CD44 Deficiency Contributes to Enhanced Experimental Autoimmune Encephalomyelitis: A Role in Immune Cells and Vascular Cells of the Blood–Brain Barrier

Adhesion molecule CD44 is expressed by multiple cell types and is implicated in various cellular and immunological processes. In this study, we examined the effect of global CD44 deficiency on myelin oligodendrocyte glycoprotein peptide (MOG)-induced experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. Compared to C57BL/6 wild-type mice, CD44-deficient mice presented with greater disease severity, increased immune cell numbers in the central nervous system, and increased anti-MOG antibody and proinflammatory cytokine production, especially those associated with T helper 17 (Th17) cells. Further, decreased numbers of peripheral CD4⁺CD25⁺FoxP3⁺ regulatory T cells (Tregs) were observed in CD44-knockout mice throughout the disease course. CD44-knockout CD4 T cells exhibited reduced transforming growth factor-β receptor type I (TGF-β RI) expression that did not impart a defect in Treg polarization in vitro, but did correlate with enhanced Th17 polarization in vitro. Further, EAE in bone marrow–chimeric animals suggested CD44 expression on both circulating and noncirculating cells limited disease severity. Endothelial expression of CD44 limited T-cell adhesion to and transmigration through murine endothelial monolayers in vitro. Importantly, we also identified increased permeability of the blood–brain barrier in vivo in CD44-deficient mice before and following immunization. These data suggest that CD44 has multiple protective roles in EAE, with effects on cytokine production, T-cell differentiation, T-cell–endothelial cell interactions, and blood–brain barrier integrity.Multiple sclerosis (MS) is an autoimmune, demyelinating disease resulting from chronic inflammation in the central nervous system (CNS). Experimental autoimmune encephalomyelitis (EAE), the primary and long-used animal model of MS, produces immune processes relevant to the human disease.¹ The progression and pathogenesis of EAE is complex and depends on multiple cell types and processes.^2–4T helper 17 (Th17) cells and their distinctive cytokine, IL-17, play pivotal roles in EAE/MS pathogenesis.^5–7 Th17 cells, members of a CD4 T-cell effector subset, are generated from naive CD4 T-cell precursors in response to cytokines TGF-β and IL-6, whereas IL-23 expands this population and increases pathogenicity.^8,9 In EAE, Th17 cells first infiltrate and initiate recruitment to the CNS,^5,6 and Th17-produced IL-17 induces neuronal death⁶ and increases permeability of the blood–brain barrier (BBB), allowing continued influx of immune cells by disrupting endothelial cell (EC) junctions.^6,10Regulatory T cells (Tregs), the primary suppressors of the immune system, play a pivotal role in EAE that is opposite to Th17 cells. Treg depletion exacerbates disease symptoms, whereas supplementation with additional Tregs ameliorates the disease.^11,12 Identified by the expression pattern CD4⁺CD25⁺FoxP3⁺, Tregs are generally divided into two principal subsets: naturally occurring Tregs, which arise in the thymus during development, and induced Tregs (iTregs), which can be generated in the periphery from naive CD4 T cells in response to TGF-β.^13,14Vascular EC also contribute to the complex pathogenesis of EAE. EC regulate leukocyte adhesion and extravasation, maintain vascular integrity, and limit injury and immune-mediated vascular permeability. The CNS vasculature, the primary constituent of the BBB, is especially unique and plays a critical role in protecting the CNS microenvironment. In MS/EAE, there is a characteristic breakdown of the BBB followed by accumulation of inflammatory infiltrates.^15,16CD44, a ubiquitously expressed type I transmembrane glycoprotein, has been implicated in a wide variety of cellular processes within and outside of the immune system.^17,18Alternative splicing and multiple posttranslational modifications generate various structural and functional versions of CD44 and are thought to be responsible for its large range of diverse and sometimes seemingly contradictory cellular functions.Although CD44 has been studied in several immunological contexts as a positive or negative regulator of inflammation, the many results are confounded by use of different mouse strains, inflammatory models, and experimental approaches. CD44 has been implicated as a proinflammatory molecule in several studies that identified an anti-inflammatory effect of a CD44 monoclonal antibody in multiple immune-mediated processes and diseases such as lymphocyte extravasation,¹⁹collagen- or proteoglycan-induced arthritis, respectively,^20,21 type 1 diabetes,²² asthma,²³ and EAE.²⁴ However, most studies in CD44-knockout (KO) mice suggest an anti-inflammatory role for this molecule in various immunological processes instead. CD44-KO mice experience enhanced inflammation in several models of pulmonary inflammation that suggest various roles of CD44 in immune cell clearance, TGF-β signaling, and repression of Toll-like receptor (TLR) signaling and inflammatory gene expression.^25–28 Further, CD44-KO mice show increased septic responses to lipopolysaccharide²⁹ and enhanced inflammatory responses following myocardial infarction³⁰ or hepatic injury.³¹ CD44 deficiency also led to increased collagen-induced arthritis severity with up-regulation of inflammatory genes in arthritic CD44-KO T cells.³² Clearly, antibody-mediated interference can have very different effects than genetic disruption of CD44. Hutas et al³³ in 2008 reported disparate effects of CD44 monoclonal antibody treatment versus CD44 deficiency on leukocyte recruitment during proteoglycan-induced arthritis.Despite antibody-mediated interference studies, the role of CD44 in EAE/MS remains poorly understood. In active MS lesions, there is an increase in CD44 expression and accumulation of hyaluronan (HA), a major CD44 ligand.^34,35 Previously, CD44 was shown to facilitate uptake of HA,³⁶ promoting resolution of tissue-injury signals and inflammation.^25,37 By contrast, a conditional mouse model of oligodendrocyte-specific overexpression of CD44 found a correlation between CD44 expression and enhanced HA accumulation, prevention of oligodendrocyte differentiation, and subsequent inflammation-independent demyelination.³⁵Until recently, EAE had not been examined in CD44-KO mice. This report demonstrates that CD44-KO mice present with increased EAE disease severity. This was associated with loss of CD44 on circulating immune cells, and also on noncirculating cells, specifically vascular EC of the BBB. We illustrate a more proinflammatory T-cell profile in CD44-KO mice with a reduction in Treg numbers throughout the disease that is accompanied by increased permeability of the BBB. Further, we illustrate a previously unidentified role for CD44 in the baseline integrity of the BBB, which has far-reaching implications beyond MS/EAE.     

The Glucagon-Like Peptide-1 Receptor Agonist Exendin 4 Has a Protective Role in Ischemic Injury of Lean and Steatotic Liver by Inhibiting Cell Death and Stimulating Lipolysis

Nonalcoholic fatty liver disease is an increasingly prevalent spectrum of conditions characterized by excess fat deposition within hepatocytes. Affected hepatocytes are known to be highly susceptible to ischemic insults, responding to injury with increased cell death, and commensurate liver dysfunction. Numerous clinical circumstances lead to hepatic ischemia. Mechanistically, specific means of reducing hepatic vulnerability to ischemia are of increasing clinical importance. In this study, we demonstrate that the glucagon-like peptide-1 receptor agonist Exendin 4 (Ex4) protects hepatocytes from ischemia reperfusion injury by mitigating necrosis and apoptosis. Importantly, this effect is more pronounced in steatotic livers, with significantly reducing cell death and facilitating the initiation of lipolysis. Ex4 treatment leads to increased lipid droplet fission, and phosphorylation of perilipin and hormone sensitive lipase – all hallmarks of lipolysis. Importantly, the protective effects of Ex4 are seen after a short course of perioperative treatment, potentially making this clinically relevant. Thus, we conclude that Ex4 has a role in protecting lean and fatty livers from ischemic injury. The rapidity of the effect and the clinical availability of Ex4 make this an attractive new therapeutic approach for treating fatty livers at the time of an ischemic insult.The incidence of obesity and fatty liver disease is increasing worldwide. Non alcoholic fatty liver disease (NAFLD) includes a spectrum of liver abnormalities ranging from simple steatosis with preserved synthetic function to end-stage liver disease requiring transplantation.^{1, 2} The cause of hepatic dysfunction related to steatosis remains incompletely defined.³ However, it is known that a steatotic liver has increased susceptibility to ischemic insults, such as those induced during liver resections and liver surgery,^{4, 5, 6} heart failure,⁷ and shock.⁸ In addition, steatotic livers are known to weather the ischemic insult of transplantation poorly,⁹ resulting in increased rates of primary nonfunction and initial graft dysfunction.^{10, 11} As such, fatty livers are routinely turned down for transplantation and this impacts transplant wait list morbidity and mortality.¹² Thus, liver steatosis contributes to the public health burden and methods to mollify the adverse effects of liver steatosis are relevant across a large spectrum of hepatic diseases.The inability of a steatotic liver to withstand ischemic insult is directly related to increased post ischemic cell death, which can occur through necrosis and apoptosis. The fundamental connection between intracellular fat and poor hepatic cell survival¹³ is incompletely understood. However, it has been suggested that methods that decrease intracellular fat reverse this susceptibility and the use of glucagon-like peptide-1 (GLP-1) analogues is one such approach. GLP-1 is secreted from the L cells of the small intestine and its cognate receptor (GLP-1R) is present in several organs, such as the pancreas, brain, heart, kidney, and liver. Although it is well known for its incretin action,¹⁴ it also has pleotropic effects.^{15, 16, 17, 18, 19} In the liver we have shown that GLP-1 or its homologue Exendin 4 (Ex4) acts directly on steatotic hepatocytes to decrease their lipid content.^{20, 21} In addition, a cytoprotective action of Ex4 with improvement in cell survival has also been reported.²² Thus, we hypothesize that anti-steatotic effects of Ex4 in hepatocytes and cytoprotective effects in other organs make it a rational target for investigation in steatotic livers undergoing ischemia reperfusion injury (IRI), a common clinical scenario in people with NAFLD. In this study, we explore the role of Ex4 in protecting against necrosis and apoptosis, the two forms of cell death encountered in hepatic IRI, and we provide evidence to show that Ex4 stimulates lipolysis with a short course of treatment. To our knowledge, this is the first study showing a direct and rapid action of Ex4 in acutely reversing the vulnerability of a steatotic liver to ischemic insults, supporting the investigation of Ex4 as a potential therapeutic agent for treatment of people with NAFLD undergoing ischemic injury and at the time of procurement of a fatty liver for transplantation.     

