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Lymphocyte differentiation is regulated by coordinated actions of cytokines and signaling pathways. IL-21 activates STAT1, STAT3, and STAT5 and is fundamental for the differentiation of human B cells into memory cells and antibody-secreting cells. While STAT1 is largely nonessential and STAT3 is critical for this process, the role of STAT5 is unknown.This study sought to delineate unique roles of STAT5 in activation and differentiation of human naive and memory B cells.STAT activation was assessed by phospho-flow cytometry cell sorting. Differential gene expression was determined by RNA-sequencing and quantitative PCR. The requirement for STAT5B in B-cell and CD4IL-21 activated STAT5 and strongly induced SOCS3 in human naive, but not memory, B cells. Deletion of STAT5B in B-cell lines diminished IL-21-mediated SOCS3 induction. PBMCs from STAT5B-null individuals contained expanded populations of immunoglobulin class-switched B cells, CD21These findings reveal novel roles for STAT5B in regulating IL-21-induced human B-cell differentiation. This is achieved by inducing SOCS3 to attenuate IL-21 signaling, and BCL6 to repress class switching and plasma cell generation. Thus, STAT5B is critical for restraining IL-21-mediated B-cell differentiation. These findings provide insights into mechanisms underpinning B-cell responses during primary and subsequent antigen encounter and explain autoimmunity and dysfunctional humoral immunity in STAT5B deficiency.

The association between cancer and autoimmune disease is unexplained, exemplified by T cell large granular lymphocytic leukemia (T-LGL) where gain-of-function (GOF) somatic STAT3 mutations correlate with co-existing autoimmunity. To investigate whether these mutations are the cause or consequence of CD8

SUMMARYThe association between cancer and autoimmune disease is unexplained, exemplified by T-cell large granular lymphocytic leukemia (T-LGL) where gain-of-function somatic mutations inSTAT3correlate with co-existing autoimmunity. To resolve whether these mutations are the cause or consequence of CD8 clonal expansions and autoimmunity, here we analyse patients with germlineSTAT3GOF syndrome and mice with the T-LGL mutationSTAT3K658Nor the most common germline mutation,STAT3T716M. STAT3 GOF mutations drove accumulation of effector CD8 T cell clones highly expressing the NKG2D receptor for MHC-I-related molecules expressed on stressed cells, and the genes for inflammatory/cytotoxic granzymes, perforin, interferon-γ andCcl5/Rantes. CD8 cells were essential to lethal disease inStat3K658Nmice and their accumulation required NKG2D and the receptor for IL-15 and IL-2, IL2RB. These results demonstrate thatSTAT3GOF mutations cause effector CD8 T cell oligoclonal accumulation and that these rogue T cells contribute to autoimmune pathology, supporting the hypothesis that somatic mutations in leukemia/lymphoma driver genes contribute to autoimmune disease.IN BRIEFLeukemia and autoimmune-associatedSTAT3gain-of-function mutations dysregulate CD8 T cells to cause autoimmune pathology and oligoclonal expansion of cytotoxic killer CD8 T cells, that depend upon NKG2D and IL2RB receptors for signals displayed on stressed, damaged, infected, or mutated tissues.

We studied a child with severe viral, bacterial, fungal, and parasitic diseases, who was homozygous for a loss-of-function mutation of REL, encoding c-Rel, which is selectively expressed in lymphoid and myeloid cells. The patient had low frequencies of NK, effector memory cells reexpressing CD45RA (Temra) CD8(+) T cells, memory CD4(+) T cells, including Th1 and Th1*, Tregs, and memory B cells, whereas the counts and proportions of other leukocyte subsets were normal. Functional deficits of myeloid cells included the abolition of IL-12 and IL-23 production by conventional DC1s (cDC1s) and monocytes, but not cDC2s. c-Rel was also required for induction of CD86 expression on, and thus antigen-presenting cell function of, cDCs. Functional deficits of lymphoid cells included reduced IL-2 production by naive T cells, correlating with low proliferation and survival rates and poor production of Th1, Th2, and Th17 cytokines by memory CD4(+) T cells. In naive CD4(+) T cells, c-Rel is dispensable for early IL2 induction but contributes to later phases of IL2 expression. The patient’s naive B cells displayed impaired MYC and BCL2L1 induction, compromising B cell survival and proliferation and preventing their differentiation into Ig-secreting plasmablasts. Inherited c-Rel deficiency disrupts the development and function of multiple myeloid and lymphoid cells, compromising innate and adaptive immunity to multiple infectious agents.

Heterozygous in-frame mutations in coding regions of human STAT3 underlie the only known autosomal dominant form of hyper IgE syndrome (AD HIES). About 5% of familial cases remain unexplained. The mutant proteins are loss-of-function and dominant-negative when tested following overproduction in recipient cells. However, the production of mutant proteins has not been detected and quantified in the cells of heterozygous patients. We report a deep intronic heterozygous STAT3 mutation, c.1282-89C>T, in 7 relatives with AD HIES. This mutation creates a new exon in the STAT3 complementary DNA, which, when overexpressed, generates a mutant STAT3 protein (D427ins17) that is loss-of-function and dominant-negative in terms of tyrosine phosphorylation, DNA binding, and transcriptional activity. In immortalized B cells from these patients, the D427ins17 protein was 2 kDa larger and 4-fold less abundant than wild-type STAT3, on mass spectrometry. The patients’ primary B and T lymphocytes responded poorly to STAT3-dependent cytokines. These findings are reminiscent of the impaired responses of leukocytes from other patients with AD HIES due to typical STAT3 coding mutations, providing further evidence for the dominance of the mutant intronic allele. These findings highlight the importance of sequencing STAT3 introns in patients with HIES without candidate variants in coding regions and essential splice sites. They also show that AD HIES-causing STAT3 mutant alleles can be dominant-negative even if the encoded protein is produced in significantly smaller amounts than wild-type STAT3.

