Renal tubular epithelial cells are constitutive non-cognate stimulators of resident T cells.

Understanding the roles of different cell types in regulating T cell homeostasis in various tissues is critical for understanding adaptive immunity. Here, we show that RTECs (renal tubular epithelial cells) are intrinsically programmed to polyclonally stimulate proliferation of kidney αß T cells by a cell-cell contact mechanism that is major histocompatibility complex (MHC) independent and regulated by CD155, αVß3-integrin, and vitronectin. Peripheral CD4 and CD8 are resistant to RTEC-mediated stimulation, while the minor subset of double-negative (DN) T cells are responsive. This functional property of RTEC is discovered by using a coculture system that recapitulates spontaneous in vivo polyclonal proliferation of kidney T cells, which are mainly comprised of central memory T (TCM) and effector memory T (TEM) cells. This robust cell-intrinsic stimulatory role of RTECs could be underlying the steady-state spontaneous proliferation of kidney T cells. The results have conceptual implications for understanding roles of different cell types in regulating systemic and organ-specific T cell homeostasis.

WDR82-Mediated H3K4me3 Is Associated with Tumor Proliferation and Therapeutic Efficacy in Pediatric High-Grade Gliomas.

Pediatric high-grade gliomas (pHGGs) are common malignant brain tumors without effective treatment and poor patient survival. Abnormal posttranslational modification at the histone H3 tail plays critical roles in tumor cell malignancy. We have previously shown that the trimethylation of lysine 4 at histone H3 (H3K4me3) plays a significant role in pediatric ependymoma malignancy and is associated with tumor therapeutic sensitivity. Here, we show that H3K4me3 and its methyltransferase WDR82 are elevated in pHGGs. A reduction in H3K4me3 by downregulating WDR82 decreases H3K4me3 promoter occupancy and the expression of genes associated with stem cell features, cell proliferation, the cell cycle, and DNA damage repair. A reduction in WDR82-mediated H3K4me3 increases the response of pediatric glioma cells to chemotherapy. These findings suggest that WDR82-mediated H3K4me3 is an important determinant of pediatric glioma malignancy and therapeutic response. This highlights the need for a more thorough understanding of the potential of WDR82 as an epigenetic target to increase therapeutic efficacy and improve the prognosis for children with malignant gliomas.

Itraconazole Exerts Its Antitumor Effect in Esophageal Cancer By Suppressing the HER2/AKT Signaling Pathway.

Itraconazole, an FDA-approved antifungal, has antitumor activity against a variety of cancers. We sought to determine the effects of itraconazole on esophageal cancer and elucidate its mechanism of action. Itraconazole inhibited cell proliferation and induced G1-phase cell-cycle arrest in esophageal squamous cell carcinoma and adenocarcinoma cell lines. Using an unbiased kinase array, we found that itraconazole downregulated protein kinase AKT phosphorylation in OE33 esophageal adenocarcinoma cells. Itraconazole also decreased phosphorylation of downstream ribosomal protein S6, transcriptional expression of the upstream receptor tyrosine kinase HER2, and phosphorylation of upstream PI3K in esophageal cancer cells. Lapatinib, a tyrosine kinase inhibitor that targets HER2, and siRNA-mediated knockdown of HER2 similarly suppressed cancer cell growth in vitro Itraconazole significantly inhibited growth of OE33-derived flank xenografts in mice with detectable levels of itraconazole and its primary metabolite, hydroxyitraconazole, in esophagi and tumors. HER2 total protein and phosphorylation of AKT and S6 proteins were decreased in xenografts from itraconazole-treated mice compared to xenografts from placebo-treated mice. In an early phase I clinical trial (NCT02749513) in patients with esophageal cancer, itraconazole decreased HER2 total protein expression and phosphorylation of AKT and S6 proteins in tumors. These data demonstrate that itraconazole has potent antitumor properties in esophageal cancer, partially through blockade of HER2/AKT signaling.