Antibodies to Gliomedin Cause Peripheral Demyelinating Neuropathy and the Dismantling of the Nodes of Ranvier

Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) are conditions that affect peripheral nerves. The mechanisms that underlie demyelination in these neuropathies are unknown. Recently, we demonstrated that the node of Ranvier is the primary site of the immune attack in patients with GBS and CIDP. In particular, GBS patients have antibodies against gliomedin and neurofascin, two adhesion molecules that play a crucial role in the formation of nodes of Ranvier. We demonstrate that immunity toward gliomedin, but not neurofascin, induced a progressive neuropathy in Lewis rats characterized by conduction defects and demyelination in spinal nerves. The clinical symptoms closely followed the titers of anti-gliomedin IgG and were associated with an important deposition of IgG at nodes. Furthermore, passive transfer of antigliomedin IgG induced a severe demyelinating condition and conduction loss. In both active and passive models, the immune attack at nodes occasioned the loss of the nodal clusters for gliomedin, neurofascin-186, and voltage-gated sodium channels. These results indicate that primary immune reaction against gliomedin, a peripheral nervous system adhesion molecule, can be responsible for the initiation or progression of the demyelinating form of GBS. Furthermore, these autoantibodies affect saltatory propagation by dismantling nodal organization and sodium channel clusters. Antibodies reactive against nodal adhesion molecules thus likely participate in the pathologic process of GBS and CIDP.Guillain-Barré syndrome (GBS) is a group of inflammatory neuropathies that affect peripheral nerves. In Europe, acute inflammatory demyelinating polyneuropathy (AIDP) is the most common form of GBS. Autopsy and biopsy studies indicated that both humoral and cellular immune reaction against Schwann cell or axonal antigens are implicated in GBS etiology.¹ Early investigations have found that conduction defects closely correlate with myelin retraction and macrophage invasion in many patients.^{2, 3, 4, 5} Some GBS cases also involve acute demyelination without immune cell invasion and are primarily humorally mediated.^{6, 7} In particular, deposition of complement on the abaxonal surface of the Schwann cells has been shown during the early stage of GBS^{8, 9, 10} and in experimental allergic neuritis (EAN).¹¹ In a recent study, we demonstrated that nodes of Ranvier and paranodes are the targets of the immune attack in GBS and in chronic inflammatory demyelinating polyneuropathy (CIDP).¹² Notably, cell adhesion molecules (CAMs) at nodes or paranodes (gliomedin, neurofascin, and contactin) were recognized by IgG antibodies in patients with GBS or CIDP.^{12, 13} Autoantibodies against neurofascin and gliomedin were also detected in a rat model of AIDP and correlated with important conduction defects.¹⁴ This finding suggested that antibodies to nodal CAMs may participate to the pathogenesis of AIDP and CIDP. However, the exact mechanisms by which these humoral factors mediate demyelination and conduction defects are still elusive.Several CAMs are implicated in node formation and are responsible for the enrichment of voltage-gated sodium (Nav) channels at the nodes of Ranvier.¹⁵ At peripheral, nodes gliomedin and NrCAM are secreted into the nodal gap lumen and interact with neurofascin-186 (NF186) expressed at nodal axolemma.^{16, 17, 18, 19} This interaction is crucial for Nav channel aggregation at nodes.^{19, 20, 21} In addition, the paranodal axoglial junctions are made by the association of contactin and contactin-associated protein (Caspr) with neurofascin-155 (NF155), a variant expressed in glia.²² This adhesive junction forms a barrier to the lateral diffusion of nodal channels.^{19, 21, 23} In a rat model of AIDP, we found that the loss of NF186 and gliomedin at nodes preceded paranodal demyelination and the diffusion of Nav channels in demyelinated segments.¹⁴ This finding indicated that antibodies to nodal CAMs may participate to conduction defects by dismantling axoglial attachment at nodes and paranodes.We investigated whether immunity toward gliomedin and NF186 can trigger peripheral neuropathies and be responsible for demyelination in GBS patients. We found that immunization against gliomedin induced a biphasic condition associated with conduction loss and demyelination. Passive transfer of antibodies to gliomedin exacerbated the clinical signs of EAN and resulted in the disorganization of the nodes of Ranvier. Altogether, these results demonstrate that humoral immune response directed against nodal CAMs participates in conduction abnormalities in peripheral nerves and in the etiology of GBS and CIDP.     

Interaction of MCM7 and RACK1 for Activation of MCM7 and Cell Growth

MCM7 is one of the pivotal DNA replication licensing factors in controlling DNA synthesis and cell entry into S phase. Its expression and DNA copy number are some of the most predictive factors for the growth and behavior of human malignancies. In this study, we identified that MCM7 interacts with the receptor for activated protein kinase C 1 (RACK1), a protein kinase C (PKC) adaptor, in vivo and in vitro. The RACK1 binding motif in MCM7 is located at the amino acid 221-248. Knocking down RACK1 significantly reduced MCM7 chromatin association, DNA synthesis, and cell cycle entry into S phase. Activation of PKC by 12-O-tetradecanoylphorbol-13-acetate dramatically decreased MCM7 DNA replication licensing and induced cell growth arrest. Activation of PKC induced redistribution of RACK1 from nucleus to cytoplasm and decreased RACK1-chromatin association. The MCM7 mutant that does not bind RACK1 has no DNA replication licensing or oncogenic transformation activity. As a result, this study demonstrates a novel signaling mechanism that critically controls DNA synthesis and cell cycle progression.Miniature chromosome maintenance (MCM) proteins were initially identified from autonomously replicating sequence in Saccharomyces cerevisiae. Mutations of some of these proteins, such as MCM7 or MCM3 result in loss of the large chunk of yeast chromosomes in yeast. MCM7 cDNA encodes a 543-amino acid protein and is ubiquitously expressed in all tissues. A large body of studies indicate that MCM7 is a critical component of DNA replication licensing complex in the yeast and xenopus.^1–4 Some studies suggest that MCM4, MCM6, and MCM7 complex contains DNA helicase activity.^5,6 DNA replication licensing complex is multimeric and phase specific. In yeast, DNA replication licensing proteins, such as MCM2-7 and several replication origin binding proteins, such as Cdc6, germinin, and Cdt1, form DNA replication licensing complex in G1 phase to enable DNA replication and to promote cell cycle entry into S phase. Initial implication of MCM7 involvement in human malignancies came from positive immunostaining of MCM7 in several human malignancies, including endometrial carcinoma,⁷ melanoma,⁸ esophageal adenocarcinoma,⁹ colorectal adenocarcinoma,¹⁰ oral squamous cell carcinoma,¹¹ glioblastoma,¹² and thyroid cancer.¹³ The first study addressing the oncogenic role of MCM7 in prostate cancer came from genome analysis of prostate cancer by performing a genome wide copy number analysis using biotin-labeled genome DNA on Affymetrix U95av2 chip.¹⁴ The DNA copy number of MCM7 was found to increase severalfold accompanied with a concomitant increase of MCM7 mRNA level. Subsequent validation analyses suggest that either copy number and/or protein level increase of MCM7 are associated with prostate cancer relapse and metastasis. Amplification of MCM7 was also found in esophageal carcinoma.⁹ The magnitude of MCM7 amplification correlates with the expression of MCM7, tumor grades, and the aggressiveness of esophageal cancer.⁹ It is presumed that amplification of MCM7 is the driving force of MCM7 overexpression in primary human malignancies. MCM7 is probably the primary target of Rb, the tumor suppressor that controls cell entry into S phase.¹⁵ There is growing evidence that other signaling pathways also regulate MCM7 activity.Receptor for activated protein kinase C 1 (RACK1), was initially identified as an adaptor of several protein kinase C (PKC) isoforms.¹⁶ The binding of RACK1 and PKC anchor PKC to its substrate to initiate second messenger signaling. It is suggested, according to recent studies that RACK1 interacts with a variety of other signaling molecules, including ras-GTPase activating protein,¹⁷ dynamin-1,¹⁸ src,¹⁹ integrins,²⁰ PTPμ,²¹ phosphodiesterase,²² hypoxia induced factor-1,²³ and so forth, that play an important role in several physiological processes, including, growth, hypoxia response, migration, adhesion, and cell differentiation. RACK1 only binds PKC activated by diacylglycerol or phorbol ester, but not quiescent PKC. In this study, we showed that RACK1 binds with MCM7 N-terminus. The MCM7/RACK1 interaction appears essential for DNA replication activity of MCM7.     

