Bevacizumab continuation versus no continuation after first-line chemotherapy plus bevacizumab in patients with metastatic colorectal cancer: a randomized phase III non-inferiority trial (SAKK 41/06).

BACKGROUND: Chemotherapy plus bevacizumab is a standard option for first-line treatment in metastatic colorectal cancer (mCRC) patients. We assessed whether no continuation is non-inferior to continuation of bevacizumab after completing first-line chemotherapy. PATIENTS AND METHODS: In an open-label, phase III multicentre trial, patients with mCRC without disease progression after 4-6 months of standard first-line chemotherapy plus bevacizumab were randomly assigned to continuing bevacizumab at a standard dose or no treatment. CT scans were done every 6 weeks until disease progression. The primary end point was time to progression (TTP). A non-inferiority limit for hazard ratio (HR) of 0.727 was chosen to detect a difference in TTP of 6 weeks or less, with a one-sided significance level of 10% and a statistical power of 85%. RESULTS: The intention-to-treat population comprised 262 patients: median follow-up was 36.7 months. The median TTP was 4.1 [95% confidence interval (CI) 3.1-5.4] months for bevacizumab continuation versus 2.9 (95% CI 2.8-3.8) months for no continuation; HR 0.74 (95% CI 0.58-0.96). Non-inferiority could not be demonstrated. The median overall survival was 25.4 months for bevacizumab continuation versus 23.8 months (HR 0.83; 95% CI 0.63-1.1; P = 0.2) for no continuation. Severe adverse events were uncommon in the bevacizumab continuation arm. Costs for bevacizumab continuation were estimated to be ∼30,000 USD per patient. CONCLUSIONS: Non-inferiority could not be demonstrated for treatment holidays versus continuing bevacizumab monotheray, after 4-6 months of standard first-line chemotherapy plus bevacizumab. Based on no impact on overall survival and increased treatment costs, bevacizumab as a single agent is of no meaningful therapeutic value. More efficient treatment approaches are needed to maintain control of stabilized disease following induction therapy. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, number NCT00544700.

Unintentional pediatric submersion-injury-related hospitalizations in the United States, 2003.

The objective of this study was to examine the demographic characteristics and hospital resource utilization of submersion-injury-related hospitalizations among persons < or =20 years of age in the USA in 2003. All 1475 pediatric submersion-injury-related hospital discharges in the Kids' Inpatient Database were identified by ICD-9-CM diagnosis code or external cause of injury code. These cases represent an estimated 2490 pediatric submersion-injury-related hospitalizations nationwide. Inpatient costs for these estimated hospitalizations were approximately $10 million. The overall pediatric submersion-injury-related rate of hospitalization was 3.0 per 100,000 persons. Children aged 0-4 years had the highest rate of hospitalization (7.7 per 100,000 persons). Children with permanent submersion-injury-related morbidity accounted for 5.8% of hospital admissions and 37.3% of hospital costs in our study, and children with submersion-injury-related in-hospital death accounted for 11.6% of hospital admissions and 20.0% of hospital costs in our study. Prevention of submersion injury using focused, proven strategies deserves increased attention.

Transmembrane proteins of tight junctions.

Tight junctions from a morphological and functional boundary between the apical and basolateral cell surface domains of epithelia and endothelia, and regulate selective diffusion along the paracellular space. Two types of four-span transmembrane proteins, occludin and claudins, as well as the single-span protein JAM are associated with tight junctions. The functional analysis of these proteins starts to reveal how they are involved in the functions of tight junctions, which of their domains are important for these functions, and how they interact with each other to form the junctional diffusion barriers.

Multiple domains of occludin are involved in the regulation of paracellular permeability.

