Optimal resource allocation in colonoscopy: timing of follow-up colonoscopies in relation to adenoma detection rates.

BACKGROUND AND STUDY AIMS: The assessment of indications for follow-up colonoscopy may help to improve the allocation of available endoscopy resources. The aim of this study was to assess the timing of early follow-up colonoscopy and surveillance utilization in relation to adenoma detection rate (ADR) at follow-up. METHODS: An assessment of the timing and yield of follow-up colonoscopies was performed in patients with non-inflammatory bowel disease (IBD) in a Dutch multicenter study. The primary outcome was the number of patients with a prior (index) colonoscopy. The necessity for follow-up procedures was assessed using the ADR. RESULTS: Of 4800 consecutive patients undergoing a colonoscopy, 1249 non-IBD patients had undergone an index colonoscopy. Of these, follow-up procedures were performed within 1 year in 27 % (331/1249). Excluding incomplete colonoscopy, incomplete polypectomy, or poor bowel preparation on index, the ADR on early follow-up was 4 % for symptomatic and 26 % for asymptomatic patients. Among the asymptomatic patients with a follow-up colonoscopy at > 1 year (n = 463), an ADR of 23 % (108/463) was found. In 27 % of these patients, the observed surveillance intervals were in accordance with American Gastroenterological Association (AGA) surveillance recommendations; 60 % were classified as over-utilization and 13 % as under-utilization according to the AGA. Optimal utilization follow-up colonoscopies had higher ADRs on follow-up compared with over-utilized procedures (31 % vs. 17 %; P < 0.001). CONCLUSIONS: Follow-up colonoscopy in symptomatic patients within a year has limited value in terms of adenoma detection. A considerable proportion of surveillance colonoscopies are performed too early according to current guidelines, resulting in low detection rates. Both aspects can be targeted for optimal usage in endoscopic capacity.

Lysophosphatidic acid signalling.

Lysophosphatidic acid is an intercellular phospholipid messenger that is released from platelets (and probably other cells) and evokes multiple biological responses, ranging from induction of mitogenesis to neurite retraction, by activating a specific G protein coupled receptor. Recent studies indicate that the lysophosphatidic acid receptor acts via the small GTP-binding proteins Ras and Rho to stimulate cell proliferation and to trigger actin-based cytoskeletal events, respectively.

Lysophosphatidic acid: G-protein signalling and cellular responses.

Lysophosphatidic acid (LPA) is a serum-borne phospholipid that activates a specific G protein coupled receptor to evoke multiple cellular responses. Recent work has identified two cDNAs encoding putative LPA receptors, various LPA-like agonists that act on distinct receptors, and new pathways that link the receptor(s) to such diverse events as Ras signalling, cytoskeletal remodelling and membrane depolarization.

LPA: a novel lipid mediator with diverse biological actions.

Lysophosphatidic acid (LPA), the smallest and structurally simplest phospholipid, is a platelet-derived serum factor that evokes a wide range of biological effects, including stimulation of fibroblast proliferation, platelet aggregation, cellular motility, tumour cell invasiveness and neurite retraction. This review summarizes recent insights into the mode of action of LPA. LPA appears to activate its own G-protein-coupled receptor(s) to initiate both classic and novel signal cascades. Of particular interest is LPA's ability to activate the Ras pathway and to stimulate protein tyrosine phosphorylation in concert with remodelling of the actin cytoskeleton.

Thrombin receptor activation causes rapid neural cell rounding and neurite retraction independent of classic second messengers.

The protease thrombin is a potent activator of various cell types. Thrombin cleaves and thereby activates its own seven-transmembrane-domain receptor which couples to G proteins. Thrombin also can inhibit neuronal differentiation, supposedly by degrading components of the extracellular matrix. Here we report that active thrombin induces immediate cell rounding and neurite retraction in differentiating N1E-115 and NG108-15 neural cells in serum-free culture. Serum (0.5-5% vol/vol) evokes similar responses, but the cell-rounding and neurite-retracting activity of serum is not attributable to thrombin. Neural cell rounding is transient, subsiding after 10-15 min, and subject to homologous desensitization, whereas retracted neurites rapidly degenerate. Thrombin action is inhibited by cytochalasin, but not colchicine. A novel 14-amino acid peptide agonist of the thrombin receptor fully mimics thrombin's morphoregulatory activity, indicating that thrombin-induced shape changes are receptor-mediated and not secondary to extracellular matrix degradation. Although thrombin receptors couple to phosphoinositide hydrolysis and Ca2+ mobilization, thrombin-induced shape changes appear to depend neither on the Ca2+/protein kinase C- nor the cyclic nucleotide-mediated signal transduction pathways; however, the morphological response to thrombin is blocked by pervanadate, an inhibitor of tyrosine phosphatases, and by broad-specificity kinase inhibitors. Our results suggest that the thrombin receptor communicates to an as-yet-uncharacterized effector to reorganize the actin cytoskeleton and to reverse the differentiated phenotype of neural cells.