Analysis of mesenchymal stem cell proteomes in situ in the ischemic heart.

Rationale: Cell therapy for myocardial infarction is promising but largely unsuccessful in part due to a lack of mechanistic understanding. Techniques enabling identification of stem cell-specific proteomes in situ in the injured heart may shed light on how the administered cells respond to the injured microenvironment and exert reparative effects. Objective: To identify the proteomes of the transplanted mesenchymal stem cells (MSCs) in the infarcted myocardium, we sought to target a mutant methionyl-tRNA synthetase (MetRSL274G) in MSCs, which charges azidonorleucine (ANL), a methionine analogue and non-canonical amino acid, to tRNA and subsequently to nascent proteins, permitting isolation of ANL-labeled MSC proteomes from ischemic hearts by ANL-alkyne based click reaction. Methods and Results: Murine MSCs were transduced with lentivirus MetRSL274G and supplemented with ANL; the ANL-tagged nascent proteins were visualized by bio-orthogonal non-canonical amino-acid tagging, spanning all molecular weights and by fluorescent non-canonical amino-acid tagging, displaying strong fluorescent signal. Then, the MetRSL274G-transduced MSCs were administered to the infarcted or Sham heart in mice receiving ANL treatment. The MSC proteomes were isolated from the left ventricular protein lysates by click reaction at days 1, 3, and 7 after cell administration, identified by LC/MS. Among all identified proteins (in Sham and MI hearts, three time-points each), 648 were shared by all 6 groups, accounting for 82±5% of total proteins in each group, and enriched under mitochondrion, extracellular exosomes, oxidation-reduction process and poly(A) RNA binding. Notably, 26, 110 and 65 proteins were significantly up-regulated and 11, 28 and 19 proteins were down-regulated in the infarcted vs. Sham heart at the three time-points, respectively; these proteins are pronounced in the GO terms of extracellular matrix organization, response to stress and regulation of apoptotic process and in the KEGG pathways of complements and coagulation cascades, apoptosis, and regulators of actin cytoskeleton. Conclusions: MetRSL274G expression allows successful identification of MSC-specific nascent proteins in the infarcted hearts, which reflect the functional states, adaptive response, and reparative effects of MSCs that may be leveraged to improve cardiac repair.

GEAMP, a novel gastroesophageal junction carcinoma cell line derived from a malignant pleural effusion.

Gastroesophageal junction (GEJ) cancer remains a clinically significant disease in Western countries due to its increasing incidence, which mirrors that of esophageal cancer, and poor prognosis. To develop novel and effective approaches for prevention, early detection, and treatment of patients with GEJ cancer, a better understanding of the mechanisms driving pathogenesis and malignant progression of this disease is required. These efforts have been limited by the small number of available cell lines and appropriate preclinical animal models for in vitro and in vivo studies. We have established and characterized a novel GEJ cancer cell line, GEAMP, derived from the malignant pleural effusion of a previously treated GEJ cancer patient. Comprehensive genetic analyses confirmed a clonal relationship between GEAMP cells and the primary tumor. Targeted next-generation sequencing identified 56 nonsynonymous alterations in 51 genes including TP53 and APC, which are commonly altered in GEJ cancer. In addition, multiple copy-number alterations were found including EGFR and K-RAS gene amplifications and loss of CDKN2A and CDKN2B. Histological examination of subcutaneous flank xenografts in nude and NOD-SCID mice showed a carcinoma with mixed squamous and glandular differentiation, suggesting GEAMP cells contain a subpopulation with multipotent potential. Finally, pharmacologic inhibition of the EGFR signaling pathway led to downregulation of key downstream kinases and inhibition of cell proliferation in vitro. Thus, GEAMP represents a valuable addition to the limited number of bona fide GEJ cancer cell lines.

