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.     

Autophagy Is a Renoprotective Mechanism During in Vitro Hypoxia and in Vivo Ischemia-Reperfusion Injury

Autophagy mediates bulk degradation and recycling of cytoplasmic constituents to maintain cellular homeostasis. In response to stress, autophagy is induced and may either contribute to cell death or serve as a cell survival mechanism. Very little is known about autophagy in renal pathophysiology. This study examined autophagy and its pathological role in renal cell injury using in vitro and in vivo models of ischemia−reperfusion. We found that hypoxia (1% O₂) induced autophagy in cultured renal proximal tubular cells. Blockade of autophagy by 3-methyladenine or small-interfering RNA knockdown of Beclin-1 and ATG5 (two key autophagic genes) sensitized the tubular cells to hypoxia-induced apoptosis. In an in vitro model of ischemia−reperfusion, autophagy was not induced by anoxic (0% O₂) incubation in glucose-free buffer, but was induced during subsequent recovery/reperfusion period. In this model, suppression of autophagy also enhanced apoptosis. In vivo, autophagy was induced in kidney tissues during renal ischemia−reperfusion in mice. Autophagy was not obvious during the ischemia period, but was significantly enhanced during reperfusion. Inhibition of autophagy by chloroquine and 3-methyladenine worsened renal ischemia/reperfusion injury, as indicated by renal function, histology, and tubular apoptosis. Together, the results demonstrated autophagy induction during hypoxic and ischemic renal injury. Under these pathological conditions, autophagy may provide a protective mechanism for cell survival.Autophagy is a cellular process of “self-eating” wherein various cytoplasmic constituents are broken down and recycled through the lysosomal degradation pathway.¹ This process consists of several sequential steps, including sequestration of cytoplasmic portions by isolation membrane to form autophagosome, fusion of the autophagosome with lysosome to create an autolysosome, and degradation of the engulfed material to generate monomeric units such as amino acids.² Identification of the autophagy-related genes (ATG) in yeast and their orthologs in other organisms including mammals demonstrates that autophagy is evolutionarily conserved in all eukaryotic cells. The ATG genes constitute the core molecular machinery of autophagy and function at the different levels to regulate autophagy induction, progression, and completion.¹Autophagy occurs at basal level in most cells and contributes to the turnover of long-lived proteins and organelles to maintain intracellular homeostasis. In response to cellular stress, autophagy is up-regulated and can provide an adaptive strategy for cell survival, but may also directly or indirectly lead to cell demise.^3–6 With the dual role in life and death, autophagy is involved in various physiological processes, and more importantly, linked to the pathogenesis of a wide array of diseases, such as neurodegeneration, cancer, heart disease, aging, and infections.^1,2,6,7 However, it remains largely unknown how autophagy makes the life and death decisions of a stressed cell. Moreover, the conundrum is further complicated by the cross talk and coordinated regulation between autophagy and apoptosis.^4,5,8Despite rapid progress of autophagy research in other organ systems, the role of autophagy in the pathogenesis of renal diseases was not recognized until very recently. In 2007, Chien et al⁹ suggested the first evidence of autophagy during renal ischemia−reperfusion in rats. Subsequent work by Suzuki et al¹⁰ further showed autophagy in ischemic mouse kidneys and notably, in transplanted human kidneys. In nephrotoxic models of acute kidney injury, we and others have demonstrated autophagy during cisplatin nephrotoxicity and have suggested a role for autophagy in renoprotection.^11,12 A prosurvival role of autophagy was also shown in tubular cells during cyclosporine A nephrotoxicity.¹³ In contrast, Gozuacik et al¹⁴ suggested that autophagy may serve as a second cell killing mechanism that acts in concert with apoptosis to trigger kidney damage in tunicamycin-treated mice. A cell killing role for autophagy was also suggested by Suzuki et al¹⁰ during H₂O₂-induced renal tubular cell injury. As a result, whether autophagy is a mechanism of cell death or survival in renal pathology remains unclear.In this study, we have determined the role of autophagy in renal tubular cell injury using in vitro and in vivo models of renal ischemia−reperfusion. We show that autophagy is induced in these models. Importantly, blockade of autophagy sensitizes renal cells and tissues to injury by hypoxia and ischemia−reperfusion, suggesting a prosurvival role for autophagy.     

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.     