Lack of association between receptor protein tyrosine phosphatase RPTP mu and cadherins.

RPTP mu is a receptor-like protein tyrosine phosphatase that mediates homophilic cell-cell interactions. Surface expression of RPTP mu is restricted to cell-cell contacts and is upregulated with increasing cell density, suggesting a role for RPTP mu in contact-mediated signaling. It was recently reported (Brady-Kalnay, S.M., D.L. Rimm, and N.K. Tonks. 1995. J. Cell Biol. 130:977-986) that RPTP mu binds directly to cadherin/catenin complexes, and thus may regulate the tyrosine phosphorylation of such complexes. Here we report that this concept needs revision. Through reciprocal precipitations using a variety of antibodies against RPTP mu, cadherins, and catenins, we show that RPTP mu does not interact with cadherin/catenin complexes, even when assayed under very mild lysis conditions. We find that the anti-RPTP mu antiserum used by others precipitates cadherins in a nonspecific manner independent of RPTP mu. We conclude that, contrary to previous claims, RPTP mu does not interact with cadherin complexes and thus is unlikely to directly regulate cadherin/catenin function.

Inhibition of lysophosphatidate- and thrombin-induced neurite retraction and neuronal cell rounding by ADP ribosylation of the small GTP-binding protein Rho.

Addition of the bioactive phospholipid lysophosphatidic acid (LPA) or a thrombin receptor-activating peptide (TRP) to serum-starved N1E-115 or NG108-15 neuronal cells causes rapid growth cone collapse, neurite retraction, and transient rounding of the cell body. These shape changes appear to be driven by receptor-mediated contraction of the cortical actomyosin system independent of classic second messengers. Treatment of the cells with Clostridium botulinum C3 exoenzyme, which ADP-ribosylates and thereby inactivates the Rho small GTP-binding protein, inhibits LPA- and TRP-induced force generation and subsequent shape changes. C3 also inhibits LPA-induced neurite retraction in PC12 cells. Biochemical analysis reveals that the ADP-ribosylated substrate is RhoA. Prolonged C3 treatment of cells maintained in 10% serum induces the phenotype of serum-starved cells, with initial cell flattening being followed by neurite outgrowth; such C3-differentiated cells fail to retract their neurites in response to agonists. We conclude that RhoA is essential for receptor-mediated force generation and ensuing neurite retraction in N1E-115 and PC12 cells, and that inactivation of RhoA by ADP-ribosylation abolishes actomyosin contractility and promotes neurite outgrowth.

Cell surface expression of receptor protein tyrosine phosphatase RPTP mu is regulated by cell-cell contact.

RPTP mu is a transmembrane protein tyrosine phosphatase with an adhesion molecule-like ectodomain. It has recently been shown that RPTP mu mediates homophilic interactions when expressed in insect cells. In this study, we have examined how RPTP mu may function as a cell contact receptor in mink lung epithelial cells, which express RPTPmu endogenously, as well as in transfected 3T3 cells. We find that RPTP mu has a relatively short half-life (3-4 hours) and undergoes posttranslational cleavage into two noncovalently associated subunits, with both cleaved and uncleaved molecules being present on the cell surface (roughly at a 1:1 ratio); shedding of the ectodomain subunit is observed in exponentially growing cells. Immunofluorescence analysis reveals that surface expression of RPTPmu is restricted to regions of tight cell-cell contact. RPTPmu surface expression increases significantly with increasing cell density. This density-induced upregulation of RPTP mu is independent of its catalytic activity and is also observed when transcription is driven by a constitutive promoter, indicating that modulation of RPTPmu surface expression occurs posttranscriptionally. Based on our results, we propose the following model of RPTP mu function: In the absence of cell-cell contact, newly synthesized RPTP mu molecules are rapidly cleared from the cell surface. Cell-cell contact causes RPTPmu to be trapped at the surface through homophilic binding, resulting in accumulation of RPTP mu at intercellular contact regions. This contact-induced clustering of RPTPmu may then lead to tyrosine dephosphorylation of intracellular substrates at cell-cell contacts.