A Public BCR Present in a Unique Dual-Receptor-Expressing Lymphocyte from Type 1 Diabetes Patients Encodes a Potent T Cell Autoantigen.

T and B cells are the two known lineages of adaptive immune cells. Here, we describe a previously unknown lymphocyte that is a dual expresser (DE) of TCR and BCR and key lineage markers of both B and T cells. In type 1 diabetes (T1D), DEs are predominated by one clonotype that encodes a potent CD4 T cell autoantigen in its antigen binding site. Molecular dynamics simulations revealed that this peptide has an optimal binding register for diabetogenic HLA-DQ8. In concordance, a synthetic version of the peptide forms stable DQ8 complexes and potently stimulates autoreactive CD4 T cells from T1D patients, but not healthy controls. Moreover, mAbs bearing this clonotype are autoreactive against CD4 T cells and inhibit insulin tetramer binding to CD4 T cells. Thus, compartmentalization of adaptive immune cells into T and B cells is not absolute, and violators of this paradigm are likely key drivers of autoimmune diseases.

Global Reduction of H3K4me3 Improves Chemotherapeutic Efficacy for Pediatric Ependymomas.

BACKGROUND: Ependymomas (EPNs) are the third most common brain tumor in children. These tumors are resistant to available chemotherapeutic treatments, therefore new effective targeted therapeutics must be identified. Increasing evidence shows epigenetic alterations including histone posttranslational modifications (PTMs), are associated with malignancy, chemotherapeutic resistance and prognosis for pediatric EPNs. In this study we examined histone PTMs in EPNs and identified potential targets to improve chemotherapeutic efficacy. METHODS: Global histone H3 lysine 4 trimethylation (H3K4me3) levels were detected in pediatric EPN tumor samples with immunohistochemistry and immunoblots. Candidate genes conferring therapeutic resistance were profiled in pediatric EPN tumor samples with micro-array. Promoter H3K4me3 was examined for two candidate genes, CCND1 and ERBB2, with chromatin-immunoprecipitation coupled with real-time PCR (ChIP-PCR). These methods and MTS assay were used to verify a relationship between H3K4me3 levels and CCND1 and ERBB2, and to investigate cell viability in response to chemotherapeutic drugs in primary cultured pediatric EPN cells. RESULTS: H3K4me3 levels positively correlate with WHO grade malignancy in pediatric EPNs and are associated with progression free survival in patients with posterior fossa group A EPNs (PF-EPN-A). Reduction of H3K4me3 by silencing its methyltransferase SETD1A, in primary cultured EPN cells increased cell response to chemotherapy. CONCLUSIONS: Our results support the development of a novel treatment that targets H3K4me3 to increase chemotherapeutic efficacy in pediatric PF-EPN-A tumors.

Autophagy, Cell Viability, and Chemoresistance Are Regulated By miR-489 in Breast Cancer.

It is postulated that the complexity and heterogeneity in cancer may hinder most efforts that target a single pathway. Thus, discovery of novel therapeutic agents targeting multiple pathways, such as miRNAs, holds promise for future cancer therapy. One such miRNA, miR-489, is downregulated in a majority of breast cancer cells and several drug-resistant breast cancer cell lines, but its role and underlying mechanism for tumor suppression and drug resistance needs further investigation. The current study identifies autophagy as a novel pathway targeted by miR-489 and reports Unc-51 like autophagy activating kinase 1 (ULK1) and lysosomal protein transmembrane 4 beta (LAPTM4B) to be direct targets of miR-489. Furthermore, the data demonstrate autophagy inhibition and LAPTM4B downregulation as a major mechanism responsible for miR-489-mediated doxorubicin sensitization. Finally, miR-489 and LAPTM4B levels were inversely correlated in human tumor clinical specimens, and more importantly, miR-489 expression levels predict overall survival in patients with 8q22 amplification (the region in which LAPTM4B resides).Implications: These findings expand the understanding of miR-489-mediated tumor suppression and chemosensitization in and suggest a strategy for using miR-489 as a therapeutic sensitizer in a defined subgroup of resistant breast cancer patients. Mol Cancer Res; 16(9); 1348-60. ©2018 AACR.