Interleukin-11 in Endometrial Adenocarcinoma Is Regulated by Prostaglandin F2α-F-Prostanoid Receptor Interaction via the Calcium-Calcineurin-Nuclear Factor of Activated T Cells Pathway and Negatively Regulated by the Regulator of Calcineurin-1

Interleukin-11 (IL-11) up-regulates the proliferative and invasive capacity of many cancers. Coexpression of glycoprotein 130 (GP130) and IL-11 receptor α (IL-11Rα) is necessary for high-affinity binding of IL-11 to IL-11Rα. This study investigated the expression of IL-11 and role of prostaglandin F_2α-F-prostanoid receptor (FP receptor) signaling in the modulation of IL-11 expression in endometrial adenocarcinoma cells. Localization of IL-11, IL-11Rα, and GP130 expression was performed by immunohistochemistry. IL-11 and regulator of calcineurin 1 isoform 4 (RCAN1-4) mRNA and protein expression were determined by real-time RT-PCR and/or enzyme-linked immunosorbent assay/Western blot analysis using Ishikawa endometrial adenocarcinoma cells stably expressing the FP receptor (FPS cells) and endometrial adenocarcinoma explants. IL-11 mRNA expression was significantly elevated in endometrial adenocarcinoma samples compared with normal endometrium and increased with tumor grade. IL-11 protein expression localized with FP receptor, IL-11Rα, and GP130 in the neoplastic glandular epithelium of endometrial adenocarcinomas. Prostaglandin F_2α-FP receptor signaling significantly elevated the expression of IL-11 mRNA and protein in a Gq-protein kinase C-calcium-calcineurin-nuclear factor of activated T cells-dependent manner in FPS cells. The calcineurin signaling pathway is known to be controlled by the RCAN (RCAN1-4). Indeed, RCAN1-4 expression was significantly elevated in well-differentiated endometrial adenocarcinoma compared with normal endometrium and was found to decrease with tumor grade and negatively regulate IL-11 expression in vitro. This study has highlighted a new mechanism regulating IL-11 expression in endometrial adenocarcinoma cells by the FP receptor via the calcium-calcineurin-nuclear factor of activated T cells pathway.Endometrial cancer is the most common female gynecological malignancy in the Western world, ranking fourth in incidence among invasive tumors in women.^1–3 Most cases of endometrial carcinomas are sporadic estrogen-dependent disorders that occur in pre- or postmenopausal women as low-grade (well differentiated, type I) endometrioid adenocarcinomas.⁴ However, ∼20% of tumors in postmenopausal women are not estrogen dependent and have a poor prognosis.^1–4 In these patients, predominantly high-grade (poorly differentiated, type II) tumors arise either as endometrioid adenocarcinomas, uterine papillary serous carcinomas, or clear cell carcinomas with a high frequency of myometrial invasion and spread into the pelvic lymph nodes.⁵Although the mechanisms regulating endometrial adenocarcinomas are still poorly defined, there is much evidence for a role for cyclooxygenase (COX) enzymes and prostaglandins (PGs) in uterine pathology. We and others^6,7 have demonstrated elevated expression of COX-2, biosynthesis of PG, and elevated expression of nuclear⁶ and membrane-bound G protein-coupled receptors^6,8 like the F-prostanoid (FP) receptor⁸ in endometrial adenocarcinomas. Moreover, we have shown that elevated PGF_2α-FP receptor signaling, via the Gq activation of inositol-1,4,5-trisphosphate, leads to up-regulation of tumorigenic and angiogenic genes including COX-2,⁹ fibroblast growth factor 2,¹⁰ and vascular endothelial growth factor,¹¹ indicating that PGF_2α-FP receptor signaling can promote endometrial tumor growth by regulating vascular function. Furthermore, FP receptor can regulate the proliferation of endometrial epithelial cells and can alter their adhesiveness to extracellular matrix and motility via the reorganization of the actin cytoskeleton and activation of focal adhesion kinase.^8,12,13 These findings suggest that PGF_2α-FP receptor signaling plays a multifactorial role in regulating endometrial adenocarcinoma by promoting an environment for angiogenesis and tissue remodeling to facilitate tumor growth.In addition to the regulation of cell architecture and growth factors by the COX-PG axis, a link between PG and chemoattractive cytokines (chemokines) such as CXCL1 has been demonstrated in colorectal cancer,¹⁴ where PGE₂ signaling has been shown to induce CXCL1 expression in colorectal cancer cells to enhance tumor growth. Similarly in COX-2-overexpressing breast cancer cells that had metastasized to bone, Singh et al¹⁵ have shown recently that the pleiotropic cytokine interleukin-11 (IL-11) is significantly elevated.¹⁵ IL-11 mediates its function via the IL-11 receptor α (IL-11Rα). On ligand binding to IL-11Rα, the glycoprotein (GP) 130 subunit, critical for signal transduction of IL-11, is recruited to form a IL-11/IL-11Rα/GP130 complex.¹⁶ Once activated, the IL-11/IL-11Rα/GP130 complex can activate signal transduction pathways¹⁷ to modulate target gene expression. IL-11 and IL-11Rα expression has been shown to correlate with cellular growth, differentiation, invasiveness, tumor progression, and poor prognosis in breast and colorectal cancer;^18–20 however, the expression and regulation of IL-11 in endometrial cancer has yet to be reported.Here we investigated the expression profile of IL-11, IL-11Rα, and GP130 in endometrial adenocarcinomas compared with normal proliferative-phase endometrium and its regulation in an in vitro model of endometrial adenocarcinoma cells by PGF_2α via the FP receptor.     