BACKGROUND: Gain-of-function (GOF) mutations in PIK3CD cause a primary immunodeficiency characterized by recurrent respiratory tract infections, susceptibility to herpesvirus infections, and impaired antibody responses. Previous work revealed defects in CD8+ T and B cells that contribute to this clinical phenotype, but less is understood about the role of CD4+ T cells in disease pathogenesis. OBJECTIVE: We sought to dissect the effects of increased phosphoinositide 3-kinase (PI3K) signaling on CD4+ T-cell function. METHODS: We performed detailed ex vivo, in vivo, and in vitro phenotypic and functional analyses of patients' CD4+ T cells and a novel murine disease model caused by overactive PI3K signaling. RESULTS: PI3K overactivation caused substantial increases in numbers of memory and follicular helper T (TFH) cells and dramatic changes in cytokine production in both patients and mice. Furthermore, PIK3CD GOF human TFH cells had dysregulated phenotype and function characterized by increased programmed cell death protein 1, CXCR3, and IFN-γ expression, the phenotype of a TFH cell subset with impaired B-helper function. This was confirmed in vivo in which Pik3cd GOF CD4+ T cells also acquired an aberrant TFH phenotype and provided poor help to support germinal center reactions and humoral immune responses by antigen-specific wild-type B cells. The increase in numbers of both memory and TFH cells was largely CD4+ T-cell extrinsic, whereas changes in cytokine production and TFH cell function were cell intrinsic. CONCLUSION: Our studies reveal that CD4+ T cells with overactive PI3K have aberrant activation and differentiation, thereby providing mechanistic insight into dysfunctional antibody responses in patients with PIK3CD GOF mutations. ispartof: JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY vol:144 issue:1 pages:236-253 ispartof: location:United States status: published

The pathophysiology of adverse events following programmed cell death protein 1 (PD-1) blockade, including tuberculosis (TB) and autoimmunity, remains poorly characterized. We studied a patient with inherited PD-1 deficiency and TB who died of pulmonary autoimmunity. The patient’s leukocytes did not express PD-1 or respond to PD-1-mediated suppression. The patient’s lymphocytes produced only small amounts of interferon (IFN)-γ upon mycobacterial stimuli, similarly to patients with inborn errors of IFN-γ production who are vulnerable to TB. This phenotype resulted from a combined depletion of Vδ2+ γδ T, mucosal-associated invariant T and CD56bright natural killer lymphocytes and dysfunction of other T lymphocyte subsets. Moreover, the patient displayed hepatosplenomegaly and an expansion of total, activated and RORγT+ CD4−CD8− double-negative αβ T cells, similar to patients with STAT3 gain-of-function mutations who display lymphoproliferative autoimmunity. This phenotype resulted from excessive amounts of STAT3-activating cytokines interleukin (IL)-6 and IL-23 produced by activated T lymphocytes and monocytes, and the STAT3-dependent expression of RORγT by activated T lymphocytes. Our work highlights the indispensable role of human PD-1 in governing both antimycobacterial immunity and self-tolerance, while identifying potentially actionable molecular targets for the diagnostic and therapeutic management of TB and autoimmunity in patients on PD-1 blockade. Dysregulated immune features in a patient with a homozygous loss-of-function mutation in PDCD1 suggest that IL-6, IL-23, STAT3 and RORγT might be potential targets for treatment of PD-1 blockade-induced autoimmunity.

Human inborn errors of IFN-γ immunity underlie mycobacterial diseases. We describe patients with Mycobacterium bovis (BCG) disease who are homozygous for loss-of-function mutations of SPPL2A. This gene encodes a transmembrane protease that degrades the N-terminal fragment (NTF) of CD74 (HLA invariant chain) in antigen-presenting cells. The CD74 NTF therefore accumulates in the HLA class II+ myeloid and lymphoid cells of SPPL2a-deficient patients. This toxic fragment selectively depletes IL-12- and IL-23-producing CD1c+ conventional dendritic cells (cDC2s) and their circulating progenitors. Moreover, SPPL2a-deficient memory TH1* cells selectively fail to produce IFN-γ when stimulated with mycobacterial antigens in vitro. Finally, Sppl2a–/– mice lack cDC2s, have CD4+ T cells that produce small amounts of IFN-γ after BCG infection, and are highly susceptible to infection with BCG or Mycobacterium tuberculosis. These findings suggest that inherited SPPL2a deficiency in humans underlies mycobacterial disease by decreasing the numbers of cDC2s and impairing IFN-γ production by mycobacterium-specific memory TH1* cells. Primary immunodeficiency can predispose patients to mycobacterial disease. Casanova and colleagues identify novel human mutations in the enzyme SPPL2A that result in selective accumulation of CD74 in a dendritic cell subset and lead to their death and the failure to mount effective TH1 responses.

Genetic etiologies of chronic mucocutaneous candidiasis (CMC) disrupt human IL-17A/F-dependent immunity at mucosal surfaces, whereas those of connective tissue disorders (CTDs) often impair the TGF-β-dependent homeostasis of connective tissues. The signaling pathways involved are incompletely understood. We report a three-generation family with an autosomal dominant (AD) combination of CMC and a previously undescribed form of CTD that clinically overlaps with Ehlers-Danlos syndrome (EDS). The patients are heterozygous for a private splice-site variant of MAPK8, the gene encoding c-Jun N-terminal kinase 1 (JNK1), a component of the MAPK signaling pathway. This variant is loss-of-expression and loss-of-function in the patients' fibroblasts, which display AD JNK1 deficiency by haploinsufficiency. These cells have impaired, but not abolished, responses to IL-17A and IL-17F. Moreover, the development of the patients' TH17 cells was impaired ex vivo and in vitro, probably due to the involvement of JNK1 in the TGF-β-responsive pathway and further accounting for the patients' CMC. Consistently, the patients' fibroblasts displayed impaired JNK1- and c-Jun/ATF-2-dependent induction of key extracellular matrix (ECM) components and regulators, but not of EDS-causing gene products, in response to TGF-β. Furthermore, they displayed a transcriptional pattern in response to TGF-β different from that of fibroblasts from patients with Loeys-Dietz syndrome caused by mutations of TGFBR2 or SMAD3, further accounting for the patients' complex and unusual CTD phenotype. This experiment of nature indicates that the integrity of the human JNK1-dependent MAPK signaling pathway is essential for IL-17A- and IL-17F-dependent mucocutaneous immunity to Candida and for the TGF-β-dependent homeostasis of connective tissues.

International audience; Inherited IL-12Rβ1 and TYK2 deficiencies impair both IL-12- and IL-23-dependent IFN-γ immunity and are rare monogenic causes of tuberculosis, each found in less than 1/600,000 individuals. We show that homozygosity for the common TYK2 P1104A allele, which is found in about 1/600 Europeans and between 1/1000 and 1/10,000 individuals in regions other than East Asia, is more frequent in a cohort of patients with tuberculosis from endemic areas than in ethnicity-adjusted controls (P = 8.37 × 10-8; odds ratio, 89.31; 95% CI, 14.7 to 1725). Moreover, the frequency of P1104A in Europeans has decreased, from about 9% to 4.2%, over the past 4000 years, consistent with purging of this variant by endemic tuberculosis. Surprisingly, we also show that TYK2 P1104A impairs cellular responses to IL-23, but not to IFN-α, IL-10, or even IL-12, which, like IL-23, induces IFN-γ via activation of TYK2 and JAK2. Moreover, TYK2 P1104A is properly docked on cytokine receptors and can be phosphorylated by the proximal JAK, but lacks catalytic activity. Last, we show that the catalytic activity of TYK2 is essential for IL-23, but not IL-12, responses in cells expressing wild-type JAK2. In contrast, the catalytic activity of JAK2 is redundant for both IL-12 and IL-23 responses, because the catalytically inactive P1057A JAK2, which is also docked and phosphorylated, rescues signaling in cells expressing wild-type TYK2. In conclusion, homozygosity for the catalytically inactive P1104A missense variant of TYK2 selectively disrupts the induction of IFN-γ by IL-23 and is a common monogenic etiology of tuberculosis.