HER2 Overexpression Triggers an IL1α Proinflammatory Circuit to Drive Tumorigenesis and Promote Chemotherapy Resistance.

Systemic inflammation in breast cancer correlates with poor prognosis, but the molecular underpinnings of this connection are not well understood. In this study, we explored the relationship between HER2 overexpression, inflammation, and expansion of the mammary stem/progenitor and cancer stem-like cell (CSC) population in breast cancer. HER2-positive epithelial cells initiated and sustained an inflammatory milieu needed to promote tumorigenesis. HER2 induced a feedforward activation loop of IL1α and IL6 that stimulated NFκB and STAT3 pathways for generation and maintenance of breast CSC. In mice, Il1a genetic deficiency delayed MMTV-Her2-induced tumorigenesis and reduced inflammatory cytokine expression as well as CSC in primary tumors. In clinical specimens of human breast tumor tissues, tissue microarray analysis revealed a strong positive correlation between IL1α/IL6 expression and CSC-positive phenotype. Pharmacologic blockade of IL1α signaling reduced the CSC population and improved chemotherapeutic efficacy. Our findings suggest new therapeutic or prevention strategies for HER2-positive breast cancers.Significance: IL1α signaling driven by HER2 promotes chronic inflammation needed to support cancer stem-like cell maintenance in HER2-positive breast cancers. Cancer Res; 78(8); 2040-51. ©2018 AACR.

A novel double-negative feedback loop between miR-489 and the HER2-SHP2-MAPK signaling axis regulates breast cancer cell proliferation and tumor growth.

Human epidermal growth factor receptor 2 (HER2 or ErBb2) is a receptor tyrosine kinase overexpressed in 20-30% of breast cancers and associated with poor prognosis and outcome. Dysregulation of several microRNAs (miRNAs) plays a key role in breast cancer progression and metastasis. In this study, we screened and identified miRNAs dysregualted in HER2-positive breast cancer cells. Our molecular study demonstrated that miR-489 was specifically downregulated by the HER2-downstream signaling, especially through the MAPK pathway. Restoration or overexpression of miR-489 in HER2-positive breast cancer cells significantly inhibited cell growth in vitro and decreased the tumorigenecity and tumor growth in xenograft mice. Mechanistically, we found that overexpression of miR-489 led to the decreased levels of HER2 and SHP2 and thus attenuated HER2-downstream signaling. Furthermore, we for the first time demonstrated that HER2 is a direct target of miR-489 and therefore HER2-SHP2-MAPK and miR-489 signaling pathways form a mutually inhibitory loop. Using quantitative real-time PCR analysis and Fluorescent in situ hybridization technique (FISH), we found that miR-489 was expressed at significantly lower level in tumor tissues compared to the adjacent normal tissues. Downregulation of miR-489 in breast cancers was associated with aggressive tumor phenotypes. Overall, our results define a double-negative feedback loop involving miR-489 and the HER2-SHP2-MAPK signaling axis that can regulate breast cancer cell proliferation and tumor progression and might have therapeutic relevance for HER2-positive breast cancer.

Transplant-induced reactivation of murine cytomegalovirus immediate early gene expression is associated with recruitment of NF-κB and AP-1 to the major immediate early promoter.