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.     

Inhibiting angiotensin-converting enzyme promotes renal repair by limiting progenitor cell proliferation and restoring the glomerular architecture

We previously reported that angiotensin-converting enzyme inhibitor (ACEi) renoprotection in Munich Wistar Frömter (MWF) rats, which develop progressive glomerular injury, was associated with podocyte repopulation and preservation of glomerular architecture. Here, we studied the time course of the lesions, their cellular components, and the effect of ACEi. Early glomerular lesions were synechiae, followed by extracapillary crescents and glomerulosclerosis. The majority of cells forming crescents were claudin1⁺ parietal epithelial cells and, to a lesser extent, WT1⁺ podocytes, both in active proliferation. In crescents, cells expressing the metanephric mesenchyme marker NCAM were also found. Three distinct populations of parietal epithelial cells were identified in the rat Bowman''s capsule: NCAM⁺WT1⁻ cells, also expressing progenitor cell marker CD24, and NCAM⁺WT1⁺ and NCAM⁻WT1⁺ cells, the latter population representing parietal podocytes. After exposure to inductive medium, cultured parietal epithelial cells that were obtained by capsulated glomeruli generated podocytes, documenting their progenitor nature. Mitotic activity of cultured renal progenitors was induced by angiotensin II through the down-regulation of cell cycle inhibitor C/EBPδ expression. Treatment with ACEi reduced number and extension of crescents and glomerulosclerosis in MWF rats. Renoprotection was accomplished through the limitation of NCAM⁺ progenitor proliferation via the modulation of C/EBPδ. Thus, chaotic migration and proliferation of the Bowman''s capsule progenitor cells pave the way to crescent formation and subsequent sclerosis. ACEi, by moderating progenitor cell activation, restores glomerular architecture and prevents renal disease progression.Chronic kidney disease is a worldwide threat to public health. Estimates report that diseases of the kidney account for 830,000 global deaths every year, with a sharp rise of renal replacement therapy now exceeding 2 million patients for an aggregate cost of more than US$1 trillion.¹ The burden of chronic kidney disease is not limited to demand of renal replacement therapies, but chronic kidney disease is also a major determinant of cardiovascular diseases, with direct impact on the health of the overall population.² Since there are no specific treatments for most chronic nephropathies so far, efforts aimed at preventing renal disease progression are mandatory. Studies have documented that progressive renal function deterioration is the result of compensatory glomerular hemodynamic changes in response to nephron loss. A key player is angiotensin II³ to the extent that angiotensin-converting enzyme inhibitors (ACEi) or angiotensin II receptor antagonists slow the development of proteinuria and limit renal damage in animals.^4,5 Robust clinical evidence of remission and regression of renal disease in humans⁶ was clarified by subsequent animal studies. By three-dimensional reconstruction of glomerular capillary tufts based on kidney serial section analysis, we found that after 10 weeks of ACEi treatment in Munich Wistar Frömter (MWF) rats, studied at 60 weeks, more that 30% of glomeruli were completely free of sclerosis, whereas all glomeruli of 50-week-old untreated MWF rats had some degree of scarring.⁷ This approach did not identify glomerular cellular components.⁸ Regression of glomerulosclerosis and neoformation of glomerular tissue has been linked to progenitor/stem cells of renal or extrarenal origin.⁹ As a follow-up of previous studies,⁷ we recently showed that, in MWF rats, ACEi halted the spontaneous podocyte loss¹⁰ and restored podocyte number. Consistently, others documented recruitment of podocytes from glomerular parietal epithelium toward the capillary tuft.¹¹ A population of progenitor cells localized within the Bowman''s capsule has been recently found in human adult kidney.¹² These cells can regenerate podocytes.¹³ Renal progenitors and transitional cells—progenitor cells that additionally expressed podocyte markers—were also detected within hyperplastic lesions of human glomerulopathies.¹⁴ Mechanisms and cellular determinants of progressive nephropathies in the context of recent findings of glomerular epithelial cell activation had never been addressed in systematic fashion. Inhibiting ACE can be a selective way to potentiate the regeneration of the glomerulus, shifting the process toward kidney healing. To this end, the spontaneous glomerulopathy of MWF rats represents the most appropriate model in which to study the cellular basis for glomerular restructuring and repair. Here, we first sought to establish whether a population of progenitor cells actually exists in the rat glomerulus. We then evaluated whether renal injury in MWF could be the consequence of aberrant progenitor cell proliferation, and to what extent renoprotection by ACEi occurred via an effect of moderating progenitor cell migration and proliferation to restore the Bowman''s capsule architecture.     