International audience; Hundreds of patients with autosomal recessive, complete IL-12p40 or IL-12Rβ1 deficiency have been diagnosed over the last 20 years. They typically suffer from invasive mycobacteriosis and, occasionally, from mucocutaneous candidiasis. Susceptibility to these infections is thought to be due to impairments of IL-12-dependent IFN-γ immunity and IL-23-dependent IL-17A/IL-17F immunity, respectively. We report here patients with autosomal recessive, complete IL-12Rβ2 or IL-23R deficiency, lacking responses to IL-12 or IL-23 only, all of whom, unexpectedly, display mycobacteriosis without candidiasis. We show that αβ T, γδ T, B, NK, ILC1, and ILC2 cells from healthy donors preferentially produce IFN-γ in response to IL-12, whereas NKT cells and MAIT cells preferentially produce IFN-γ in response to IL-23. We also show that the development of IFN-γ-producing CD4 + T cells, including, in particular , mycobacterium-specific TH1* cells (CD45RA− CCR6+), is dependent on both IL-12 and IL-23. Last, we show that IL12RB1, IL12RB2, and IL23R have similar frequencies of deleterious variants in the general population. The comparative rarity of symptomatic patients with IL-12Rβ2 or IL-23R deficiency, relative to IL-12Rβ1 deficiency , is, therefore, due to lower clinical penetrance. There are fewer symptomatic IL-23R-and IL-12Rβ2-deficient than IL-12Rβ1-deficient patients, not because these genetic disorders are rarer, but because the isolated absence of IL-12 or IL-23 is, in part, compensated by the other cytokine for the production of IFN-γ, thereby providing some protection against mycobacteria. These experiments of nature show that human IL-12 and IL-23 are both required for optimal IFN-γ dependent immunity to mycobacteria, both individually and much more so cooperatively.

// Xiulong Xu 1, 2, 3 , Geetha Rao 3 , Yi Li 4 1 Institute of Comparative Medicine, Yangzhou University, Jiangsu Province, Yangzhou 225009, P.R. China 2 College of Veterinary Medicine, Yangzhou University, Jiangsu Province, Yangzhou 225009, P.R. China 3 Department of Anatomy and Cell Biology, Rush University Medical Center, Chicago, IL 60612, USA 4 Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX 77030, USA Correspondence to: Xiulong Xu, email: xxl@yzu.edu.cn , xxu@rush.edu Keywords: xanthine oxidoreductase, breast cancer, NKG2D ligand, uric acid, MAP kinase Received: January 19, 2016 Accepted: July 18, 2016 Published: August 03, 2016 ABSTRACT MICA/B (the major histocompatibility antigen-related chain A and B) and Rae I are stress-inducible ligands for the immune-receptor NKG2D. Mechanisms by which genotoxic stress and DNA damage induce the expression of NKG2D ligands remain incompletely understood. Here, we report that inhibition of xanthine oxidoreductase (XOR) activity by allopurinol or inhibition of XOR expression by gene knockdown abrogated genotoxic stress-induced expression of MICA/B and Rae I in three tumor cell lines. XOR knockdown also blocked gemcitabine-mediated antitumor activity in an orthotopic syngeneic mouse model of breast cancer. As a rate-limiting enzyme in the purine catabolic pathway, XOR generates two end-products, uric acid and reactive oxygen species (ROS). ROS scavenging had an insignificant effect on genotoxic drug-induced MICA/B expression but modestly inhibited radiation-induced MICA/B expression. Exogenous uric acid (in the form of monosodium urate) induced MICA/B expression by activating the MAP kinase pathway. Allopurinol blocked genotoxic stress-induced MAP kinase activation. Our study provides mechanistic insights into genotoxic stress-induced activation of the MAP kinase pathway and suggests that XOR is required for genotoxic stress-induced NKG2D ligand expression and gemcitabine-mediated antitumor activity.

Tangye and collaborators use a series of mutants to elucidate the pathways required to generate distinct subsets of human effector CD4+ T cells., Naive CD4+ T cells differentiate into specific effector subsets—Th1, Th2, Th17, and T follicular helper (Tfh)—that provide immunity against pathogen infection. The signaling pathways involved in generating these effector cells are partially known. However, the effects of mutations underlying human primary immunodeficiencies on these processes, and how they compromise specific immune responses, remain unresolved. By studying individuals with mutations in key signaling pathways, we identified nonredundant pathways regulating human CD4+ T cell differentiation in vitro. IL12Rβ1/TYK2 and IFN-γR/STAT1 function in a feed-forward loop to induce Th1 cells, whereas IL-21/IL-21R/STAT3 signaling is required for Th17, Tfh, and IL-10–secreting cells. IL12Rβ1/TYK2 and NEMO are also required for Th17 induction. Strikingly, gain-of-function STAT1 mutations recapitulated the impact of dominant-negative STAT3 mutations on Tfh and Th17 cells, revealing a putative inhibitory effect of hypermorphic STAT1 over STAT3. These findings provide mechanistic insight into the requirements for human T cell effector function, and explain clinical manifestations of these immunodeficient conditions. Furthermore, they identify molecules that could be targeted to modulate CD4+ T cell effector function in the settings of infection, vaccination, or immune dysregulation.

