibodies, 12,13 IL-15 mRNA is widely and abundantly expressed in multiple tissues. However, IL-15 is inefficiently translated and secreted with multiple posttranscriptional checkpoints. [14][15][16] 36,37 We therefore hypothesized that alteration of endogenous IL-15 produ...

IntroductionXenogeneic graft-versus-host disease (X-GVHD) is the only experimental model of human allogeneic GVHD that incorporates human T cells and recapitulates the T-cell expansion and tissue destruction seen in patients. Severe combined immunodeficient (SCID) mice reconstituted with human peripheral-blood lymphocytes (hu-PBL-SCID) demonstrate, albeit infrequently, lethal X-GVHD associated with high levels of human T-cell engraftment and inflammation of the liver, kidneys, spleen, and lung. 1 We have previously demonstrated that administration of continuous, lowdose recombinant human IL-2 (rhIL-2) to hu-PBL-SCID mice does not increase X-GVHD mortality. 2 IL-15 is structurally similar to IL-2 and uses identical ␤ and ␥ receptor subunits for signaling, but is produced by antigen-presenting cells and demonstrates in vivo activities distinct from IL-2. Recently, data from our laboratory and others have shown that endogenous or exogenous administration of IL-15 increases mortality in murine models of acute allogeneic GVHD. 3,4 Therefore, we have hypothesized that exogenous rhIL-15 would enhance X-GVHD mortality in hu-PBL-SCID mice in contrast to the effects of rhIL-2. Study design Hu-PBL-SCID mouse modelHuman PBLs (5 ϫ 10 7 ) were obtained from healthy donors under an institutional review board (IRB)-approved protocol and injected (intraperitoneal) into 8-to 12-week-old female CB17 SCID mice (Taconic Farms, Germantown, NY) pretreated with murine IL-2R␤ antibodies. 2,5 This regimen eliminates host natural killer cells 6 and increases human T-cell chimerism. 7 Mice were killed as described. 3 All animal research was approved by The Ohio State University. Approval for the use of human subjects was obtained from The Ohio State University IRB. Informed consent was provided according to the Declaration of Helsinki. CytokinesAnimals were dosed with 10 g/d rhIL-15 (Amgen, Thousand Oaks, CA), rhIL-2 (Proleukin; Chiron, Emeryville, CA), or phosphate-buffered saline (PBS) containing 0.05% human albumin via subcutaneous Alzet miniosmotic pumps (Model 1007D; DURECT, Cupertino, CA) beginning 1 day after injection of human PBLs. In vivo antibody treatmentAnimals were treated with anti-human CD3 (50 g/mouse, Orthoclone-OKT3; Ortho Biotech Products, Raritan, NJ) or control mouse immunoglobulin G (IgG; Sigma, St Louis, MO) via intraperitoneal injection on days 1 to 5 after injection of human PBLs. 8 HistopathologyBlinded histologic samples were prepared as described, 3 reviewed by a board-certified veterinary pathologist (D.K.), and scored for lymphocytic infiltration and tissue damage characteristic of X-GVHD. 9 Cytokine bead arrayThe Human Th1/Th2 Cytokine Bead Array (BD Pharmingen, San Diego, CA) was used to measure IL-4, IL-5, IL-10, interferon-␥ (IFN-␥), and tumor necrosis factor-␣ (TNF-␣) in plasma samples as described. 3 StatisticsMedian survival times were compared using the log-rank test. Student t test and the exact Wilcoxon rank sum test were used for the remaining comparisons. Results and discussionAdministration of r...