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.     

Knockdown of Osteopontin Reduces the Inflammatory Response and Subsequent Size of Postsurgical Adhesions in a Murine Model

Adhesions between organs after abdominal surgery remain a significant unresolved clinical problem, causing considerable postoperative morbidity. Osteopontin (OPN) is a cytokine up-regulated after cell injury and tissue repair. Our previous studies have shown that blocking OPN expression at sites of cutaneous wounding resulted in reduced granulation tissue and scarring. We hypothesize that it may be possible to similarly reduce inflammation-associated fibrosis that causes small-bowel adhesions after abdominal surgery. By using a mouse model, we deliver OPN antisense oligodeoxynucleotides via Pluronic gel to the surface of injured, juxtaposed small bowel and show a significant reduction of inflammatory cell influx to the developing adhesion and a dramatic reduction in the resulting adhesion size. A significant reduction in α-smooth muscle actin expression and collagen deposition within the mature adhesion is also demonstrated. We see no impact on mortality, and the healing of serosal injury to intact bowel appeared normal given the reduced inflammatory response. Our studies suggest that dampening OPN levels might be a potentially important target for anti-adhesion therapeutics.The peritoneum is an extensive and complex organ consisting of a layer of mesothelial cells lining the peritoneal cavity and all organs within it.¹ One of the main functions of the peritoneum is to allow friction-free movement between abdominal viscera and the peritoneal wall.² Any surgery that breaches the peritoneal lining causes injury to the peritoneum, which responds by raising inflammatory signals that attract innate immune cells in parallel with a wound repair response and subsequent fibrosis.^3–5 This almost invariably results in permanent peritoneal adhesion formation.⁶ The result can be tethering of adjacent small-bowel loops that may lead to abdominal pain⁷ and/or bowel obstruction,⁸ which is a significant cause of postoperative morbidity in clinical practice. Readmission rates secondary to adhesional complications are as high as 5% to 10% after abdominal surgery.^9,10 Adhesion prevention options in clinical practice are limited to either barrier methods¹¹ or flotation fluids,¹² which use the concept of keeping damaged peritoneal surfaces separated during their healing process; however, these options are of limited effectiveness.^13,14 Pathophysiological manipulation of the cascade events leading to fibrosis has been investigated,^15–18 but none has led to a clinically usable product. Herein, we investigate whether therapeutic strategies used to block scar formation after skin healing might also be effective during peritoneal repair. Microarray studies of wound tissues from wild-type mice versus PU.1 mice (lacking neutrophils, macrophages, and mast cells) reveal an inflammation-dependent gene, osteopontin (OPN), that is expressed by wound granulation tissue fibroblasts, coincident with a skin wound inflammatory response.^19,20 PU.1 mice heal skin wounds without the standard inflammatory cascade, which results in less fibrosis and scarring at the healed wound site.¹⁹ OPN acts both as a secreted chemokine-like protein and as part of an intracellular signaling complex.²¹ It plays key roles in several processes associated with tissue repair, including cell adhesion, migration, and survival.^21,22 Short-term local knockdown of OPN in cutaneous wounds leads to decreased granulation tissue and reduced scar formation.²³ In this study, we investigate whether these effects are transferable to peritoneal repair and also might block i.p. fibrosis.     

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.     