Reactivation of latent human cytomegalovirus is a significant infectious complication of organ transplantation and current therapies target viral replication once reactivation of latent virus has already occurred. The specific molecular pathways that activate viral gene expression in response to transplantation are not well understood. Our studies aim to identify these factors, with the goal of developing novel therapies that prevent transcriptional reactivation in transplant recipients. Murine cytomegalovirus (MCMV) is a valuable model for studying latency and reactivation of CMV in vivo. We previously demonstrated that transplantation of MCMV-latently infected kidneys into allogeneic recipients induces reactivation of immediate early (IE) gene expression and epigenetic reprogramming of the major immediate early promoter (MIEP) within 48 h. We hypothesize that these events are mediated by activation of signalling pathways that lead to binding of transcription factors to the MIEP, including AP-1 and NF-κB. Here we show that transplantation induces rapid activation of several members of the AP-1 and NF-κB transcription factor family and we demonstrate that canonical NF-κB (p65/p50), the junD component of AP-1, and nucleosome remodelling complexes are recruited to the MIEP following transplantation. Proteomic analysis of recipient plasma and transcriptome analysis of kidney RNA identified five extracellular ligands, including TNF, IL-1ß, IL-18, CD40L and IL-6, and three intracellular signalling pathways associated with reactivation of IE gene expression. Identification of the factors that mediate activation of these signalling pathways may eventually lead to new therapies to prevent reactivation of CMV and its sequelae.

Central role of ULK1 in type I interferon signaling.

We provide evidence that the Unc-51-like kinase 1 (ULK1) is activated during engagement of the type I interferon (IFN) receptor (IFNR). Our studies demonstrate that the function of ULK1 is required for gene transcription mediated via IFN-stimulated response elements (ISRE) and IFNγ activation site (GAS) elements and controls expression of key IFN-stimulated genes (ISGs). We identify ULK1 as an upstream regulator of p38α mitogen-activated protein kinase (MAPK) and establish that the regulatory effects of ULK1 on ISG expression are mediated possibly by engagement of the p38 MAPK pathway. Importantly, we demonstrate that ULK1 is essential for antiproliferative responses and type I IFN-induced antineoplastic effects against malignant erythroid precursors from patients with myeloproliferative neoplasms. Together, these data reveal a role for ULK1 as a key mediator of type I IFNR-generated signals that control gene transcription and induction of antineoplastic responses.

Recipient Myd88 Deficiency Promotes Spontaneous Resolution of Kidney Allograft Rejection.

The myeloid differentiation protein 88 (MyD88) adapter protein is an important mediator of kidney allograft rejection, yet the precise role of MyD88 signaling in directing the host immune response toward the development of kidney allograft rejection remains unclear. Using a stringent mouse model of allogeneic kidney transplantation, we demonstrated that acute allograft rejection occurred equally in MyD88-sufficient (wild-type [WT]) and MyD88(-/-) recipients. However, MyD88 deficiency resulted in spontaneous diminution of graft infiltrating effector cells, including CD11b(-)Gr-1(+) cells and activated CD8 T cells, as well as subsequent restoration of near-normal renal graft function, leading to long-term kidney allograft acceptance. Compared with T cells from WT recipients, T cells from MyD88(-/-) recipients failed to mount a robust recall response upon donor antigen restimulation in mixed lymphocyte cultures ex vivo. Notably, exogenous IL-6 restored the proliferation rate of T cells, particularly CD8 T cells, from MyD88(-/-) recipients to the proliferation rate of cells from WT recipients. Furthermore, MyD88(-/-) T cells exhibited diminished expression of chemokine receptors, specifically CCR4 and CXCR3, and the impaired ability to accumulate in the kidney allografts despite an otherwise MyD88-sufficient environment. These results provide a mechanism linking the lack of intrinsic MyD88 signaling in T cells to the effective control of the rejection response that results in spontaneous resolution of acute rejection and long-term graft protection.

Mesenchymal change and drug resistance in neuroblastoma.