Endometrial Cancer Side-Population Cells Show Prominent Migration and Have a Potential to Differentiate into the Mesenchymal Cell Lineage

Cancer stem-like cell subpopulations, referred to as “side-population” (SP) cells, have been identified in several tumors based on their ability to efflux the fluorescent dye Hoechst 33342. Although SP cells have been identified in the normal human endometrium and endometrial cancer, little is known about their characteristics. In this study, we isolated and characterized the SP cells in human endometrial cancer cells and in rat endometrial cells expressing oncogenic human K-Ras protein. These SP cells showed i) reduction in the expression levels of differentiation markers; ii) long-term proliferative capacity of the cell cultures; iii) self-renewal capacity in vitro; iv) enhancement of migration, lamellipodia, and, uropodia formation; and v) enhanced tumorigenicity. In nude mice, SP cells formed large, invasive tumors, which were composed of both tumor cells and stromal-like cells with enriched extracellular matrix. The expression levels of vimentin, α-smooth muscle actin, and collagen III were enhanced in SP tumors compared with the levels in non-SP tumors. In addition, analysis of microdissected samples and fluorescence in situ hybridization of Hec1-SP-tumors showed that the stromal-like cells with enriched extracellular matrix contained human DNA, confirming that the stromal-like cells were derived from the inoculated cells. Moreober, in a Matrigel assay, SP cells differentiated into α-smooth muscle actin-expressing cells. These findings demonstrate that SP cells have cancer stem-like cell features, including the potential to differentiate into the mesenchymal cell lineage.Recently, adult stem cells have been identified in several mature tissues, such as the adult intestine,¹ skin,² muscle,³ blood,⁴ and the nervous system^5–7 A stem cell is an undifferentiated cell that is defined by its ability to both self-renew and to produce mature progeny cells.⁸ Stem cells are classified based on their developmental potential as totipotent, pluripotent, oligopotent, and unipotent. Adult somatic stem cells were originally thought to be tissue specific and only able to give rise to progeny cells corresponding to their tissue of origin. Recent studies, however, have shown that adult mammalian stem cells are able to differentiate across tissue lineage boundaries,^9,10 although this “plasticity” of adult somatic stem cells remains controversial.Stem cell subpopulations (“side-population” (SP) cells) have been identified in many mammals, including humans, based on the ability of these cells to efflux the fluorescent dye Hoechst 33342.¹¹ Recent evidence suggests that the SP phenotype is associated with a high expression level of the ATP-binding cassette transporter protein ABCG2/Bcrp1.¹² Most recently, established malignant cell lines, which have been maintained for many years in culture, have also been shown to contain SP cells as a minor subpopulation.¹³The human endometrium is a highly dynamic tissue undergoing cycles of growth, differentiation, shedding, and regeneration throughout the reproductive life of women. Endometrial adult stem/progenitor cells are likely responsible for endometrial regeneration.¹⁴ Rare populations of human endometrial epithelial and stromal colony-forming cells¹⁵ and SP cells^16,17 have been identified. Although coexpression of CD146 and PDGFRβ isolates a population of mesenchymal stem like cells from human endometrium,¹⁸ specific stem cell markers of endometrium remain unclear. Recently, Gotte et al¹⁹ demonstrated that the adult stem cell marker Musashi-1 was coexpressed with Notch-1 in a subpopulation of endometrial cells. Furthermore, they showed that telomerase and Musashi-1-expressing cells were significantly increased in proliferative endometrium, endometriosis, and endometrial carcinoma tissue, compared with secretary endometrium, suggesting the concept of a stem cell origin of endometriosis and endometrial carcinoma.Recent evidence suggests that cancer stem-like cells exist in several malignant tumors, such as leukemia^20,21 breast cancer,²² and brain tumors,²³ and that these stem cells express surface markers similar to those expressed by normal stem cells in each tissue.^20,24Development of endometrial carcinoma is associated with a variety of genetic alterations. For example, increased expression and activity of telomerase^25,26 and frequent dysregulation of signaling pathways have been observed in endometrial carcinoma. Some of these pathways are important determinants of stem cell activity (Wnt-β-catenin and PTEN).^27–29 These suggest a stem cell contribution to endometrial carcinoma development.Recently, we isolated SP cells from the human endometrium. These SP cells showed long-term proliferating capacity in cultures and produced both gland and stromal-like cells. Additionally, they were able to function as progenitor cells.¹⁶ In this study, we isolated and characterized SP cells from human endometrial cancer cells and from rat endometrial cells expressing oncogenic [¹²Val] human K-Ras protein and demonstrated their cancer stem-like cell phenotypes.     

Cytoprotective Mitochondrial Chaperone TRAP-1 As a Novel Molecular Target in Localized and Metastatic Prostate Cancer

Molecular chaperones of the heat shock protein-90 (Hsp90) family promote cell survival, but the molecular requirements of this pathway in tumor progression are not understood. Here, we show that a mitochondria-localized Hsp90 chaperone, tumor necrosis factor receptor-associated protein-1 (TRAP-1), is abundantly and ubiquitously expressed in human high-grade prostatic intraepithelial neoplasia, Gleason grades 3 through 5 prostatic adenocarcinomas, and metastatic prostate cancer, but largely undetectable in normal prostate or benign prostatic hyperplasia in vivo. Prostate lesions formed in genetic models of the disease, including the transgenic adenocarcinoma of the mouse prostate and mice carrying prostate-specific deletion of the phosphatase tensin homolog tumor suppressor (Pten^pc−/−), also exhibit high levels of TRAP-1. Expression of TRAP-1 in nontransformed prostatic epithelial BPH-1 cells inhibited cell death, whereas silencing of TRAP-1 in androgen-independent PC3 or DU145 prostate cancer cells by small interfering RNA enhanced apoptosis. Targeting TRAP-1 with a novel class of mitochondria-directed Hsp90 inhibitors, ie, Gamitrinibs, caused rapid and complete killing of androgen-dependent or -independent prostate cancer, but not BPH-1 cells, whereas reintroduction of TRAP-1 in BPH-1 cells conferred sensitivity to Gamitrinib-induced cell death. These data identify TRAP-1 as a novel mitochondrial survival factor differentially expressed in localized and metastatic prostate cancer compared with normal prostate. Targeting this pathway with Gamitrinibs could be explored as novel molecular therapy in patients with advanced prostate cancer.Apart from skin tumors, prostate cancer is the most commonly diagnosed malignancy in men in the United States.¹ Despite progress in early diagnosis,² and prolongation of patient survival,³ the disease still carries significant morbidity and mortality, with its advanced and metastatic phase claiming over 30,000 deaths per year in the United States alone. Similar to the genetic heterogeneity of most epithelial malignancies, prostate cancer progresses through a stepwise acquisition of multiple molecular changes,⁴ of which insensitivity to androgen deprivation,⁵ emergence of an ‘osteomimetic’ phenotype responsible for metastatic tropism to the bone,⁶ and deregulated cell proliferation and cell survival,⁷ are pivotal traits.In this context, advanced prostate cancer is almost invariably associated with a heightened anti-apoptotic threshold,⁴ which may contribute to disease progression and resistance to therapy. This process often involves aberrant resistance to mitochondrial cell death,⁸ with reduced organelle permeability to solutes, and attenuated release of mitochondrial apoptogenic proteins in the cytosol.⁹ The regulators of such ‘mitochondrial permeability transition’ normally triggered by cell death stimuli are still largely elusive, but knockout data in mice have identified pro-apoptotic Bcl-2 family proteins and the mitochondrial matrix immunophilin, cyclophilin D, as pivotal effectors of this process, controlling the integrity of the mitochondrial outer membrane,⁸ and the opening a permeability transition pore,^10,11 respectively.Recent data have shown that molecular chaperones of the heat shock protein-90 (Hsp90) family,¹² may function as novel regulators of mitochondrial permeability transition,¹³ especially in tumor cells.¹⁴ Accordingly, Hsp90, and its ortholog, tumor necrosis factor receptor-associated protein-1 (TRAP-1) are abundantly localized to mitochondria of tumor, but not most normal cells, and antagonize cyclophilin D-dependent pore-forming function, potentially via a protein (re)folding mechanism.¹⁴ Consistent with a general role of Hsp90 as a drug target in prostate cancer,¹⁵ this mitochondria-compartmentalized cytoprotective pathway could provide a novel therapeutic target to enhance tumor cell apoptosis.¹⁴In the current study, we demonstrate that TRAP-1 is dramatically expressed in all lesions that comprise the entire natural history of human prostate cancer, as well as genetic disease models in rodents, but undetectable in the normal prostate. Importantly, we show that Gamitrinibs, a novel class of small molecule Hsp90 antagonists selectively engineered to target the pool of these chaperones in mitochondria,¹⁶ cause sudden prostate cancer cell death without affecting nontransformed prostatic epithelium.     