Regulation of IL-17 Family Members by Adrenal Hormones During Experimental Sepsis in Mice

Severe sepsis is a life-threatening disease that causes major morbidity and mortality. Catecholamines and glucocorticoids often have been used for the treatment of sepsis. Several recent studies have suggested a potential role of IL-17 during the development and progression of sepsis in small animal models. In this study, the cross-talk of catecholamines and glucocorticoids with members of the IL-17 family was investigated during sepsis in C57BL/6 mice. The concentrations in plasma of IL-17A, IL-17F, and the IL-17AF heterodimer all were increased greatly in mice after endotoxemia or cecal ligation and puncture as compared with sham mice. Surprisingly, when compared with IL-17A (487 pg/mL), the concentrations of IL-17F (2361 pg/mL) and the heterodimer, IL-17AF (5116 pg/mL), were much higher 12 hours after endotoxemia. After surgical removal of the adrenal glands, mice had much higher mortality after endotoxemia or cecal ligation and puncture. The absence of endogenous adrenal gland hormones (cortical and medullary) was associated with 3- to 10-fold higher concentrations of IL-17A, IL-17F, IL-17AF, and IL-23. The addition of adrenaline, noradrenaline, hydrocortisone, or dexamethasone to lipopolysaccharide-activated peritoneal macrophages dose-dependently suppressed the expression and release of IL-17s. The production of IL-17s required activation of c-Jun-N-terminal kinase, which was antagonized by both catecholamines and glucocorticoids. These data provide novel insights into the molecular mechanisms of immune modulation by catecholamines and glucocorticoids during acute inflammation.More than 600,000 cases of sepsis occur annually in the United States, with mortality rates ranging from 20% to 30%.^1,2 The pathophysiology of sepsis is poorly understood. Sepsis often involves the spread of a localized infection (bacterial, fungal), resulting in the acute systemic inflammatory response syndrome with mediator release, followed by lymphocyte apoptosis and accompanying immunosuppression.^3–6 We and others recently reported on the involvement of IL-17 during sepsis.^7–9 The first described IL-17 family member, IL-17A, is a dimeric, glycosylated protein (35 kDa) that originally was described as a product from activated T cells.¹⁰ Several other isoforms (IL-17s) also are known, with IL-17F displaying the highest homology (approximately 40%) to IL-17A.^11,12 Notably, IL-17A and IL-17F can form either disulfide-linked homodimers or heterodimers (IL-17AF). Recent data indicate a 10- to 30-fold higher biological potency for IL-17A as compared with IL-17F, with IL-17AF displaying intermediate activity.¹³ Several studies have suggested that IL-17 family members are not derived exclusively from CD4⁺ T cells (Th17); they also are expressed by a broad spectrum of immune and inflammatory cells, including polymorphonuclear leukocytes, macrophages, monocytes, lymphocyte tissue-inducer cells, invariant natural killer T cells, γδT cells, and Paneth cells.^7,8,14–16 In a recent study using IL-17A reporter mice, γδT cells were the predominant source of this cytokine during Pseudomonas pneumonia.¹⁷ In vivo neutralization of γδT cell–derived IL-17A improved survival in polymicrobial sepsis induced by cecal ligation and puncture (CLP).⁸ Likewise, blockade of IL-17A was protective after lethal endotoxemia in mice.⁷ In Bacteroides fragilis infection, neutralization of IL-17A prevented the formation of abscesses.¹⁸ On the other hand, peritoneal clearance of Escherichia coli was impaired by neutralization of IL-17A, indicating that certain levels of IL-17A are protective during the early phases of infection.⁹ Collectively, accumulating evidence suggests a role of IL-17 family members during sepsis and related diseases. However, it also is clear that these cytokines have important functions in mediating immune defenses against infections.Currently, no Food and Drug Administration–approved drugs are available to improve the outcome of sepsis. Besides modern interventions of critical care medicine (low tidal volume mechanical ventilation, fluid resuscitation, and so forth), low-dose glucocorticoids and catecholamines have been used as supportive treatments for patients with sepsis.¹⁹ Here, we report on the immune modulatory functions of glucocorticoids and catecholamines related to the appearance of IL-17 isoforms during two experimental models of sepsis in mice.     