Signal transducer and activator of transcription 3 (STAT3) regulates gene expression downstream of cell surface cytokine and hormone receptors. Heterozygous germline loss of function STAT3 mutations lead to the primary immunodeficiency hyper-IgE syndrome while somatic activating STAT3 mutations recur in human solid organ and immune malignancies. Germline heterozygous activating STAT3 mutations result in early-onset autoimmunity with aspects of immunodeficiency. Affected individuals share characteristics with autoimmune lymphoproliferative syndrome and immunodysregulation polyendocrinopathy enteropathy X-linked syndrome, including reduced T regulatory cell numbers. They present with variable autoimmune symptoms including type 1 diabetes, rheumatoid arthritis, gut enteropathies and autoimmune cytopenias. Whilst many effects of STAT3 loss of function on immune cells have been described, the mechanisms behind autoimmunity and immunodeficiency in patients with activating STAT3 mutations remain unclear. Here, we present a detailed characterisation of T cell development and maturation in young and old mice on two different backgrounds with Crispr-engineered germline activating mutations in two domains of STAT3. We use mixed chimeras, flow cytometry, T cell receptor deep sequencing and high throughput single-cell transcriptomics to reveal cell-extrinsic and -autonomous roles of overactive STAT3 in T cells in autoimmunity and immune malignancy. To our knowledge, this is the first report of mice with Stat3 germline activating mutations identical to those in autoimmunity or malignancy. We validate our key findings in humans with gain of function germline STAT3 mutations and childhood-onset autoimmunity.

Comment in :Who regulates whom: ZNF341 is an additional player in the STAT3/TH17 song. [Sci Immunol. 2018]; International audience; Heterozygosity for human signal transducer and activator of transcription 3 (STAT3) dominant-negative (DN) mutations underlies an autosomal dominant form of hyper-immunoglobulin E syndrome (HIES). We describe patients with an autosomal recessive form of HIES due to loss-of-function mutations of a previously uncharacterized gene, ZNF341 ZNF341 is a transcription factor that resides in the nucleus, where it binds a specific DNA motif present in various genes, including the STAT3 promoter. The patients' cells have low basal levels of STAT3 mRNA and protein. The autoinduction of STAT3 production, activation, and function by STAT3-activating cytokines is strongly impaired. Like patients with STAT3 DN mutations, ZNF341-deficient patients lack T helper 17 (TH17) cells, have an excess of TH2 cells, and have low memory B cells due to the tight dependence of STAT3 activity on ZNF341 in lymphocytes. Their milder extra-hematopoietic manifestations and stronger inflammatory responses reflect the lower ZNF341 dependence of STAT3 activity in other cell types. Human ZNF341 is essential for the STAT3 transcription-dependent autoinduction and sustained activity of STAT3.

© 2015 Elsevier B.V. All rights reserved. Abstract The ATP-binding cassette (ABC) transporter, ABCG1, is a lipid exporter involved in removal of cholesterol from cells that has been investigated for its role in foam cells formation and atherosclerosis. The mechanism by which ABC lipid transporters bind and recognise their substrates is currently unknown. In this study, we identify a critical region in the final transmembrane domain of ABCG1, which is essential for its export function and stabilisation by cholesterol, a post-translational regulatory mechanism that we have recently identified as dependent on protein ubiquitination. This transmembrane region contains several Cholesterol Recognition/interaction Amino acid Consensus (CRAC) motifs, and its inverse CARC motifs. Mutational analyses identify one CRAC motif in particular with Y667 at its core, that is especially important for transport activity to HDL as well as stability of the protein in the presence of cholesterol. In addition, we present a model of how cholesterol docks to this CRAC motif in an energetically favourable manner. This study identifies for the first time how ABCG1 can interact with cholesterol via a functional CRAC domain, which provides the first insight into the substrate-transporter interaction of an ABC lipid exporter.

The ABC lipid transporters, ABCA1 and ABCG1, are essential for maintaining lipid homeostasis in cells such as macrophages by exporting excess cholesterol to extracellular acceptors. These transporters are highly regulated at the post-translational level, including protein ubiquitination. Our aim was to investigate the role of the E3 ubiquitin ligase HECTD1, recently identified as associated with ABCG1, on ABCG1 and ABCA1 protein levels and cholesterol export function. Here, we show that HECTD1 protein is widely expressed in a range of human and murine primary cells and cell lines, including macrophages, neuronal cells and insulin secreting β-cells. siRNA knockdown of HECTD1 unexpectedly decreased overexpressed ABCG1 protein levels and cell growth, but increased native ABCA1 protein in CHO-K1 cells. Knockdown of HECTD1 in unloaded THP-1 macrophages did not affect ABCG1 but significantly increased ABCA1 protein levels, in wild-type as well as THP-1 cells that do not express ABCG1. Cholesterol export from macrophages to apoA-I over time was increased after knockdown of HECTD1, however these effects were not sustained in cholesterol-loaded cells. In conclusion, we have identified a new candidate, the E3 ubiquitin ligase HECTD1, that may be involved in the regulation of ABCA1-mediated cholesterol export from unloaded macrophages to apoA-I. The exact mechanism by which this ligase affects this pathway remains to be elucidated.

The sonic hedgehog (SHH) pathway is activated in several types of malignancy and plays an important role in tumor cell proliferation and tumorigenesis. SHH binding to a 12-pass transmembrane receptor, Patched (PTCH), leads to freeing of Smoothened (SMO) and subsequent activation of GLI transcription factors. In the present study, we analyzed the expression of SHH, PTCH, SMO, and GLI1 in 31 follicular thyroid adenomas (FTA), 8 anaplastic thyroid carcinomas (ATC), and 51 papillary thyroid carcinomas (PTC) by immunohistochemical staining. More than 65% of FTA, PTC, and ATC specimens stained positive for SHH, PTCH, SMO, and GLI. However, the expression of the genes encoding these four molecules did not correlate with any clinicopathologic parameters, including the age, gender, the status ofBRAFgene mutation, tumor stage, local invasion, and metastasis. Three thyroid tumor cell lines (KAT-18, WRO82, and SW1736) all expressed the genes encoding these four molecules. 5-Bromo-2-deoxyuridine labeling and MTT cell proliferation assays revealed that cyclopamine (CP), an inhibitor of the SHH pathway, was able to inhibit the proliferation of KAT-18 and WRO82 cells more effectively than SW1736 cells. CP led to the arrest of cell cycle or apoptosis. Knockdown ofSHHandGLIexpression by miRNA constructs that targetSHHorGLImRNA in KAT-18 and SW1736 cells led to the inhibition of cell proliferation. Our results suggest that the SHH pathway is widely activated in thyroid neoplasms and may have potential as an early marker of thyroid cancer or as a potential therapeutic target for thyroid cancer treatment.