Biological sample collections from minors for genetic research: a systematic review of guidelines and position papers

Stored tissue samples are an important resource for epidemiological genetic research. Genetic research on biological material from minors can yield valuable information on the development and genesis of early-onset genetic disorders and the early interaction of environmental and genetic factors. The use of such tissue raises some specific ethical and governance questions, which are not completely covered by the discussion on biological materials from adults. We have retrieved 29 guidelines and position papers pertaining to the storage and use of biological tissue samples for genetic research, originating from 27 different organizations. Five documents have an international scope, three have an European scope and 21 have a national scope. We discovered that 11 of these documents did not contain a section on biological materials from minors. The content of the remaining 18 documents was categorized according to four themes: consent, principles of non-therapeutic research on vulnerable populations, ethics committee approval and difference between anonymous and identifiable samples. We found out that these themes are not consistently mentioned by each document, but that documents discussing the same themes were mostly in agreement with their recommendations. However, a systematic reflection on the ethical and policy issues arising from the participation of minors in biobank research is missing.Stored tissue sample collections for genetic research exist in different forms. Some of these collections provide a resource for potentially unlimited genetic research, and gather samples and data from specific populations. An example is the ‘UK biobank''.¹ Other collections are stored for research on a specific disease. Collections that were originally gathered for different purposes, for example blood spot cards for newborn screening, could be reused for genetic research.²Genetic research on biological material from minors and the associated medical records can yield valuable information on the development and genesis of early-onset genetic disorders and the early interaction of environmental and genetic factors. For example, Rasmussen³ describes the incorporation of DNA sample collections into the ‘National Birth Defects Prevention Study'' in the United States to identify the risk factors for birth defects. Studies such as the ‘Avon Longitudinal Study of Parents and Children'' in Bristol (children of the nineties) use genetic, phenotypic and environmental information of 14 000 babies from their conception onwards to study the interaction between these data.⁴An extensive ethical literature exists on the collection, storage and use of biological samples for genetic research. The overwhelming majority of these documents discuss issues of privacy, confidentiality, commercialization and consent.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14} However, research on pediatric data raises specific ethical questions with regard to consent and privacy. For example, who should give consent to the inclusion of tissue and data from children? Is the general requirement that non-therapeutic research can only be done with children if it involves no more than minimal risk, applicable to biobank research? We shall review whether and how guidelines and policy documents discuss children in the context of storing biological samples and DNA for non-therapeutic research.     

Murine Cerebral Malaria Is Associated with a Vasospasm-Like Microcirculatory Dysfunction,and Survival upon Rescue Treatment Is Markedly Increased by Nimodipine

Brain hemodynamics in cerebral malaria (CM) is poorly understood, with apparently conflicting data showing microcirculatory hypoperfusion and normal or even increased blood flow in large arteries. Using intravital microscopy to assess the pial microvasculature through a closed cranial window in the murine model of CM by Plasmodium berghei ANKA, we show that murine CM is associated with marked decreases (mean: 60%) of pial arteriolar blood flow attributable to vasoconstriction and decreased blood velocity. Leukocyte sequestration further decreased perfusion by narrowing luminal diameters in the affected vessels and blocking capillaries. Remarkably, vascular collapse at various degrees was observed in 44% of mice with CM, which also presented more severe vasoconstriction. Coadministration of artemether and nimodipine, a calcium channel blocker used to treat postsubarachnoid hemorrhage vasospasm, to mice presenting CM markedly increased survival compared with artemether plus vehicle only. Administration of nimodipine induced vasodilation and increased pial blood flow. We conclude that vasoconstriction and vascular collapse play a role in murine CM pathogenesis and nimodipine holds potential as adjunctive therapy for CM.Cerebral malaria (CM) caused by Plasmodium falciparum claims the lives of nearly 1 million children every year.¹ Despite antimalarial treatment, 10% to 20% of patients die, and one in every four survivors develops neurological sequelae,^2,3 therefore adjunctive therapies are urgently needed. A number of clinical trials addressing potential adjunctive therapies for CM showed no proven benefits and some interventions were even deleterious,⁴ stressing the need for a better understanding of CM pathogenesis to develop effective therapies.An unresolved issue of CM pathogenesis regards the role of brain hemodynamic perturbations and ischemia. Sequestration of parasitized red blood cells (pRBCs) containing mature forms of the parasite in the brain microvasculature is a characteristic postmortem finding in human CM cases⁵ and together with rosetting⁶ and reduced RBC deformability⁷ may result in the obstruction of blood flow potentially leading to ischemia and hypoxia. In vivo studies of the microcirculation in human CM support this mechanism, with direct observation of retinal microvasculature showing impaired perfusion, retinal whitening, vascular occlusion, and ischemia.⁸ Accordingly, microvascular obstruction observed in the rectal mucosa of CM patients was proportional to the severity of the disease.⁹ In addition, hypovolemia, shock and intracranial hypertension, commonly associated with poor outcomes in CM,⁴ reduce tissue perfusion, and tissue hypoxia is one of the likely explanations for the acidosis frequently observed in severe malaria.^7,10 Ischemic damage has also been shown in children with CM and was associated with severe neurological sequelae.¹¹ On the other hand, transcranial Doppler sonography studies showed normal or even increased cerebral blood flow (CBF) velocities^12–15 in large arteries during CM, which associated with microcirculatory obstruction has been suggested to increase cerebral blood volume leading to intracranial hypertension.¹⁶ Alternatively, collateral flow has been proposed as a mechanism to reconcile the findings of normal or increased CBF velocities and impaired perfusion,¹⁷ an interpretation supported by findings of hyperdynamic flow in capillaries adjacent to obstructed vessels.⁹ Interventions that improve cerebral perfusion have been proposed to be beneficial in CM.^8,18The murine model of CM by Plasmodium berghei ANKA (PbA) shares many features with the human pathology,¹⁹ including the presence of multiple brain microhemorrhages and vascular obstruction, although the nature of the sequestered cell (leukocytes) differs. In murine CM, magnetic resonance imaging (MRI) and spectroscopy studies showed the presence of brain edema, decreased CBF, and ischemia.^20,21 Lack of resolution in MRI, however, precludes detailed studies of the microcirculation, which is a major target and player in CM pathogenesis. A few studies have addressed the in vivo microcirculatory changes in murine models of severe malaria,^22–24 however in sites other than the brain (cremaster muscle or skin). In the present work, we used for the first time brain intravital microscopy to follow the dynamic changes in the pial microcirculation during the course of PbA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow, and eventually vascular collapse, events similar to postsubarachnoid hemorrhage (SAH) vasospasm.²⁵ We also show that nimodipine, a calcium channel blocker used to treat post-SAH vasospasm,^25,26 markedly increased survival when given off-label to mice with CM as adjunctive therapy to artemether.     