BACKGROUND: Metastatic initiation has many phenotypic similarities to epithelial-to-mesenchymal transition, including loss of cell-cell adhesion, increased invasiveness, and increased cell mobility. We have previously demonstrated that drug resistance is associated with a metastatic phenotype in neuroblastoma (NB). The purpose of this project was to determine if the development of doxorubicin resistance is associated with characteristics of mesenchymal change in human NB cells. MATERIALS AND METHODS: Total RNA was isolated from wild type (WT) and doxorubicin-resistant (DoxR) human NB cell lines (SK-N-SH and SK-N-BE(2)C) and analyzed using the Illumina Human HT-12 version 4 Expression BeadChip. Differentially expressed genes (DEGs) were identified. Volcano plots and heat maps were generated. Genes of interest with a fold change in expression >1.5 and an adjusted P < 0.1 were analyzed. Immunofluorescence (IF) and Western blot analysis confirmed microarray results of interest. Matrigel invasion assay and migration wounding assays were performed. RESULTS: Volcano plots and heat maps visually demonstrated a similar pattern of DEGs in the SK-N-SH and SK-N-BE(2)C DoxR cell lines relative to their parental WT lines. Venn diagramming revealed 1594 DEGs common to both DoxR cell lines relative to their parental cell lines. Network analysis pointed to several significantly upregulated epithelial-to-mesenchymal transition pathways, through TGF-beta pathways via RhoA, PI3K, and ILK and via SMADs, as well as via notch signaling pathways. DoxR cell lines displayed a more invasive phenotype than respective WT cell lines. CONCLUSIONS: Human SK-N-SH and SK-N-BE(2)C NB cells display characteristics of mesenchymal change via multiple pathways in the transition to a drug-resistant state.

HOXB7 promotes malignant progression by activating the TGFß signaling pathway.

Overexpression of HOXB7 in breast cancer cells induces an epithelial-mesenchymal transition and promotes tumor progression and lung metastasis. However, the underlying mechanisms for HOXB7-induced aggressive phenotypes in breast cancer remain largely unknown. Here, we report that phosphorylation of SMAD3 was detected in a higher percentage in primary mammary tumor tissues from double-transgenic MMTV-Hoxb7/Her2 mice than tumors from single-transgenic Her2/neu mice, suggesting activation of TGFß/SMAD3 signaling by HOXB7 in breast tumor tissues. As predicted, TGFß2 was high in four MMTV-Hoxb7/Her2 transgenic mouse tumor cell lines and two breast cancer cell lines transfected with HOXB7, whereas TGFß2 was low in HOXB7-depleted cells. HOXB7 directly bound to and activated the TGFß2 promoter in luciferase and chromatin immunoprecipitation assays. Increased migration and invasion as a result of HOXB7 overexpression in breast cancer cells were reversed by knockdown of TGFß2 or pharmacologic inhibition of TGFß signaling. Furthermore, knockdown of TGFß2 in HOXB7-overexpressing MDA-MB-231 breast cancer cells dramatically inhibited metastasis to the lung. Interestingly, HOXB7 overexpression also induced tumor-associated macrophage (TAM) recruitment and acquisition of an M2 tumor-promoting phenotype. TGFß2 mediated HOXB7-induced activation of macrophages, suggesting that TAMs may contribute to HOXB7-promoted tumor metastasis. Providing clinical relevance to these findings, by real-time PCR analysis, there was a strong correlation between HOXB7 and TGFß2 expression in primary breast carcinomas. Taken together, our results suggest that HOXB7 promotes tumor progression in a cell-autonomous and non-cell-autonomous manner through activation of the TGFß signaling pathway.

E2F1 suppresses cardiac neovascularization by down-regulating VEGF and PlGF expression.