Tight junctions form selective paracellular diffusion barriers that regulate the diffusion of solutes across epithelia and constitute intramembrane diffusion barriers that prevent the intermixing of apical and basolateral lipids in the extracytoplasmic leaflet of the plasma membrane. In MDCK cells, previous expression experiments demonstrated that occludin, a tight junction protein with four transmembrane domains, is critically involved in both of these tight junction functions and that its COOH-terminal cytoplasmic domain is of functional importance. By expressing mutant and chimeric occludin that exert a dominant negative effect on selective paracellular diffusion, we now demonstrate that the extracytoplasmic domains and at least one of the transmembrane domains are also critically involved in selective paracellular permeability. Multiple domains of occludin are thus important for the regulation of paracellular permeability. Expression of chimeras containing at least one transmembrane domain of occludin also resulted in an enhanced intracellular accumulation of claudin-4, another transmembrane protein of tight junctions, suggesting that the two proteins may cooperate in the regulation of paracellular permeability.

The tight junction protein ZO-1 and an interacting transcription factor regulate ErbB-2 expression.

Epithelial tight junctions regulate paracellular diffusion and restrict the intermixing of apical and basolateral plasma membrane components. We now identify a Y-box transcription factor, ZONAB (ZO-1-associated nucleic acid-binding protein), that binds to the SH3 domain of ZO-1, a submembrane protein of tight junctions. ZONAB localizes to the nucleus and at tight junctions, and binds to sequences of specific promoters containing an inverted CCAAT box. In reporter assays, ZONAB and ZO-1 functionally interact in the regulation of the ErbB-2 promoter in a cell density-dependent manner. In stably transfected overexpressing cells, ZO-1 and ZONAB control expression of endogenous ErbB-2 and function in the regulation of paracellular permeability. These data indicate that tight junctions directly participate in the control of gene expression and suggest that they function in the regulation of epithelial cell differentiation.

Occludin modulates transepithelial migration of neutrophils.

Neutrophils cross epithelial sheets to reach inflamed mucosal surfaces by migrating along the paracellular route. To avoid breakdown of the epithelial barrier, this process requires coordinated opening and closing of tight junctions, the most apical intercellular junctions in epithelia. To determine the function of epithelial tight junction proteins in this process, we analyzed neutrophil migration across monolayers formed by stably transfected epithelial cells expressing wild-type and mutant occludin, a membrane protein of tight junctions with four transmembrane domains and both termini in the cytosol. We found that expression of mutants with a modified N-terminal cytoplasmic domain up-regulated migration, whereas deletion of the C-terminal cytoplasmic domain did not have an effect. The N-terminal cytosolic domain was also found to be important for the linear arrangement of occludin within tight junctions but not for the permeability barrier. Moreover, expression of mutant occludin bearing a mutation in one of the two extracellular domains inhibited neutrophil migration. The effects of transfected occludin mutants on neutrophil migration did not correlate with their effects on selective paracellular permeability and transepithelial electrical resistance. Hence, specific domains and functional properties of occludin modulate transepithelial migration of neutrophils.

Epithelial polarity: sorting out the sorters.

Epithelial cell polarity depends on the continuous sorting of plasma membrane proteins. While various sorting signals and pathways have been identified, only recently has a protein been identified that recognizes such sorting determinants and mediates sorting to a specific cell-surface domain.

Occludin and the functions of tight junctions.

The tight junction or zonula occludens is the most apical structure of the epithelial junctional complex. Tight junctions from semipermeable intercellular diffusion barriers that control paracellular diffusion in a regulated manner. This intercellular junction also acts as an intramembrane fence that prevents the intermixing of apical and basolateral lipids in the exocytoplasmic leaflet of the plasma membrane. Moreover, evidence suggests that tight junction components participate in the regulation of cell growth and differentiation. Occludin was the first identified transmembrane protein of this intercellular junction and received much attention since its molecular characterization. This review discusses experiments that were done with occludin and how they influenced our current thinking of the molecular functioning of tight junctions.

The cytoplasmic domains of a beta1 integrin mediate polarization in Madin-Darby canine kidney cells by selective basolateral stabilization.