Genetic testing in asymptomatic minors Background considerations towards ESHG Recommendations

Although various guidelines and position papers have discussed, in the past, the ethical aspects of genetic testing in asymptomatic minors, the European Society of Human Genetics had not earlier endorsed any set of guidelines exclusively focused on this issue. This paper has served as a background document in preparation of the development of the policy recommendations of the Public and Professional Committee of the European Society of Human Genetics. This background paper first discusses some general considerations with regard to the provision of genetic tests to minors. It discusses the concept of best interests, participation of minors in health-care decisions, parents'' responsibilities to share genetic information, the role of clinical genetics and the health-care system in communication within the family. Second, it discusses, respectively, the presymptomatic and predictive genetic testing for adult-onset disorders, childhood-onset disorders and carrier testing.Although various guidelines and position papers have discussed, in the past, the ethical aspects of genetic testing in asymptomatic minors,^{1, 2} the European Society of Human Genetics had not earlier endorsed any set of guidelines exclusively focused on this issue. This background paper was preceded by an in-depth research on the topic by Eurogentest.³ Eurogentest (http://www.eurogentest.org aims to develop the necessary infrastructure, tools, resources, guidelines and procedures that will structure, harmonize and improve the overall quality of all the EU genetic services at the molecular, cytogenetic, biochemical and clinical level.⁴ Attention has also been paid to the provision of appropriate counselling related to genetic testing, the education of patients and professionals, as well as to the ethical, legal and social issues surrounding testing. The focus of the ethics unit of Eurogentest was oriented towards the study of the ethical issues related to genetic testing in minors. This work was the starting point for this background paper, which has been prepared and supported by different types of evidence. First, research has been performed on the existing recommendations regarding predictive genetic testing in minors¹ and carrier testing,² with the intention of identifying areas of agreement and disagreement. Second, the literature on medico–ethical and medico–legal aspects of predictive genetic testing in minors,⁵ carrier testing,^{6, 7} the position of minors⁸ and patient rights⁹ was studied. Third, a systematic literature review was performed to gather information regarding the attitudes of the different stakeholders (minors, health-care professionals, parents and relatives of the affected individuals) towards genetic testing in asymptomatic minors.^{10, 11} Fourth, the attitudes of European clinical geneticists regarding genetic testing in asymptomatic minors were gathered.^{12, 13, 14}In 2007, contacts were made with the Public and Professional Policy Committee of the European Society of Human Genetics with the aim of developing policy recommendations on the issue. On the basis of a decision of the PPPC meeting during the ESHG conference in Nice (June 2007), an ad hoc committee, consisting of Pascal Borry (Eurogentest), Kris Dierickx (Eurogentest), Angus Clarke, Gerry Evers-Kiebooms (PPPC) and Martina Cornel (PPPC), was created. This ad hoc committee met on 15 November 2007 to discuss a first draft of a background paper and recommendations that were prepared by Pascal Borry under the supervision of Kris Dierickx. A revised version was discussed during a PPPC meeting in Amsterdam (April 2008) and Barcelona (June 2008). In order not to repeat issues that have been discussed elsewhere, reference will often be made to the above-referenced publications.     

Non-invasive prenatal testing: ethical issues explored

This paper explores the ethical implications of introducing non-invasive prenatal diagnostic tests (NIPD tests) in prenatal screening for foetal abnormalities. NIPD tests are easy and safe and can be performed early in pregnancy. Precisely because of these features, it is feared that informed consent may become more difficult, that both testing and selective abortion will become ‘normalized'', and that there will be a trend towards accepting testing for minor abnormalities and non-medical traits as well. In our view, however, the real moral challenge of NIPD testing consists in the possibility of linking up a technique with these features (easy, safe and early) with new genomic technologies that allow prenatal diagnostic testing for a much broader range of abnormalities than is the case in current procedures. An increase in uptake and more selective abortions need not in itself be taken to signal a thoughtless acceptance of these procedures. However, combining this with considerably enlarging the scope of NIPD testing will indeed make informed consent more difficult and challenge the notion of prenatal screening as serving reproductive autonomy. If broad NIPD testing includes later-onset diseases, the ‘right not to know'' of the future child will become a new issue in the debate about prenatal screening. With regard to the controversial issue of selective abortion, it may make a morally relevant difference that after NIPD testing, abortion can be done early. A lower moral status may be attributed to the foetus at that moment, given the dominant opinion that the moral status of the foetus progressively increases with its development.Since the discovery of cell-free foetal DNA/RNA (cffDNA/RNA) in maternal plasma in 1997,¹ the possibility to use this cffDNA/RNA for non-invasive prenatal diagnosis (NIPD) has been investigated many times.^{2, 3, 4, 5, 6} cffDNA/RNA can be obtained from a maternal blood sample, as early as 4 weeks of gestation,⁷ but currently only reliably so from 7 weeks of gestation.⁴ This development holds the promise of NIPD testing early in pregnancy and without the small, but significant risk of foetal loss that the current invasive procedures of chorionic villus sampling (CVS) and amniocentesis (AP) carry. NIPD testing for the determination of a Y-signal for pregnancies at risk of X-linked disorders and for diagnosis of Rhesus factor status in RhD-negative women is now being translated into clinical practice.⁴ In many European countries, discussion about broader applications of NIPD testing can be expected in the coming years.^{8, 9} The feasibility of NIPD for trisomy 21, 13 and 18 has already been shown,² but large-scale independent studies are still needed. Sex-chromosomal abnormalities (eg, Turner syndrome (X0) and triple X syndrome (XXX)) could in principle be diagnosed by NIPD testing as well,⁴ if reliable quantitative tests become available in the future and the maternal ‘background'' can be excluded from testing. Even if accurate NIPD testing for the mentioned abnormalities becomes possible, the clinical utility of the test remains to be assessed. This includes balancing the benefits to the harms also with regard to its psychological, ethical, legal, social and economic implications.^{10, 11} The possible ethical implications of NIPD as a new approach to prenatal testing have so far been reviewed in a few publications.^{4, 8, 9, 12, 13, 14, 15, 16, 17} Apart from clear benefits related to avoiding the miscarriage risk of present invasive methods, important potential drawbacks have been mentioned as well. For one thing, proper counselling and informed consent is argued to become more challenging when offering NIPD testing. Moreover, there is a concern that the ease and safety of NIPD may lead to prenatal screening being increasingly conceived as a matter of course, both by those making the offer and by the women undergoing the test. Related to this is the concern that selective abortion of foetuses with minor abnormalities, the wrong sex or unwanted paternity, will become normalized.This paper aims to expand and refine these ethical evaluations and will add some new ethical perspectives with regard to possible implications of NIPD at present and in the future.In our view, it is not so much the fact that foetal material used for prenatal testing can be obtained early and non-invasively (allowing easy and safe testing) that would lead to moral challenges. Rather, it is the fact that a technology with these features would be open to being used for testing a potentially much broader range of abnormalities than those included in the presently used method of microscopic chromosome analysis (karyotyping).Although NIPD testing can also be applied in high genetic-risk families and for the management of pregnancy, the focus of this paper will primarily be on the application of NIPD testing in the screening context. The reason for this focus on prenatal screening is that in the near future, the question if, and if so, in what way NIPD testing is to be applied within prenatal screening strategies should be considered and discussed by policy makers, health care professionals and society at large.To avoid confusion, a preliminary remark is needed on terminology. In medicine, ‘screening'' is often used as referring to a kind of test for risk assessment or disease discovery. However, after the convention in normative and regulatory discourse, we will use ‘screening'' as referring to any systematic and unsolicited offer of predictive testing (using whatever types of test) involving individuals who themselves have no reason (yet) to seek medical help for the condition in question.¹⁸ In this broader sense, screening stands in contrast to ‘diagnosis'' as testing on indication.     