Identification of a novel, putative Rho-specific GDP/GTP exchange factor and a RhoA-binding protein: control of neuronal morphology.

The small GTP-binding protein Rho has been implicated in the control of neuronal morphology. In N1E-115 neuronal cells, the Rho-inactivating C3 toxin stimulates neurite outgrowth and prevents actomyosin-based neurite retraction and cell rounding induced by lysophosphatidic acid (LPA), sphingosine-1-phosphate, or thrombin acting on their cognate G protein-coupled receptors. We have identified a novel putative GDP/GTP exchange factor, RhoGEF (190 kD), that interacts with both wild-type and activated RhoA, but not with Rac or Cdc42. RhoGEF, like activated RhoA, mimics receptor stimulation in inducing cell rounding and in preventing neurite outgrowth. Furthermore, we have identified a 116-kD protein, p116(Rip), that interacts with both the GDP- and GTP-bound forms of RhoA in N1E-115 cells. Overexpression of p116(Rip) stimulates cell flattening and neurite outgrowth in a similar way to dominant-negative RhoA and C3 toxin. Cells overexpressing p116(Rip) fail to change their shape in response to LPA, as is observed after Rho inactivation. Our results indicate that (a) RhoGEF may link G protein-coupled receptors to RhoA activation and ensuing neurite retraction and cell rounding; and (b) p116(Rip) inhibits RhoA-stimulated contractility and promotes neurite outgrowth.

Acute loss of cell-cell communication caused by G protein-coupled receptors: a critical role for c-Src.

Gap junctions mediate cell-cell communication in almost all tissues, but little is known about their regulation by physiological stimuli. Using a novel single-electrode technique, together with dye coupling studies, we show that in cells expressing gap junction protein connexin43, cell-cell communication is rapidly disrupted by G protein-coupled receptor agonists, notably lysophosphatidic acid, thrombin, and neuropeptides. In the continuous presence of agonist, junctional communication fully recovers within 1-2 h of receptor stimulation. In contrast, a desensitization-defective G protein-coupled receptor mediates prolonged uncoupling, indicating that recovery of communication is controlled, at least in part, by receptor desensitization. Agonist-induced gap junction closure consistently follows inositol lipid breakdown and membrane depolarization and coincides with Rho-mediated cytoskeletal remodeling. However, we find that gap junction closure is independent of Ca2+, protein kinase C, mitogen-activated protein kinase, or membrane potential, and requires neither Rho nor Ras activation. Gap junction closure is prevented by tyrphostins, by dominant-negative c-Src, and in Src-deficient cells. Thus, G protein-coupled receptors use a Src tyrosine kinase pathway to transiently inhibit connexin43-based cell-cell communication.

Ionic responses and growth stimulation induced by nerve growth factor and epidermal growth factor in rat pheochromocytoma (PC12) cells.

Rat pheochromocytoma cells (clone PC12) respond to nerve growth factor (NGF) by the acquirement of a phenotype resembling neuronal cells. In an earlier study we showed that NGF causes an increase in Na+,K+ pump activity, as monitored by ouabain-sensitive Rb+ influx. Here we show that addition of epidermal growth factor (EGF) to PC12 cells resulted in a stimulation of Na+,K+ pump activity as well. The increase of Na+,K+ pump activity by NGF or EGF was due to increased Na+ influx. This increased Na+ influx was sensitive to amiloride, an inhibitor of Na+,H+ exchange. Furthermore, no changes in membrane potential were observed upon addition of NGF or EGF. Amiloride-sensitive Na+,H+ exchange in PC12 cells was demonstrated by H+ efflux measurements and the effects of weak acids on Na+ influx. These observations suggest that both NGF and EGF activate an amiloride-sensitive, electroneutral Na+,H+ exchange mechanism in PC12 cells. These findings were surprising in view of the opposite ultimate biological effects of NGF and EGF, e.g., growth arrest vs. growth stimulation. However, within 24 h after addition, NGF was found to stimulate growth of PC12 cells, comparable to EGF. In the presence of amiloride, this stimulated growth by NGF and EGF was abolished. In contrast, amiloride did not affect NGF-induced neurite outgrowth of PC12 cells. From these observations it is concluded that in PC12 cells: (a) NGF has an initial growth stimulating effect; (b) neurite outgrowth is independent of increased amiloride-sensitive Na+ influx; and (c) growth stimulation by NGF and EGF is associated with increased amiloride-sensitive Na+ influx.