AIMS: The E2F transcription factors are best characterized for their roles in cell-cycle regulation, cell growth, and cell death. Here we investigated the potential role of E2F1 in cardiac neovascularization. METHODS AND RESULTS: We induced myocardial infarction (MI) by ligating the left anterior descending artery in wild-type (WT) and E2F1(-/-) mice. E2F1(-/-) mice demonstrated a significantly better cardiac function and smaller infarct sizes than WT mice. At infarct border zone, capillary density and endothelial cell (EC) proliferation were greater, apoptotic ECs were fewer, levels of VEGF and placental growth factor (PlGF) were higher, and p53 level was lower in E2F1(-/-) than in WT mice. Blockade of VEGF receptor 2 (VEGFR2) signalling with the selective inhibitor SU5416 or with the VEGFR2-blocking antibody DC101 abolished the differences between E2F1(-/-) mice and WT mice in cardiac function, infarct size, capillary density, EC proliferation, and EC apoptosis. In vitro, hypoxia-induced VEGF and PlGF up-regulation was significantly greater in E2F1(-/-) than in WT cardiac fibroblasts, and E2F1 overexpression suppressed PlGF up-regulation in both WT and p53(-/-) cells; however, VEGF up-regulation was suppressed only in WT cells. E2F1 interacted with and stabilized p53 under hypoxic conditions, and both E2F1 : p53 binding and the E2F1-induced suppression of VEGF promoter activity were absent in cells that expressed an N-terminally truncated E2F1 mutant. CONCLUSION: E2F1 limits cardiac neovascularization and functional recovery after MI by suppressing VEGF and PlGF up-regulation through p53-dependent and -independent mechanisms, respectively.

Hedgehog signaling regulates FOXA2 in esophageal embryogenesis and Barrett's metaplasia.

Metaplasia can result when injury reactivates latent developmental signaling pathways that determine cell phenotype. Barrett's esophagus is a squamous-to-columnar epithelial metaplasia caused by reflux esophagitis. Hedgehog (Hh) signaling is active in columnar-lined, embryonic esophagus and inactive in squamous-lined, adult esophagus. We showed previously that Hh signaling is reactivated in Barrett's metaplasia and overexpression of Sonic hedgehog (SHH) in mouse esophageal squamous epithelium leads to a columnar phenotype. Here, our objective was to identify Hh target genes involved in Barrett's pathogenesis. By microarray analysis, we found that the transcription factor Foxa2 is more highly expressed in murine embryonic esophagus compared with postnatal esophagus. Conditional activation of Shh in mouse esophageal epithelium induced FOXA2, while FOXA2 expression was reduced in Shh knockout embryos, establishing Foxa2 as an esophageal Hh target gene. Evaluation of patient samples revealed FOXA2 expression in Barrett's metaplasia, dysplasia, and adenocarcinoma but not in esophageal squamous epithelium or squamous cell carcinoma. In esophageal squamous cell lines, Hh signaling upregulated FOXA2, which induced expression of MUC2, an intestinal mucin found in Barrett's esophagus, and the MUC2-processing protein AGR2. Together, these data indicate that Hh signaling induces expression of genes that determine an intestinal phenotype in esophageal squamous epithelial cells and may contribute to the development of Barrett's metaplasia.

Two clinical phenotypes in polycythemia vera.

BACKGROUND: Polycythemia vera is the ultimate phenotypic consequence of the V617F mutation in Janus kinase 2 (encoded by JAK2), but the extent to which this mutation influences the behavior of the involved CD34+ hematopoietic stem cells is unknown. METHODS: We analyzed gene expression in CD34+ peripheral-blood cells from 19 patients with polycythemia vera, using oligonucleotide microarray technology after correcting for potential confounding by sex, since the phenotypic features of the disease differ between men and women. RESULTS: Men with polycythemia vera had twice as many up-regulated or down-regulated genes as women with polycythemia vera, in a comparison of gene expression in the patients and in healthy persons of the same sex, but there were 102 genes with differential regulation that was concordant in men and women. When these genes were used for class discovery by means of unsupervised hierarchical clustering, the 19 patients could be divided into two groups that did not differ significantly with respect to age, neutrophil JAK2 V617F allele burden, white-cell count, platelet count, or clonal dominance. However, they did differ significantly with respect to disease duration; hemoglobin level; frequency of thromboembolic events, palpable splenomegaly, and splenectomy; chemotherapy exposure; leukemic transformation; and survival. The unsupervised clustering was confirmed by a supervised approach with the use of a top-scoring-pair classifier that segregated the 19 patients into the same two phenotypic groups with 100% accuracy. CONCLUSIONS: Removing sex as a potential confounder, we identified an accurate molecular method for classifying patients with polycythemia vera according to disease behavior, independently of their JAK2 V617F allele burden, and identified previously unrecognized molecular pathways in polycythemia vera outside the canonical JAK2 pathway that may be amenable to targeted therapy. (Funded by the Department of Defense and the National Institutes of Health.).