The Role of Vascular Endothelial Growth Factor-Induced Activation of NADPH Oxidase in Choroidal Endothelial Cells and Choroidal Neovascularization

Rac1, a subunit of NADPH oxidase, plays an important role in directed endothelial cell motility. We reported previously that Rac1 activation was necessary for choroidal endothelial cell migration across the retinal pigment epithelium, a critical step in the development of vision-threatening neovascular age-related macular degeneration. Here we explored the roles of Rac1 and NADPH oxidase activation in response to vascular endothelial growth factor treatment in vitro and in a model of laser-induced choroidal neovascularization. We found that vascular endothelial growth factor induced the activation of Rac1 and of NADPH oxidase in cultured human choroidal endothelial cells. Further, vascular endothelial growth factor led to heightened generation of reactive oxygen species from cultured human choroidal endothelial cells, which was prevented by the NADPH oxidase inhibitors, apocynin and diphenyleneiodonium, or the antioxidant, N-acetyl-l-cysteine. In a model of laser-induced injury, inhibition of NADPH oxidase with apocynin significantly reduced reactive oxygen species levels as measured by dihydroethidium fluorescence and the volume of laser-induced choroidal neovascularization. Mice lacking functional p47phox, a subunit of NADPH oxidase, had reduced dihydroethidium fluorescence and choroidal neovascularization compared with wild-type controls. Taken together, these results indicate that vascular endothelial growth factor activates Rac1 upstream from NADPH oxidase in human choroidal endothelial cells and increases generation of reactive oxygen species, contributing to choroidal neovascularization. These steps may contributed to the pathology of neovascular age-related macular degeneration.Age-related macular degeneration (AMD) is a leading cause of visual impairment in elderly individuals^1–4 and is estimated to affect approximately 14 million people worldwide.⁵ Ninety percent of legal blindness from AMD is due to neovascularization that originates from endothelial cells in the choroid and grows into neurosensory retina as choroidal neovascularization (CNV).⁵ Although it is recognized that genetic polymorphisms, such as those involved in the alternative pathway of the complement system, have been strongly associated with increased risk of advanced AMD,^6–11 AMD does not manifest at birth but rather in the seventh or eighth decades of life. The current thinking is that a genetic predisposition accompanied by exogenous, chronic, repeated stresses could lead to tissue damage and dysfunction that becomes apparent only later in life.^10,12,13 In support of this theory is clinical evidence of independent and sometimes additive risk of advanced AMD from environmental factors.¹⁴Several environmental factors associated with increased risk of AMD also increase oxidative stress. Examples include blue light-induced photochemically released oxidants¹⁵ and cigarette smoke-related compounds.¹⁶ Other clinical evidence supports oxidative stress in advanced AMD. Proteomic analysis of Bruch''s membrane from human eyes with AMD revealed the presence of oxidized compounds.¹⁷ The Eye Disease Case Control Study found an association between reduced prevalence of AMD and high dietary intake of antioxidants.¹⁸ The placebo-controlled, double-masked multicenter clinical Age-Related Eye Disease Study provided strong support for the relationship between AMD and oxidants by finding reduced progression to advanced AMD and vision loss in subjects who took certain antioxidants and zinc supplements.¹⁹Laboratory evidence provides mechanistic support for oxidative stress in the pathophysiology of AMD. Conditioned media from hydrogen peroxide-treated retinal pigment epithelium (RPE) induced an angiogenic phenotype in cocultured choroidal endothelial cells.²⁰ Glutathione-depleted RPE, thus susceptible to oxidative damage, had increased expression of vascular endothelial growth factor (VEGF) and its receptors compared with the control.²⁰ A mouse model lacking the antioxidant enzyme, CuZn superoxide dismutase, developed features of AMD, namely drusen, CNV, and dysfunction in RPE cell junctions and barrier properties.²¹ Photo-oxidation of bis-retinoid lipofuscin in cultured RPE cells resulted in released complement activation fragments, providing evidence that oxidation activated complement and also linked photo-oxidative stress and the complement system.²² Although evidence supports oxidative damage in AMD, gaps remain in our understanding of the steps involved in the pathophysiology of advanced vision-threatening AMD.We reported previously that activation of the small Rho GTPase, Rac1, was necessary for choroidal endothelial cell migration across the RPE, a critical step in the development of neovascular AMD.²³ Further, the activation of Rac1 was found downstream of VEGF-VEGF receptor 2 signaling.²⁴ Rac1 is important in directed endothelial cell motility and is also an important subunit of the enzyme complex, NADPH oxidase. In its inactive state, NADPH oxidase consists of membrane-bound subunits, ie, gp91phox and p22phox, and cytosolic subunits, p67phox and p47phox, in addition to Rac1 in endothelial cells.²⁵ When activated, the subunits aggregate and the active enzyme generates reactive oxygen species (ROS) and triggers angiogenic signaling in endothelial cells (ECs).²⁵ In this study, we investigated the role of NADPH oxidase in the pathogenesis of CNV.     

Neutrophil Elastase Is Produced by Pulmonary Artery Smooth Muscle Cells and Is Linked to Neointimal Lesions

Previously, we reported that murine gammaherpesvirus-68 (M1-MHV-68) induces pulmonary artery (PA) neointimal lesions in S100A4-overexpressing, but not in wild-type (C57), mice. Lesions were associated with heightened lung elastase activity and PA elastin degradation. We now investigate a direct relationship between elastase and PA neointimal lesions, the nature and source of the enzyme, and its presence in clinical disease. We found an association exists between the percentage of PAs with neointimal lesions and elastin fragmentation in S100A4 mice 6 months after viral infection. Confocal microscopy documented the heightened susceptibility of S100A4 versus C57 PA elastin to degradation by elastase. A transient increase in lung elastase activity occurs in S100A4 mice, 7 days after M1-MHV-68, unrelated to inflammation or viral load and before neointimal lesions. Administration of recombinant elafin, an elastase-specific inhibitor, ameliorates early increases in serine elastase and attenuates later development of neointimal lesions. Neutrophils are the source of elevated elastase (NE) in the S100A4 lung, and NE mRNA and protein levels are greater in PA smooth muscle cells (SMC) from S100A4 mice than from C57 mice. Furthermore, elevated NE is observed in cultured PA SMC from idiopathic PA hypertension versus that in control lungs and localizes to neointimal lesions. Thus, PA SMC produce NE, and heightened production and activity of NE is linked to experimental and clinical pulmonary vascular disease.Pulmonary arterial (PA) neointimal lesions are observed in patients with PA hypertension that is idiopathic (IPAH) or associated with other medical conditions. These vascular abnormalities cause narrowing and even obliteration of the vessel lumen and contribute to the progressive increase in pulmonary vascular resistance that can lead to right ventricular failure (reviewed in Ref. ¹). Only a few murine or rodent models recapitulate this pathological feature, eg, mice exposed to ovalbumin or aspergillus² or to schistosomiasis,³ rats treated with the vascular endothelial receptor blocker Sugen 5416, exposed to chronic hypoxia and recovered in room air,⁴ mice that overexpress IL-6 and are subjected to chronic hypoxia,⁵ or mice that overexpress S100A4.⁶ The latter mice, when over 1 year of age, can on rare occasions “spontaneously” develop severe neointimal lesions.⁶ However, these lesions are observed consistently following infection with murine gammaherpesvirus-68 (MHV-68).⁷ S100A4 is also known as metastasin-1 (mts-1), and is a member of the calcium binding family of proteins that clusters on chromosome 1 and that has been related to cancer and inflammation.⁸The S100A4-overexpressing mouse infected with MHV-68 is relevant to clinical PAH. Increased immunoreactivity for S100A4 is observed in vascular lesions in patients with advanced PAH,⁶ and human immunodeficiency virus (HIV) and HHV-8/Kaposi''s sarcoma virus, the human homologue of MHV-68, have been implicated in clinical PAH. Specifically, the viral protein for HHV-8 has been detected in neointimal and plexiform lesions in lung tissues from some,⁹ albeit not all,¹⁰ series of PAH patients.Pulmonary vascular neointimal and plexiform lesions in S100A4 mice are associated with fragmentation of elastic laminae and with heightened activity of a serine elastase.⁷ Fragmentation of PA elastin has been observed in PAs of PAH patients,¹¹ and heightened activity of a serine elastase has been identified in the PA in a variety of experimental forms of PAH^12–15 and in cultured PA smooth muscle cells (SMC).^16–19 Moreover, inhibition of this elastase can attenuate or prevent^12–14 and even reverse¹⁵ experimental pulmonary vascular disease in rodents. In all rodent models where elastase inhibitors were used, the pulmonary vascular lesions were characterized by loss or increased muscularization of normally nonmuscular peripheral arteries at the alveolar wall and duct level, and medial hypertrophy of proximal muscular arteries. Neointimal lesion formation, however, was not present. Elastase inhibition should, however, attenuate these lesions since proliferation and migration of SMC in the neointima are likely the consequences of elastase-mediated release of growth factors from the extracellular matrix^16,17 and activation of growth factor receptors.²⁰We therefore hypothesized, and subsequently demonstrated in this study, that in the S100A4 overexpressing versus C57 mouse, heightened susceptibility of elastin to fragmentation, coupled to elevated serine elastase activity following M1-MHV-68 infection, can contribute to the development of neointimal lesions. We identified the elastase involved as neutrophil elastase (NE) produced by PA SMC, suggesting that it is the endogenous vascular elastase previously related to PAH in other experimental models.^12,16,18,19 Moreover, we showed that NE is produced in significantly greater amounts by cultured murine S100A4 versus C57 PA SMC, and by human PA SMC from IPAH versus control lungs, and we localized NE to neointimal and plexiform lesions in human lung specimens.     