High-affinity epidermal growth factor binding is specifically reduced by a monoclonal antibody, and appears necessary for early responses.

We have tested the effects of an mAb directed against the protein core of the extracellular domain of the human EGF receptor (mAb108), on the binding of EGF, and on the early responses of cells to EGF presentation. We used NIH 3T3 cells devoid of murine EGF receptor, transfected with a cDNA encoding the full-length human EGF receptor gene, and fully responsive to EGF. The binding to saturation of mAb108 to the surface of these cells at 4 degrees C and at other temperatures specifically reduced high-affinity binding of EGF, but did not change the dissociation constant or the estimated number of binding sites for low-affinity binding of EGF. The kinetics of EGF binding to the transfected cells were measured to determine the effects of the mAb on the initial rate of EGF binding at 37 degrees C. Interestingly, high-affinity EGF receptor bound EGF with an intrinsic on-rate constant 40-fold higher (9.8 x 10(6) M-1.s-1) than did low-affinity receptor (2.5 x 10(5) M-1.s-1), whereas the off-rate constants, measured at 4 degrees C were similar. Cells treated with the mAb or with phorbol myristate acetate displayed single on-rate constants similar to that for the low-affinity receptors. At low doses of EGF ranging from 0.4 to 1.2 nM, pretreatment of cells with mAb108 inhibited by 50-100% all of the early responses tested, including stimulation of tyrosine-specific phosphorylation of the EGF receptor, turnover of phosphatidyl inositol, elevation of cytoplasmic pH, and release of Ca2+ from intracellular stores. At saturating doses of EGF (20 nM) the inhibition of these early responses by prebinding of mAb108 was overcome. On the basis of these results, we propose that the high-affinity EGF receptors are necessary for EGF receptor signal transduction.

Histamine as a growth factor and chemoattractant for human carcinoma and melanoma cells: action through Ca2(+)-mobilizing H1 receptors.

Histamine receptors are present on the surface of various normal and tumor-derived cell types, where their biological function is incompletely understood. Here we report that histamine not only stimulates cell proliferation under serum-free conditions, but also is chemotactic for human carcinoma (Hela and A431) and melanoma (A875) cells expressing H1 type receptors. Histamine was found to be a potent activator of phospholipase C, leading to polyphosphoinositide hydrolysis and subsequent intracellular Ca2+ mobilization. In addition, histamine also causes the protein kinase C-mediated activation of Na+/H+ exchange, as evidenced by an amiloride-sensitive rise in cytoplasmic pH. All histamine-induced responses, including chemotaxis and DNA synthesis, are completely inhibited by the H1 receptor antagonist pyrilamine, but not by cimetidine, an inhibitor of histamine H2 type receptors. Our results suggest that histamine may have a previously unrecognized role in the migration and proliferation of cells expressing H1 receptors.

Signal transduction by epidermal growth factor occurs through the subclass of high affinity receptors.

Many cell types display two classes of epidermal growth factor receptor (EGFR) as judged from EGF binding studies; i.e., a major class of low affinity EGFR and a minor class of high affinity EGFR. We have studied their respective contribution to the cascade of events elicited by EGF in human A431 carcinoma cells, using anti-EGFR mAb 2E9. This antibody specifically blocks EGF binding to low affinity EGFR, without activating receptors in intact cells, and thus enables us to study the effects of exclusive EGF binding to high affinity EGFR. We show that blocking of low affinity EGFR by mAb 2E9 has almost no effect on the activation of the receptor protein-tyrosine kinase by EGF, suggesting that EGFR kinase activation occurs exclusively through the subclass of high affinity EGFR (5-10%). In addition, we provide evidence that high affinity EGFR exists both in monomeric and dimeric forms, and that cross-phosphorylation of low affinity EGFR by high affinity EGFR may take place in dimers of both receptor types. We demonstrate that the following early cellular response to EGF are also unimpaired in the presence of mAb 2E9: (a) inositol phosphate production, (b) release of Ca2+ from intracellular stores, (c) rise in intracellular pH, (d) phosphorylation of EGF on threonine residue 654, (e) induction of c-fos gene expression, and (f) alteration in cell morphology. As possible nonspecific side effects, we observed that the EGF induced Ca2+ influx and fluid-phase pinocytosis were inhibited in A431 cells in the presence of mAb 2E9. We conclude, therefore, that the activation of the EGFR signal transduction cascade can occur completely through exclusive binding of EGF to the subclass of high affinity EGFR.