Heparanase-1 (HPR1), an endoglycosidase that specifically degrades heparan sulfate (HS) proteoglycans, is overexpressed in a variety of malignancies. Our present study sought to determine whether oncogene BRAF and RAS mutations lead to increased HPR1 expression. Reverse transcription-polymerase chain reaction analysis revealed that HPR1 gene expression was increased in HEK293 cells transiently transfected with a mutant BRAF or RAS gene. Flow cytometric analysis revealed that B-Raf activation led to loss of the cell surface HS, which could be blocked by two HPR1 inhibitors: heparin and PI-88. Cotransfection of a BRAF or RAS mutant gene with HPR1 promoter-driven luciferase reporters increased luciferase reporter gene expression in HEK293 cells. Knockdown of BRAF expression in a BRAF-mutated KAT-10 tumor cell line led to the suppression of HPR1 gene expression, subsequently leading to increased cell surface HS levels. Truncational and mutational analyses of the HPR1 promoter revealed that the Ets-relevant elements in the HPR1 promoter were critical for BRAF activation-induced HPR1 expression. Luciferase reporter gene expression driven by a four-copy GA binding protein (GABP) binding site was significantly lower in BRAF siRNA-transfected KAT-10 cells than in the control siRNA-transfected cells. We further showed that BRAF knockdown led to suppression of the expression of the GABPβ, an Ets family transcription factor involved in regulating HPR1 promoter activity. Taken together, our study suggests that B-Raf kinase activation plays an important role in regulating HPR1 expression. Increased HPR1 expression may contribute to the aggressive behavior of BRAF-mutated cancer.

Heparan sulfate proteoglycans (HSPGs) function as a co-receptor for heparin-binding growth factors, such as fibroblast growth factors (FGFs) and heparin-bound epidermal growth factor (HB-EGF). The HS side chain of HSPGs can be cleaved by HPR1 (heparanase-1), an endoglycosidase that is overexpressed in many types of malignancies. In the present study, we demonstrated that HPR1 expression in pancreatic adenocarcinomas inversely correlated with the presence of heparan sulfate (HS) in the basement membrane. In vitro cell culture study revealed that cell surface HS levels inversely correlated with HPR1 activity in five pancreatic cancer cell lysates and their conditioned media. Heparin and PI-88, two HPR1 inhibitors, were able to increase cell surface HS levels in PANC-1 cells in a dose-dependent manner. The ability of HPR1 to degrade cell surface HS was confirmed by showing that cell surface HS levels were increased in HT1080 cells stably transfected with the HPR1 antisense gene but was decreased in the cells overexpressing HPR1. Further studies showed that PI-88 and heparin were able to stimulate PANC-1 cell proliferation in the absence or presence of exogenous FGF2, whereas exogenous HPR1 was able to inhibit PANC-1 cell proliferation in a dose-dependent manner. Modulation of PANC-1 cell proliferation by HPR1 or HPR1 inhibitors corresponded with the inhibition or activation of the mitogen-activated protein kinase. Our results suggest that HPR1 expressed in pancreatic adenocarcinomas can suppress the proliferation of pancreatic tumor cells in response to the growth factors that require HSPGs as their co-receptors.

The ATP-binding cassette transporter ABCG1 has an essential role in cellular cholesterol homeostasis, and dysregulation has been associated with a number of high burden diseases. Previous studies reported that ABCG1 is ubiquitinated and degraded via the ubiquitin proteasome system. However, so far the molecular mechanism, including the identity of any of the rate-limiting ubiquitination enzymes, or E3 ligases, is unknown. Using liquid chromatography mass spectrometry, we identified two HECT domain E3 ligases associated with ABCG1, named HUWE1 (HECT, UBA, and WWE domain containing 1, E3 ubiquitin protein ligase) and NEDD4-1 (Neural precursor cell-expressed developmentally down regulated gene 4), of which the latter is the founding member of the NEDD4 family of ubiquitin ligases. Silencing both HUWE1 and NEDD4-1 in cells overexpressing human ABCG1 significantly increased levels of the ABCG1 monomeric and dimeric protein forms, however ABCA1 protein expression was unaffected. In addition, ligase silencing increased ABCG1-mediated cholesterol export to HDL in cells overexpressing the transporter as well as in THP-1 macrophages. Reciprocally, overexpression of both ligases resulted in a significant reduction in protein levels of both the ABCG1 monomeric and dimeric forms. Like ABCG1, ABCG4 protein levels and cholesterol export activity were significantly increased after silencing both HUWE1 and NEDD4-1 in cells overexpressing this closely related ABC half-transporter. In summary, we have identified for the first time two E3 ligases that are fundamental enzymes in the post-translational regulation of ABCG1 and ABCG4 protein levels and cellular cholesterol export activity.

Background Follicular helper T (T FH ) cells underpin T cell–dependent humoral immunity and the success of most vaccines. T FH cells also contribute to human immune disorders, such as autoimmunity, immunodeficiency, and malignancy. Understanding the molecular requirements for the generation and function of T FH cells will provide strategies for targeting these cells to modulate their behavior in the setting of these immunologic abnormalities. Objective We sought to determine the signaling pathways and cellular interactions required for the development and function of T FH cells in human subjects. Methods Human primary immunodeficiencies (PIDs) resulting from monogenic mutations provide a unique opportunity to assess the requirement for particular molecules in regulating human lymphocyte function. Circulating follicular helper T (cT FH ) cell subsets, memory B cells, and serum immunoglobulin levels were quantified and functionally assessed in healthy control subjects, as well as in patients with PIDs resulting from mutations in STAT3 , STAT1 , TYK2 , IL21 , IL21R , IL10R , IFNGR1/2 , IL12RB1 , CD40LG , NEMO , ICOS , or BTK . Results Loss-of-function (LOF) mutations in STAT3 , IL10R , CD40LG , NEMO , ICOS , or BTK reduced cT FH cell frequencies. STAT3 and IL21/R LOF and STAT1 gain-of-function mutations skewed cT FH cell differentiation toward a phenotype characterized by overexpression of IFN-γ and programmed death 1. IFN-γ inhibited cT FH cell function in vitro and in vivo , as corroborated by hypergammaglobulinemia in patients with IFNGR1/2 , STAT1 , and IL12RB1 LOF mutations. Conclusion Specific mutations affect the quantity and quality of cT FH cells, highlighting the need to assess T FH cells in patients by using multiple criteria, including phenotype and function. Furthermore, IFN-γ functions in vivo to restrain T FH cell–induced B-cell differentiation. These findings shed new light on T FH cell biology and the integrated signaling pathways required for their generation, maintenance, and effector function and explain the compromised humoral immunity seen in patients with some PIDs.