PTEN,PIK3CA,p-AKT,and p-p70S6K Status: Association with Trastuzumab Response and Survival in Patients with HER2-Positive Metastatic Breast Cancer

Phosphatase and tensin homolog (PTEN) is a key modulator of trastuzumab sensitivity in HER2-overexpressing breast cancer. Because PTEN opposes the downstream signaling of phosphoinositide 3-kinase (PI3K), we investigated the role of PTEN and other components of the PI3K pathway in trastuzumab resistance. We analyzed the status of PTEN, p-AKT-Ser473, and p-p70S6K-Thr389 using immunohistochemistry. PIK3CA mutation status was analyzed by direct sequencing. Primary tumor tissue was available from 137 patients with HER2-overexpressing metastatic breast cancer who had received trastuzumab-based chemotherapy. We observed that each of the four biomarkers alone did not significantly correlate with trastuzumab response, whereas PTEN loss alone significantly correlated with shorter survival times (P = 0.023). PI3K pathway activation, defined as PTEN loss and/or PIK3CA mutation, was associated with a poor response to trastuzumab (P = 0.047) and a shorter survival time (P = 0.015). PTEN loss was significantly associated with a poor response to trastuzumab (P = 0.028) and shorter survival time (P = 0.008) in patients who had received first-line trastuzumab and in patients with estrogen receptor- (P = 0.029) and progesterone receptor-negative tumors (P = 0.033). p-AKT-Ser473 and p-p70S6K-Thr389 each had a limited correlation with trastuzumab response. When these markers were combined with PTEN loss, an increased correlation with patient outcome was observed. In conclusion, PI3K pathway activation plays a pivotal role in trastuzumab resistance. Our findings may facilitate the evaluation of tumor response to trastuzumab-based and targeted therapies.Human epidermal growth factor receptor 2 (HER2) is overexpressed in 20% to 25% of invasive breast cancers. Patients with HER2-overexpressing tumors experience a shorter time to relapse and shorter overall survival than patients with tumors of normal HER2 levels.^1,2 HER2 overexpression can lead to activation of many downstream signaling molecules, including Ras-Gap, Src, phosphoinositide 3-kinase (PI3K)/AKT, and many other signaling events.^3,4 Trastuzumab (Herceptin; Genentech, CA), a humanized monoclonal antibody that directly targets the extracellular domain of HER2, has a remarkable therapeutic efficacy in treating patients with HER2-expressing metastatic breast cancer (MBC)⁵ and patients with HER2-positive early-stage disease in adjuvant settings.^6,7 Trastuzumab treatment, when combined with chemotherapy, resulted in a significant improvement in patients'' response rate, time to progression, and duration of response.⁸ The underlying mechanisms of trastuzumab''s antitumor activities include, but are not limited to, inducing antibody-dependent cellular cytotoxicity,⁹ inhibiting HER2 extracellular domain cleavage,¹⁰ activating phosphatase and tensin homolog (PTEN),¹¹ and inhibiting PI3K/AKT survival signaling.¹² However, the overall response rate to trastuzumab is low, and almost half of patients with HER2-positive breast cancer exhibit an initial resistance to trastuzumab-based therapy.^11,13 Despite the large amounts of preclinical and clinical data, the causes of trastuzumab resistance are still poorly understood.¹⁴The PI3K pathway, downstream of HER2, plays a central role in regulating a number of cellular processes, such as apoptosis, migration, angiogenesis, cell proliferation, and glucose metabolism, and it is involved in trastuzumab resistance.^15,16 PI3K phosphorylates phosphatidylinositols on the cell membrane, generating phosphatidylinositol-3,4,5-trisphosphate (PIP3) from phosphatidylinositol-4,5-bisphosphate (PIP2). Then, at the cell membrane, PIP3 recruits protein kinases and activates protein kinase B (PKB)/AKT.¹⁷ In breast cancer cells, HER2 overexpression can lead to activation of the PI3K/AKT pathway.¹⁸ The activation of AKT and its downstream signaling have been demonstrated to inhibit cell cycle arrest and block trastuzumab-mediated apoptosis.¹² AKT phosphorylation and AKT kinase activities were found to be increased in trastuzumab-resistant cells, derived from BT474 HER2-overexpressing breast cancer cells, when compared with parental cells.¹⁹ These data provide insight into the trastuzumab-resistance mechanism of PI3K/AKT signaling.¹⁵Aberrations in the components of the PI3K pathway have been reported in most solid tumors, including breast cancer.¹⁶ PTEN is a tumor suppressor that dephosphorylates the D3 position of PIP3 and inhibits the PI3K/AKT pathway.²⁰ Loss of PTEN function as a result of mutation, deletion, or promoter methylation has been reported in nearly 50% of breast cancers.¹¹ In addition, the gene encoding one of the PI3K catalytic subunits, p110α (PIK3CA), has been found to be mutated in about 25% of breast cancers.^21,22,23 Most of the reported mutations are localized to hotspots in exons 9 and 20 of the PIK3CA gene, which result in increased PI3K pathway signaling.^22,24 We previously discovered that PTEN activation is a novel mechanism of trastuzumab antitumor function, and PTEN loss confers trastuzumab resistance in HER2-overexpressing breast cancer cells.¹¹ PTEN loss significantly predicted poor response to trastuzumab-based therapy in a small cohort of HER2-positive patients with MBC.¹¹ Later, it was reported that both low PTEN levels and PI3K-activating PIK3CA mutations contribute to trastuzumab resistance in HER2-overexpressing breast cancer.^25,26 PTEN loss or PIK3CA mutations, which indicate activation of the PI3K pathway, are considered as markers for poor response to trastuzumab in patients with HER2-overexpressing breast cancer.²⁵ On the other hand, some studies found no correlation between PTEN expression and trastuzumab response or survival in patients with HER2-positive breast cancer.^27,28 These contradictory findings prompted us to further investigate the association between PTEN status and clinical outcomes in a large cohort of patients with MBC who were treated with trastuzumab-based therapy. We tested the hypothesis that a comprehensive assessment of PI3K pathway activation status provides biomarkers that can identify patients who may not benefit from trastuzumab-based therapy.     

Chitinase 3-Like-1 Expression in Colonic Epithelial Cells as a Potentially Novel Marker for Colitis-Associated Neoplasia