Plasma protein biomarkers enhance the clinical prediction of kidney injury recovery in patients undergoing liver transplantation.

UNLABELLED: Biomarkers predictive of recovery from acute kidney injury (AKI) after liver transplantation (LT) could enhance decision algorithms regarding the need for liver-kidney transplantation or renal sparing regimens. Multianalyte plasma/urine kidney injury protein panels were performed immediately before and 1 month post-LT in an initial test group divided by reversible pre-LT AKI (rAKI = post-LT renal recovery) versus no AKI (nAKI). This was followed by a larger validation set that included an additional group: irreversible pre-LT AKI (iAKI = no post-LT renal recovery). In the test group (n = 16), six pre-LT plasma (not urine) kidney injury proteins (osteopontin [OPN], neutrophil gelatinase-associated lipocalin, cystatin C, trefoil factor 3, tissue inhibitor of metalloproteinase [TIMP]-1, and ß-2-microglobulin) were higher in rAKI versus nAKI (P < 0.05) and returned to normal values with renal recovery post-LT. In the validation set (n = 46), a number of proteins were significantly higher in both rAKI and iAKI versus nAKI. However, only pre-LT plasma OPN (P = 0.009) and TIMP-1 (P = 0.019) levels were significantly higher in rAKI versus iAKI. Logistic regression modeling was used to correlate the probability of post-LT rAKI, factoring in both pre-LT protein markers and clinical variables. A combined model including elevated OPN and TIMP-1 levels, age <57, and absence of diabetes had the highest area under the curve of 0.82, compared to protein-only and clinical variable-only models. CONCLUSION: These data suggest that plasma protein profiles might improve the prediction of pre-LT kidney injury recovery after LT. However, multicenter, prospective studies are needed to validate these findings and ultimately test the value of such protein panels in perioperative management and decision making.

A prospective study evaluating the role of donor-specific anti-endothelial crossmatch (XM-ONE assay) in predicting living donor kidney transplant outcome.

Anti-endothelial cell antibodies (AECAs) may play a role in allograft rejection. We prospectively tested 150 consecutive living donor kidney transplant recipients, with transplants performed at Northwestern Memorial Hospital between January and December 2010, using the donor-specific endothelial (XM-ONE) crossmatch. 88/150 Patients received standard of care (SOC) immunosuppression and analyzed separately, in addition to the complete study cohort. Patients were followed for one year and XM-ONE results were analyzed in relation to occurrence of acute rejection, proteinuria, serum creatinine levels, and biopsy proven fibrosis. No correlation was found between XM-ONE results and protocol or "for-cause" biopsy proven acute rejection or vasculopathy at 12 months. When IgG+ and IgM+ results of the XM-ONE assay were combined, a correlation with proteinuria at 12 months was observed (p=0.047). Although IgG+XM-ONE results were associated with significantly higher creatinine at 6 months (p=0.018), significance was lost at 12 months. Conversely, patients with an IgM+XM-ONE crossmatch had significantly lower creatinine at 1 month (p=0.019), 3 months (p=0.0045), and 6 months (p=0.038) post-transplant, but lost statistical significance at 12 months (p=0.67) post-transplant. In summary, the presence of AECAs as determined by a positive XM-ONE result was not predictive of overall poorer graft outcome after one year in our center.