Major histocompatibility complex class I-related chains A and B (MICA/B) are two stress-inducible ligands for the immunoreceptor NKG2D that is expressed on cytotoxic T cells and natural killer (NK) cells. It is not known whether MICA/B expression is up-regulated in thyroid cancer as a result of oncogene activation.The objective of the investigation was to study MICA/B expression and regulation in thyroid cancer and its role in mediating the cytotoxicity of NK cells.MICA/B expression in thyroid cancer was analyzed by immunohistochemical staining. Cell surface MICA/B levels in thyroid tumor cell lines and fresh tumor cells were analyzed by flow cytometric analysis. The susceptibility of thyroid tumor cells to NK cell killing was tested by using (51)Cr release assay.MICA/B was expressed at moderate or high levels in 18 of 39 papillary thyroid carcinomas and four of eight anaplastic thyroid carcinomas. MICA/B expression was confirmed by flow cytometric analysis in three fresh thyroid neoplasms. MICA/B expression was detected in eight of 10 thyroid tumor cell lines and correlated with their sensitivity to killing by the NKG2/D-positive NK-92 cells. Blocking of NKG2D and MICA/B interaction by specific antibodies partially led to the inhibition of NK-92 cell-mediated cytotoxicity. The MAPK inhibitors were able to block MICA/B expression in MRO87 and HeLa cells. Transient transfection of mutant BRAF and RAS oncogenes led to increased MICA/B expression in 293 cells and WRO82 cells.MICA/B expression is up-regulated in thyroid cancer, probably due to the activation of the MAPK pathway. MICA/B in thyroid cancer plays an important role in NK-92 cell-mediated cytotoxicity.

The molecular mechanisms of heparan sulfate proteoglycan downregulation in the glomerular basement membrane (GBM) of the kidneys with diabetic nephropathy remain controversial. In the present study, we showed that the expression of heparanase-1 (HPR1), a heparan sulfate–degrading endoglycosidase, was upregulated in the renal epithelial cells in the kidney with diabetic nephropathy. Urinary HPR1 levels were elevated in patients with diabetic nephropathy. In vitro cell culture studies revealed that HPR1 promoter–driven luciferase reporter gene expression, HPR1 mRNA, and protein were upregulated in renal epithelial cells under high glucose conditions. Induction of HPR1 expression by high glucose led to decreased cell surface heparan sulfate expression. HPR1 inhibitors were able to restore cell surface heparan sulfate expression. Functional analysis revealed that renal epithelial cells grown under high glucose conditions resulted in an increase of basement membrane permeability to albumin. Our studies suggest that loss of heparan sulfate in the GBM with diabetic nephropathy is attributable to accelerated heparan sulfate degradation by increased HPR1 expression.

Wilson et al. show that individuals with loss-of-function mutations in STAT3 have reduced numbers of peripheral blood MAIT and NKT cells, but not γδ T cells. Residual MAIT cells had normal expression of RORγt, but displayed impaired secretion of IL-17A and IL-17F., Unconventional T cells such as γδ T cells, natural killer T cells (NKT cells) and mucosal-associated invariant T cells (MAIT cells) are a major component of the immune system; however, the cytokine signaling pathways that control their development and function in humans are unknown. Primary immunodeficiencies caused by single gene mutations provide a unique opportunity to investigate the role of specific molecules in regulating human lymphocyte development and function. We found that individuals with loss-of-function mutations in STAT3 had reduced numbers of peripheral blood MAIT and NKT but not γδ T cells. Analysis of STAT3 mosaic individuals revealed that this effect was cell intrinsic. Surprisingly, the residual STAT3-deficient MAIT cells expressed normal levels of the transcription factor RORγt. Despite this, they displayed a deficiency in secretion of IL-17A and IL-17F, but were able to secrete normal levels of cytokines such as IFNγ and TNF. The deficiency in MAIT and NKT cells in STAT3-deficient patients was mirrored by loss-of-function mutations in IL12RB1 and IL21R, respectively. Thus, these results reveal for the first time the essential role of STAT3 signaling downstream of IL-23R and IL-21R in controlling human MAIT and NKT cell numbers.

The role of mesenchymal stem cells (MSCs) on breast cancer progression, growth and tumorigenesis remains controversial or unknown. In the present study, we investigated the role of MSCs on breast tumor induction and growth in a clinically relevant somatic breast cancer model. We first conducted in vitro studies and found that conditioned media (CM) of RCAS-Neu and RCAS-PyMT breast cancer cell lines and tumor cells themselves dramatically increased the proliferation and motility of MSCs and induced morphological changes of MSCs and differentiation into fibroblast-like cells. In contrast, the CM of MSCs inhibited the proliferation of two breast cancer cell lines by arresting the cell cycle at the G0/G1 phase. In vivo studies revealed that fluorescence dye-labeled MSCs migrated into tumor tissues. Unexpectedly, single or multiple intravenous injections of MSCs did not affect the latency of breast cancer in TVA- transgenic mice induced by intraductal injection of the RCAS vector encoding polyoma middle-T antigen (PyMT) or Neu oncogenes. Moreover, MSCs had no effect on RCAS-Neu tumor growth in a syngeneic ectopic breast cancer model. While our studies consistently demonstrated the ability of breast cancer cells to profoundly induce MSCs migration, differentiation, and proliferation, the anti-proliferative effect of MSCs on breast tumor cells observed in vitro could not be translated into an antitumor activity in vivo, probably reflecting the antagonizing or complex effects of MSCs on tumor environment and tumor cells themselves.

Item does not contain fulltext AIMS/HYPOTHESIS: The content of heparan sulphate is reduced in the endothelium under hyperglycaemic conditions and may contribute to the pathogenesis of atherosclerosis. Heparanase-1 (HPR1) specifically degrades heparan sulphate proteoglycans. We therefore sought to determine whether: (1) heparan sulphate reduction in endothelial cells is due to increased HPR1 production through increased reactive oxygen species (ROS) production; and (2) HPR1 production is increased in vivo in endothelial cells under hyperglycaemic and/or atherosclerotic conditions. METHODS: HPR1 mRNA and protein levels in endothelial cells were analysed by RT-PCR and Western blot or HPR1 enzymatic activity assay, respectively. Cell surface heparan sulphate levels were analysed by FACS. HPR1 in the artery from control rats and a rat model of diabetes, and from patients under hyperglycaemic and/or atherosclerotic conditions was immunohistochemically examined. RESULTS: High-glucose-induced HPR1 production and heparan sulphate degradation in three human endothelial cell lines, both of which were blocked by ROS scavengers, glutathione and N-acetylcysteine. Exogenous H(2)O(2) induced HPR1 production, subsequently leading to decreased cell surface heparan sulphate levels. HPR1 content was significantly increased in endothelial cells in the arterial walls of a rat model of diabetes. Clinical studies revealed that HPR1 production was increased in endothelial cells under hyperglycaemic conditions, and in endothelial cells and macrophages in atherosclerotic lesions. CONCLUSIONS/INTERPRETATION: Hyperglycaemia induces HPR1 production and heparan sulphate degradation in endothelial cells through ROS. HPR1 production is increased in endothelial cells from a rat model of diabetes, and in macrophages in the atherosclerotic lesions of diabetic and non-diabetic patients. Increased HPR1 production may contribute to the pathogenesis and progression of atherosclerosis. 01 juni 2011