Chitinase 3-like-1 (CHI3L1/YKL-40) is a protein secreted from restricted cell types including colonic epithelial cells (CECs) and macrophages. CHI3L1 is an inflammation-associated molecule, and its expression is enhanced in persons with colitis and colon cancer. The biological function of CHI3L1 on CECs is unclear. In this study, we investigated the role of CHI3L1 on CECs during the development of colitis-associated neoplasia. We analyzed colonic samples obtained from healthy persons and from persons with ulcerative colitis with or without premalignant or malignant changes. DNA microarray and RT-PCR analyses significantly increased CHI3L1 expression in non-dysplastic mucosa from patients with inflammatory bowel disease (IBD) who had dysplasia/adenocarcinoma compared with that in healthy persons and in patients with IBD who did not have dysplasia. As determined by IHC, CHI3L1 was expressed in specific cell types in the crypts of colonic biopsies obtained from patients with ulcerative colitis who have remote dysplasia. Purified CHI3L1 efficiently activated the NF-κB signaling pathway and enhanced the secretion of IL-8 and TNF-α in SW480 human colon cancer cells. In addition, colon cancer cell proliferation and migration were significantly promoted in response to CHI3L1 in these cells. In summary, CHI3L1 may contribute to the proliferation, migration, and neoplastic progression of CECs under inflammatory conditions and could be a useful biomarker for neoplastic changes in patients with IBD.Chitinase 3-like-1 (CHI3L1, also known as YKL-40 or HC-gp39) is classified in the glycosyl hydrolases 18 family based on the structural similarity with other chitinases.^1,2 However, functionally, CHI3L1 lacks enzymatic activity and belongs to the family of chi-lectins (chitinase-like lectins) that includes Ym-1³ and stabilin-1-interacting chitinase-like protein.⁴ CHI3L1 is a 40 kDa protein and is produced by restricted cell types, including colonic epithelial cells (CECs) and macrophages.^5–7 CHI3L1 can be detected in the Golgi apparatus and the endoplasmic reticulum,⁸ but its major sites of action seems to be extracellular as a secreted protein.⁹ The secreted form of CHI3L1 has growth-stimulating effects in connective tissue cells, including synoviocytes and chondrocytes.¹⁰ In addition, CHI3L1 shows dose-dependent growth-stimulating effects in human fibroblasts and shows similar and synergistic effects with well-characterized mitogen, insulin-like growth factor 1 (IGF-1).¹¹ However, the exact biological function of CHI3L1 in CECs remains uncertain.It is well documented that elevated levels of CHI3L1 can be detected in the sera of persons with rheumatoid arthritis, bronchial asthma, or inflammatory bowel disease (IBD).^12–15 Serum CHI3L1 is significantly increased in active but not quiescent IBD.^5,9,15 In agreement with this observation, approximately 64% of persons with Crohn''s disease (CD) who have extra-intestinal manifestations such as erythema nodosum and fistulas showed significantly increased serum levels of CHI3L1.^15,16 In addition, patients with CD who had stenotic disease had higher serum CHI3L1 than did patients with non-stenotic disease.¹⁷ Of note, the colonic CHI3L1 mRNA level was increased in persons with active ulcerative colitis (UC) and CD but was in the normal range in persons with quiescent UC and the uninvolved regions of CD.⁵ In addition, CHI3L1 serum concentrations seem to be not only highly up-regulated in persons with active CD and UC but also correlated with poor prognosis of solid tumors, including breast cancer and colon cancer.⁹Patients with chronic IBD have an increased risk of developing colitis-associated cancer, which increases by 0.5% to 1% annually after 10 years of chronic inflammation.¹⁸ A growing amount of evidence indicates that various soluble factors produced by epithelial cells and immune cells play a pathogenic role in the carcinogenic change of CECs.^19,20 CHI3L1 seems to be one of the soluble factors that play a pivotal role in protecting cancer cells from undergoing apoptosis, as well as promoting tissue remodeling by interacting with the extracellular matrix and by stimulating angiogenesis.²¹ However, little is know about the role of CHI3L1 in IBD-associated colon cancer.In this study we show that CHI3L1 expression in CECs is significantly and specifically increased in non-dysplastic mucosa of patients with UC who have dysplasia that is away from the non-dysplastic mucosa (remote dysplasia) as well as colorectal adenocarcinoma; we also show that it may be a reliable biological marker of neoplasia in high-risk individuals. In addition, we demonstrate a new mechanism by which CHI3L1 may contribute to IBD-associated neoplasia through a growth-stimulating effect on enhancing the production of NF-κB–induced IL-8 and tumor necrosis factor (TNF)-α, which presumably are associated with chronic inflammation-mediated malignant transformation in CECs.     

Aberrant CD8+ T-cell responses and memory differentiation upon viral infection of an ataxia-telangiectasia mouse model driven by hyper-activated Akt and mTORC1 signaling

Immune system-related pathology is common in ataxia-telangiectasia (A-T) patients and mice that lack the protein kinase, A-T mutated (ATM). However, it has not been studied how ATM influences immune responses to a viral infection. Using the lymphocytic choriomeningitis virus (LCMV) infection model, we show that ATM^−/− mice, despite having fewer naïve CD8⁺ T cells, effectively clear the virus. However, aberrant CD8⁺ T-cell responses are observed, including defective expansion and contraction, effector-to-memory differentiation, and a switch in viral-epitope immunodominance. T-cell receptor-activated, but not naïve, ATM^−/− splenic CD8⁺ T cells have increased ribosomal protein S6 and Akt phosphorylation and do not proliferate well in response to IL-15, a cytokine important for memory T-cell development. Accordingly, pharmacological Akt or mammalian target of rapamycin complex 1 (mTORC1) inhibition during T-cell receptor activation alone rescues the IL-15 proliferation defect. Finally, rapamycin treatment during LCMV infection in vivo increases the number of memory T cells in ATM^−/− mice. Altogether, these results show that CD8⁺ T cells lacking ATM have hyperactive Akt and mTORC1 signaling in response to T-cell receptor activation, which results in aberrant cytokine responses and memory T-cell development. We speculate that similar signaling defects contribute to the immune system pathology of A-T, and that inhibition of Akt and/or mTORC1 may be of therapeutic value.Ataxia-telangiectasia (A-T) is a human disease caused by mutations in the gene encoding the PI3-kinase-like protein kinase A-T mutated (ATM).¹ A-T is a multifaceted disease with complex pathology. Cerebellar degeneration underlies the hallmark ataxia symptoms, but another prominent issue is immune system-related pathology, including immunodeficiency and lymphoid cancers.² A-T patients commonly acquire hematological malignancies (eg, leukemia and lymphoma) that together with recurrent bronchial infections account for most of the mortality from the disease.³ ATM gene knock-out mouse models of A-T exhibit many features of the human disease,^4–6 including sexual immaturity, immune system defects, hematopoietic stem cell defects, and thymic lymphoma, the latter of which is the most common cause of death in these animals.^4,7Immunodeficiency associated with decreased production of immunoglobulins A, E, and G2, and thymic hypoplasia has been documented in A-T patients.^8,9 The latter involves decreased peripheral CD4⁺ and CD8⁺ T-lymphocyte pools resulting from developmental defects in the thymic microenvironment.⁹ Because ATM is recruited to double-strand breaks, it is likely that defects in the V(D)J recombination process, which results in a block in differentiation at the CD4⁺/CD8⁺ double-positive stage in the thymus, cause lower thymic output of mature CD4⁺ and CD8⁺ cells. This is corroborated by the ability of a functional T-cell receptor (TCR)-αβ transgene to rescue the deficit in peripheral T cells in ATM^−/− mice.¹⁰ Despite the defective thymic development of T cells in A-T patients, the immune function of mature T cells has been reported to be essentially normal.¹¹ However, to date, there have been no studies of how deficiency of ATM affects the response to an infection in A-T patients or in the mouse models of the disease.The best-defined role for ATM is in the nuclear DNA damage response^2,12; however, other functions for ATM have been described.^12,13 For example, ATM is important for mitochondrial homeostasis,^14,15 insulin signaling,^13,16 phosphorylation of 5''-AMP-activated protein kinase (AMPK),^17–19 and activation of Akt.^16,20 In addition, ATM signals to TSC2 in response to reactive oxygen species,²¹ to inhibit mammalian target of rapamycin complex 1 (mTORC1) that itself is regulated by AMPK and Akt.^22–24 Finally, treatment of mice with the mTORC1 inhibitor rapamycin significantly increases the life span of ATM^−/− mice by delaying development of thymic lymphoma.²⁵ Altogether, these results highlight how the loss of ATM might disrupt the integration of signals that feed into the nutrient-sensing mTORC1 pathway.The CD8⁺ T-cell response is a crucial arm of the adaptive immune system. In response to an infection, these cells are activated through the TCR, proliferate, and differentiate into cytotoxic effector cells that kill infected cells. Most of these cells die after clearance of the pathogen, but a subpopulation survives, loses effector cell properties, and become memory T cells.^26,27 Memory T cells are important for fighting recurrent infections, as they are programmed to respond faster and more effectively to the pathogen. The CD8⁺ T-cell response to infection involves differentiation into short-lived effector cells and memory-precursor cells^26,28 that can be monitored based on surface expression of KLRG1 and CD127 markers. Effector CD8⁺ T cells are preferentially represented in the KLRG1^hi population,^29,30 whereas cells that are CD127^hi, which is the receptor for IL-7, and KLRG1^lo preferentially become long-lived memory T cells.^31–33The response of CD8⁺ T cells to TCR activation and the pathways involved in effector and memory cell differentiation are well-documented.³⁴ These include roles of the AMPK and mTORC1 pathways at several levels. For example, TCR activation leads to rapid activation of AMPK in response to Ca²⁺ signaling, presumably in anticipation of the enormous energy demand required for T-cell expansion.³⁵ In addition, we have shown that AMPK/mTORC1 signaling dynamically regulates mitochondrial biogenesis during TCR activation.³⁶ Finally, treatment of mice with the AMPK activator metformin or the mTORC1 inhibitor rapamycin enhances memory T-cell differentiation by boosting fatty acid oxidation.^33,37 Similarly, mTORC1 regulates differentially effector and memory T-cell commitment,³⁸ and it is a negative regulator of memory T-cell differentiation in mice.^33,37,39The goal of this study was to determine how loss of ATM affects normal CD8⁺ T-cell activation and differentiation upon viral infection and to understand how alterations in mTORC1 and related pathways due to lack of ATM might contribute to the immune-related pathology of A-T using ATM^−/− mice as a model of the disease and the well-characterized murine lymphocytic choriomeningitis virus (LCMV) infection paradigm.