Molecular dissection of the Rho-associated protein kinase (p160ROCK)-regulated neurite remodeling in neuroblastoma N1E-115 cells.

A critical role for the small GTPase Rho and one of its targets, p160ROCK (a Rho-associated coiled coil-forming protein kinase), in neurite remodeling was examined in neuroblastoma N1E-115 cells. Using wild-type and a dominant-negative form of p160ROCK and a p160ROCK-specific inhibitor, Y-27632, we show here that p160ROCK activation is necessary and sufficient for the agonist-induced neurite retraction and cell rounding. The neurite retraction was accompanied by elevated phosphorylation of myosin light chain and the disassembly of the intermediate filaments and microtubules. Y-27632 blocked both neurite retraction and the elevation of myosin light chain phosphorylation in a similar concentration-dependent manner. On the other hand, suppression of p160ROCK activity by expression of a dominant-negative form of p160ROCK induced neurites in the presence of serum by inducing the reassembly of the intermediate filaments and microtubules. The neurite outgrowth by the p160ROCK inhibition was blocked by coexpression of dominant-negative forms of Cdc42 and Rac, indicating that p160ROCK constitutively and negatively regulates neurite formation at least in part by inhibiting activation of Cdc42 and Rac. The assembly of microtubules and intermediate filaments to form extended processes by inhibitors of the Rho-ROCK pathway was also observed in Swiss 3T3 cells. These results indicate that Rho/ROCK-dependent tonic inhibition of cell process extension is exerted via activation of the actomysin-based contractility, in conjunction with a suppression of assembly of intermediate filaments and microtubules in many cell types including, but not exclusive to, neuronal cells.

Membrane currents examined under voltage clamp in cultured neuroblastoma cells.

Examination of ionic membrane currents in a voltage-clamped neuronal cell line derived from the mouse C1300 neuroblastoma disclosed four kinetically different components: sodium, potassium, calcium, and leakage current. The kinetics, voltage dependence, and pharmacological properties of the sodium and potassium currents qualitatively resemble those of the corresponding currents in squid giant axon and frog myelinated nerve fiber, suggesting that the molecular structures of the sodium and potassium channels in neuroblastoma are similar to those of the non-mammalian preparations.

Variable workup calls for guideline development for type 2A hereditary haemochromatosis.

BACKGROUND: Type 2A hereditary haemochromatosis (type 2A HH) is a rare iron-loading disorder caused by mutations in the HFE2 gene, which encodes the HJV protein. We present characteristics, treatment and follow-up of subjects diagnosed with type 2A HH in the Netherlands to increase awareness of the disease and its treatment, and to define knowledge gaps. METHODS: We collected clinical, biochemical and genetic data from seven patients (two female; five probands) from six families genetically diagnosed with type 2A HH at the Expertise Center for Iron Disorders, Radboud University Medical Centre between 2006 and 2016. RESULTS: The five probands presented with heterogeneous complaints between the ages of 19 and 39. One of two patients with delayed clinical diagnosis developed hypogonadism and Y. enterocolitica sepsis. Diagnostic workup and follow-up varied. When assessed, elevated transferrin saturation (79-98%), ferritin (1400-6200 µg/l) and severely elevated liver iron levels were found, and in all subjects, phlebotomies were initiated. One subject was switched to erythrocytapheresis. Target ferritin levels varied. Despite long-term iron depletion, two subjects developed clinical complications. Sanger sequencing revealed two pathogenic HFE2 variants (homozygous or compound heterozygous) for the five families of Dutch descent and one new pathogenic variant in the family of non-Dutch descent. CONCLUSION: Three genetic variants caused type 2A HH in six families. Clinical diagnosis was delayed in two subjects. We observed variance in presentation, workup, follow-up and treatment. We found new complications in long-term iron-depleted patients. We recommend research and guidelines for optimal workup, follow-up and treatment of type 2A HH.