BACKGROUND: This study seeks to determine whether estrogen is able to regulate the expression of heparanase-1 (HPR1) in human endometrium. METHODS: HPR1 expression and heparan sulphate (HS) deposition in the endometrium collected in various menstrual phases were analysed by immunohistochemical and immunofluorescence staining, respectively. HPR1 expression in the endometrial cells unexposed or exposed to estradiol was analysed by using RT-PCR and luciferase reporter assay. HPR1 activity was analysed by using a novel enzyme-linked immunosorbent assay (ELISA). Cell surface HS levels were analysed by flow cytometry. Serum HPR1 activity in women receiving follicle-stimulating hormone (FSH) for IVF was measured by ELISA. RESULTS: HPR1 expression was rarely detected in the endometrium in the early and mid-proliferative phases but was increased in the late proliferative phase and in the secretory phases. HPR1 expression was negatively associated with HS in the basement membrane (BM) of the endometrial glands. HPR1 gene expression, HPR1 promoter activity and HPR1 enzymatic activity were increased in the endometrial cells when exposed to 17b-estradiol (E2), whereas cell surface HS levels showed a decrease which could be blocked by PI-88, an HPR1 inhibitor. Serum HPR1 levels were increased in women with moderately elevated blood estrogen levels after receiving FSH. CONCLUSIONS: HPR1 is differentially expressed in the endometrium in different menstrual phases. Estrogen plays an important role in inducing HPR1 expression, subsequently leading to HS degradation on the endometrial cell surface and in the BM of the endometrium.

The first time Ashok Jhunjhunwala and his team helped install an ATM in rural South India, around 2005, the villagers didn’t like what they saw: They knew that the new bank notes it issued would be suspected as counterfeit, in contrast to soiled notes, considered authentic because they'd been in circulation.

Heparan sulfate proteoglycans (HSPGs) function as a co-receptor for heparin-binding growth factors, such as fibroblast growth factors (FGFs) and heparin-bound epidermal growth factor (HB-EGF). The HS side chain of HSPGs can be cleaved by HPR1 (heparanase-1), an endoglycosidase that is overexpressed in many types of malignancies. In the present study, we demonstrated that HPR1 expression in pancreatic adenocarcinomas inversely correlated with the presence of heparan sulfate (HS) in the basement membrane. In vitro cell culture study revealed that cell surface HS levels inversely correlated with HPR1 activity in five pancreatic cancer cell lysates and their conditioned media. Heparin and PI-88, two HPR1 inhibitors, were able to increase cell surface HS levels in PANC-1 cells in a dose-dependent manner. The ability of HPR1 to degrade cell surface HS was confirmed by showing that cell surface HS levels were increased in HT1080 cells stably transfected with the HPR1 antisense gene but was decreased in the cells overexpressing HPR1. Further studies showed that PI-88 and heparin were able to stimulate PANC-1 cell proliferation in the absence or presence of exogenous FGF2, whereas exogenous HPR1 was able to inhibit PANC-1 cell proliferation in a dose-dependent manner. Modulation of PANC-1 cell proliferation by HPR1 or HPR1 inhibitors corresponded with the inhibition or activation of the mitogen-activated protein kinase. Our results suggest that HPR1 expressed in pancreatic adenocarcinomas can suppress the proliferation of pancreatic tumor cells in response to the growth factors that require HSPGs as their co-receptors.

BACKGROUND It has previously been shown that heparanase-1 (HPR1), an endoglycosidase, is up-regulated in pancreatic carcinoma. The purpose of this study was to test whether serum HPR1 levels in pancreatic carcinoma patients are elevated, and whether higher serum HPR1 levels are associated with a shortened survival. METHODS Serum HPR1 levels in 40 healthy donors, 31 pancreatic carcinoma patients, and 11 patients treated with gemcitabine were measured by a novel enzyme-linked immunoadsorbent assay. HPR1 expression in tumors was analyzed by immunohistochemical staining. Patient overall survival time was determined according to the Kaplan–Meier method, and their difference was evaluated by the log-rank test. A P value < 0.05 was considered statistically significant. RESULTS The mean serum HPR1 activity in pancreatic carcinoma patients was 439 ± 14 units/mL, compared with 190 ± 4 units/mL in the control serum samples from healthy donors. Serum HPR1 levels were significantly higher in patients with HPR1-positive tumors (660 ± 62 units/mL) compared with those with HPR1-negative tumors (241 ± 14 units/mL). The mean survival of 19 pancreatic carcinoma patients with serum HPR1 activity > 300 units/mL was 7.9 ± 0.2 months, whereas the mean survival of 12 patients with serum HPR1 activity < 300 units/mL was 13.3 ± 0.6 months. A Kaplan–Meier plot of the patient survival curve followed by log-rank test revealed that patients in the high serum HPR1 group had a significantly shorter survival compared with those in the low serum HPR1 group. Mean serum HPR1 activity decreased by 64% in 11 pancreatic carcinoma patients after 2 weeks of treatment with gemcitabine. CONCLUSIONS Serum HPR1 activity in pancreatic carcinoma patients was found to be significantly elevated, in particular in those with HPR1-positive tumors. Increased serum HPR1 activity was associated with a shorter survival in patients with pancreatic carcinoma patients. Cancer 2006. © 2005 American Cancer Society.

The molecular mechanisms of heparan sulfate proteoglycan downregulation in the glomerular basement membrane (GBM) of the kidneys with diabetic nephropathy remain controversial. In the present study, we showed that the expression of heparanase-1 (HPR1), a heparan sulfate-degrading endoglycosidase, was upregulated in the renal epithelial cells in the kidney with diabetic nephropathy. Urinary HPR1 levels were elevated in patients with diabetic nephropathy. In vitro cell culture studies revealed that HPR1 promoter-driven luciferase reporter gene expression, HPR1 mRNA, and protein were upregulated in renal epithelial cells under high glucose conditions. Induction of HPR1 expression by high glucose led to decreased cell surface heparan sulfate expression. HPR1 inhibitors were able to restore cell surface heparan sulfate expression. Functional analysis revealed that renal epithelial cells grown under high glucose conditions resulted in an increase of basement membrane permeability to albumin. Our studies suggest that loss of heparan sulfate in the GBM with diabetic nephropathy is attributable to accelerated heparan sulfate degradation by increased HPR1 expression.