On the Etiology of Type 1 Diabetes: A New Animal Model Signifying a Decisive Role for Bacteria Eliciting an Adverse Innate Immunity Response

The cause of type 1 diabetes (T1D) remains unknown; however, a decisive role for environmental factors is recognized. The increased incidence of T1D during the last decades, as well as regional differences, is paralleled by differences in the intestinal bacterial flora. A new animal model was established to test the hypothesis that bacteria entering the pancreatic ductal system could trigger β-cell destruction and to provide new insights to the immunopathology of the disease. Obtained findings were compared with those present in two patients dying at onset of T1D. Different bacterial species, present in the human duodenum, instilled into the ductal system of the pancreas in healthy rats rapidly induced cellular infiltration, consisting of mainly neutrophil polymorphonuclear cells and monocytes/macrophages, centered around the pancreatic ducts. Also, the islets of Langerhans attracted polymorphonuclear cells, possibly via release of IL-6, IL-8, and monocyte chemotactic protein 1. Small bleedings or large dilatations of the capillaries were frequently found within the islets, and several β-cells had severe hydropic degeneration (ie, swollen cytoplasm) but with preserved nuclei. A novel rat model for the initial events in T1D is presented, revealing marked similarities with the morphologic findings obtained in patients dying at onset of T1D and signifying a decisive role for bacteria in eliciting an adverse innate immunity response. The present findings support the hypothesis that T1D is an organ-specific inflammatory disease.Our understanding of the etiology of type 1 diabetes (T1D) remains limited and originates to a large extent from two animal models: the nonobese diabetic mouse and the BioBreeding-diabetes prone rat.¹ In both models a progressive T-cell–mediated destruction of the β-cells occurs; however, the immunopathologic findings reveal limited similarities with the human disease.^2–5 In human pancreatic specimens, insulitis is discrete, affects only a few islets, and is heterogeneously distributed within the gland. In a recent meta-analysis, insulitis was reported in only 29% of patients with onset between 15 and 39 years of age and with a disease duration of <1 month.⁶ At the time of diagnosis, autoantibodies were only present in approximately 70% to 80% of affected patients.⁷ Likewise, attempts to prevent disease progression with immunosuppression^8–11 or immunointerventions^12–14 cause no or only transient preservation of β-cell function.The fact that the exocrine pancreas gets affected in patients with T1D is underappreciated, and several studies have found autoantibodies in the exocrine cells before the onset of T1D.^15–18 Mild to moderate exocrine pancreatic insufficiency is an early event in T1D,¹⁹ and a substantial reduction (32%) in pancreatic volume is already present 3 to 4 months after onset.²⁰ Also, in the classic report by Gepts,⁴ lesions of the acinar tissue were reported to occur frequently in patients with recent onset of T1D. The findings comprised mostly focal or diffuse lesions of acute pancreatitis with accumulation of leukocytes, often centered around the excretory canals.^2–5 In a more recent study of patients with long-term T1D, 40% had periductal fibrosis and 60% of cases had periductal fibrosis that extended to the interlobular pancreas.²¹ Collectively, these observations suggest that the injurious process that causes T1D affects both the exocrine and endocrine components of the pancreas and challenge the view that T1D is a β-cell–specific autoimmune disease.The low concordance rate for the development of T1D in identical twins and the current rapid increase in incidence of T1D argue against a decisive role for genetic factors. Notably, there is a close to sixfold gradient in the incidence of T1D between Russian and Finland Karelia, although the population is homogenous and the predisposing HLA genotypes are equally frequent.²² In addition, children born in Finland by immigrants from Somalia, a low incidence country for T1D, acquire the same risk for T1D as the native Finish population.²³ On the basis of these and similar observations, it is generally assumed that environmental factors may act as triggers of T1D. For decades different enteroviruses have been implicated in the pathogenesis of T1D²⁴; however, evidence of causality remains missing.Bacterial colonization of the infantile gut is influenced by environmental factors and has changed markedly in developed countries during the last decades.²⁵ Interestingly, the increased incidence of T1D²⁶ and the difference in incidence of T1D in Sweden, Italy, and Africa^26–28 are paralleled by reported frequencies of intestinal Staphylococcus aureus.^29–32 Bacteria entering the ductal system of the pancreas would be exposed to the pancreatic juice–containing substances, with marked antibacterial effects initiating release of bacterial components, such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), and various toxins. Notably, these substances have been implicated in the etiology of neurogenerative diseases and neural cell death because they stimulate microglia to produce proinflammatory cytokines (IL-1b, IL-6, tumor necrosis factor-α), nitric oxide, and reactive oxygen species, causing significant cell death in neighboring neural cells.³³The present study was conducted to establish an animal model for the initial events in T1D to test the hypothesis that bacteria entering into the ductal system of the pancreas could elicit an adverse innate immunity response. Different bacterial species present transiently or continuously in the human duodenum were instilled into the ductal system of the pancreas in healthy rats. To examine the clinical relevance of the experimental model, obtained findings were compared with those present in the pancreases of two patients dying at onset of T1D.     

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.     

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.     

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.