The gap junction protein connexin43 interacts with the second PDZ domain of the zona occludens-1 protein.

Gap junctions mediate cell-cell communication in almost all tissues and are composed of channel-forming integral membrane proteins, termed connexins [1-3]. Connexin43 (Cx43) is the most widely expressed and the most well-studied member of this family. Cx43-based cell-cell communication is regulated by growth factors and oncogenes [3-5], although the underlying mechanisms are poorly understood as cellular proteins that interact with connexins have yet to be identified. The carboxy-terminal cytosolic domain of Cx43 contains several phosphorylation sites and potential signalling motifs. We have used a yeast two-hybrid protein interaction screen to identify proteins that bind to the carboxy-terminal tail of Cx43 and thereby isolated the zona occludens-1 (ZO-1) protein. ZO-1 is a 220 kDa peripheral membrane protein containing multiple protein interaction domains including three PDZ domains and a Src homology 3 (SH3) domain [6-9]. The interaction of Cx43 with ZO-1 occurred through the extreme carboxyl terminus of Cx43 and the second PDZ domain of ZO-1. Cx43 associated with ZO-1 in Cx43-transfected COS7 cells, as well as endogenously in normal Rat-1 fibroblasts and mink lung epithelial cells. Confocal microscopy revealed that endogenous Cx43 and ZO-1 colocalised at gap junctions. We suggest that ZO-1 serves to recruit signalling proteins into Cx43-based gap junctions.

Regulating c-Ras function. cholesterol depletion affects caveolin association, GTP loading, and signaling.

Cholesterol-rich and caveolin-containing microdomains of the plasma membrane, termed "caveolae," have been implicated in signal transduction. However, the role of caveolae in regulating the Ras-MAP kinase cascade is incompletely understood. The mammalian Ras isoforms (H, N, and K) use different membrane anchors to attach to the plasma membrane and thereby may localize to functionally distinct microdomains, which might explain isoform-specific signaling. Here, we show that, in Cos epithelial cells, endogenous K-Ras colocalizes largely with caveolin, whereas N-Ras localizes to both caveolar and noncaveolar subdomains; H-Ras localization was below detection limits. We find that epidermal growth factor (EGF) activates N-Ras but fails to activate K-Ras in these cells. Extraction of cholesterol with methyl-beta-cyclodextrin disrupts complex formation between caveolin and K- and N-Ras and, strikingly, enables EGF to activate both K-Ras and N-Ras. While cholesterol depletion enhances GTP-loading on total c-Ras, activation of the downstream MEK-MAP kinase cascade by EGF and lysophosphatidic acid but not that by phorbol ester is inhibited. Thus, plasma membrane cholesterol is essential for negative regulation of c-Ras isoforms (complexed to caveolin), as well as for mitogenic signaling downstream of receptor-activated c-Ras.

Galpha(13) mediates activation of a depolarizing chloride current that accompanies RhoA activation in both neuronal and nonneuronal cells.

Loss of membrane potential (membrane depolarization) is one of the earliest and most striking responses of quiescent cells to stimulation with serum or G protein-coupled receptor (GPCR) agonists such as lysophosphatidic acid and thrombin. Membrane depolarization is due to the activation of a chloride conductance. While this response has received relatively little attention in the past, it is clear that the acute loss of membrane potential may have important physiological consequences. However, the dissection of the underlying G protein pathway and the establishment of cause-effect relationships have remained elusive to date. Here we report that, in neuronal cells, the depolarizing chloride current invariably accompanies GPCR-induced activation of RhoA and subsequent neurite retraction, and neither of these events requires phosphoinositide hydrolysis or Ca2+ mobilization. Through antibody microinjections and a genetic approach, we demonstrate that activation of the chloride conductance is mediated by Galpha(13) in a RhoA-independent manner in both neuronal cells and fibroblasts. We further show that, in neuronal cells, this newly described Galpha(13) pathway may profoundly modulate membrane excitability during RhoA-regulated neurite remodeling.