OBJECTIVES: To measure family members' preferences for nutrition interventions to improve the oral food and fluid intake of their relatives in a nursing home. DESIGN: Cross-sectional descriptive. SETTING: Three skilled nursing facilities in Southern California. PARTICIPANTS: One hundred five residents from the three skilled nursing facilities and their respective family members. MEASUREMENTS: A mailed questionnaire to family members that consisted of 15 forced-choice comparisons between six nutrition interventions. An assessment of oral food and fluid intake during mealtime for 3 days (nine meals) for each participant using direct observations and estimations of percentage consumed (0% to 100%) by trained research staff. RESULTS: In order of most to least desirable, the family members preferred the following interventions to improve their relative's oral food and fluid intake: (1) improve quality of food; (2) improve quality and quantity of feeding assistance; (3) provide multiple small meals and snacks throughout the day; (4) place resident in preferred dining location; (5) provide an oral liquid nutritional supplement between meals; and (6) provide a medication to stimulate appetite. The average ± standard deviation total percentage intake for residents whose family members reported that they thought their relative had a problem with their intake was 50%± 16%. CONCLUSIONS: Family members prefer that other nutrition interventions be attempted before the use of oral supplements or pharmacological approaches. Family members perceive a need for interventions when residents consume, on average, only half of the food and fluid items provided during mealtime.

Background: The impact of mesenchymal stem cells (MSCs) on breast cancer (BC) progression and growth remains controversial. Some studies using xenograft BC models in nude mice suggest that MSCs stimulate BC metastases, whereas other studies in syngeneic rat BC models suggest that MSCs suppress BC development and growth. In addition, whether MSCs have a role in BC initiation has not been tested. The objective of this study was to determine if bone marrow-derived MSCs affect BC initiation and progression in vitro and in clinically relevant somatic and synegeneic BC mouse models. Materials and Methods: MSCs were isolated from bone marrows of FVB wild-type mice, cultured, and characterized for their potential to differentiate into adipocytes and express flow-cytometric cell surface markers. MSC-conditioned medium (CM) was used to culture RCAS-Neu and RCAS-PyMT BC cell lines, derived from TVA-transgenic mice infected with an avian retroviral vector encoding Neu or polyoma middle T antigen (PyMT). Cell proliferation was analyzed with CellTiter 96 proliferation assay and 5-bromo-2-deoxyuridine labeling. The effect of RCAS-Neu and RCAS-PyMT cells and their CM on MSC migration was determined with Boyden chamber migration assay. In a somatic BC model, TVA-transgenic mice expressing the receptor for an avian retrovirus vector, RCAS, were infected with RCAS-PyMT vector by intraductal injection and treated with MSC (2x106cells/mouse) by i.v. injection. Mice were observed for BC development by palpation. In a syngeneic BC model, RCAS-Neu BC cells were co-implanted with MSCs (5x105/gland) at the ratios 1:0, 1:0.2, or 1:1 into the fat pad of FVB female mice. BC growth was monitored for 9 weeks. Results: CM from MSC did not significantly affect the proliferation of RCAS-Neu and RCAS-PyMT cells. However, RCAS-Neu and RCAS-PyMT cells and their CM induced morphologic changes in MSC and dramatically increased their motility. In somatic model, MSCs had no effect on BC initiation or growth with the mean tumor latency 27.5±7.5 days in MSC-treated mice and 29±5.5 days in control mice treated with PBS. In syngeneic BC model, there was no significant difference in the growth of RCAS-Neu alone or RCAS-Neu cells co-implanted with MSCs. Since BC in these models does not metastasize to distant organs, it could not be determined whether MSCs affect BC metastases. Discussion: Our results demonstrated that BC cell lines and their CM are able to induce MSCs migration and possibly differentiation. However, MSCs had no effect on BC initiation in a somatic BC model and on tumor growth in a syngeneic BC model. It is possible that the number of MSCs and schedule of MSC injection were not optimized or strong PyMT oncogene rapidly induced BC and overcame the effect of MSCs on BC formation. Experiments are ongoing to determine if multiple administrations of MSCs affect BC development induced by PyMT and Neu oncogenes. Lack of effect of MSCs in BC initiation and progression, if confirmed, will suggest that MSCs are safe for delivering novel antitumor agents for BC treatment. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-05-04.

A comparative study of transesophageal echocardiography with single-photon emission computed tomography (SPECT) and coronary artereography was performed in a community outpatient setting to determine accuracy and feasibility of the technique. Forty-one of 55 patients underwent all 3 procedures within a 90-day period. Fourteen patients underwent only SPECT and were compared with transesophageal echocardiography with pacing (TEEP). Atrial esophageal pacing was performed with transesophageal echocardiography to increase double product and induce ischemia, which would manifest as abnormal wall motion. The results in these patients indicated a sensitivity and specificity of 92% and 87% for TEEP and 96% and 82% for SPECT, respectively, using angiography as the gold standard. In 14 patients, the sensitivity of TEEP using SPECT as standard was 80% and the specificity was 87%. The 1 view that appeared to pick up the highest yield of abnormalities was the transgastric short-axis view. Thus, TEEP is indicated in the detection of chronotropically incompetent patients and those unable to exercise whose transthoracic images are not optimal. It is highly accurate compared with angiography or SPECT.

Introduction: Major histocompatibility complex class I chain-related A (MICA) is a stress-inducible glycoprotein that is expressed as a transmembrane protein or released as a soluble protein. MICA binds to NKG2D, an activation receptor expressed in natural killer cells and various T cells. MICA expression is increased in a variety of epithelial malignancies, including melanomas, breast, gastric, and colon cancers. We investigated whether serum MICA levels are increased in patients with pancreatic cancer. Methods: Serum of 61 pancreatic cancer patients and 33 healthy volunteers were analyzed for MICA expression using an ELISA kit. Serum MICA levels were correlated with patient demographic and clinicopathologic parameters. Mann-Whitney U test was used to test siginificance. Results: Serum MICA levels of pancreatic cancer patients were significantly higher than in controls (1107 pg/mL vs. 215 pg/ml, p 0.05). Serum MICA levels in patients with resected tumors (n = 24) were significantly lower than in unresected patients (n = 37)(390 pg/mL vs. 1572 pg/mL, p < 0.05). Survival in resected patients (n = 23) was significantly better than in unresected patients (n = 34)(17 months vs. 11 months, p < 0.05). Conclusions: Serum MICA levels are significantly increased in pancreatic cancer patients, and correlate with extent of tumor burden. Surgery may decrease serum MICA levels and improve survival.