In Madin-Darby canine kidney cells, newly synthesized apical and basolateral membrane proteins are generally transported directly to their respective cell surface domain due to targeting determinants that mediate sorting in the Golgi complex. In several basolateral membrane proteins, these targeting determinants reside in the cytoplasmic domains. We show here that basolateral expression of the human alpha5beta1 integrin in stably transfected Madin-Darby canine kidney cells is also mediated by the cytoplasmic domains. Distinct regions in both cytoplasmic domains were found to be sufficient to mediate basolateral expression independently from one another. Unexpectedly, newly synthesized wild-type alpha5beta1 and basolaterally expressed chimeras containing the cytoplasmic domain of either alpha5 or beta1 were integrated into both cell surface domains, preferentially apically, during biosynthesis. The apical pools of wild-type integrin and chimeric subunits were found to become quickly degraded, whereas the basolateral pools were stabilized. Thus, the cytoplasmic domains of the alpha5beta1 integrin are independently sufficient to mediate sorting by selective basolateral stabilization.

Fc gamma RII-B1 regulates the presentation of B cell receptor-bound antigens.

Fc gamma receptors (Fc gamma RII) on B lymphocytes negatively regulate B cell receptor (BCR)-dependent activation upon cross-linking of the two receptors. The mechanism reflects the ability of the Fc gamma RII cytoplasmic tail to recruit specific phosphatases that inactivate elements of the BCR-signaling cascade. We now show that cross-linking also blocks the processing and presentation of BCR-bound Ag. This occurs because the Fc gamma RII isoform typically expressed by B cells (Fc gamma RII-B1) is incompetent for endocytosis. When cross-linked, Fc gamma RII-B1 acts as a dominant negative inhibitor of BCR endocytosis. In contrast, cross-linking of endocytosis-competent Fc gamma RII isoforms did not inhibit endocytosis or processing of BCR-bound Ag. Thus, Fc gamma RII-B1 acts not only to prevent B cell activation under conditions of Ab excess, but also to prevent clonotypic T cell activation by inhibiting the ability of B cells to generate specific MHC class II-bound TCR ligands.

Carbohydrate-mediated Golgi to cell surface transport and apical targeting of membrane proteins.

Polarized expression of most epithelial plasma membrane proteins is achieved by selective transport from the Golgi apparatus or from endosomes to a specific cell surface domain. In Madin-Darby canine kidney (MDCK) cells, basolateral sorting generally depends on distinct cytoplasmic targeting determinants. Inactivation of these signals often resulted in apical expression, suggesting that apical transport of transmembrane proteins occurs either by default or is mediated by widely distributed characteristics of membrane glycoproteins. We tested the hypothesis of N-linked carbohydrates acting as apical targeting signals using three different membrane proteins. The first two are normally not glycosylated and the third one is a glycoprotein. In all three cases, N-linked carbohydrates were clearly able to mediate apical targeting and transport. Cell surface transport of proteins containing cytoplasmic basolateral targeting determinants was not significantly affected by N-linked sugars. In the absence of glycosylation and a basolateral sorting signal, the reporter proteins accumulated in the Golgi complex of MDCK as well as CHO cells, indicating that efficient transport from the Golgi apparatus to the cell surface is signal-mediated in polarized and non-polarized cells.

Tight junctions.

Tight junctions are the most apical intercellular junctions of epithelial and endothelial cells and create a regulatable semipermeable diffusion barrier between individual cells. On a cellular level, they form an intramembrane diffusion fence that restricts the intermixing of apical and basolateral membrane components. In addition to these well defined functions, more recent evidence suggests that tight junctions are also involved in basic cellular processes like the regulation of cell growth and differentiation.

Biogenesis of tight junctions: the C-terminal domain of occludin mediates basolateral targeting.

Tight junctions form a morphological and physical border between the apical and the basolateral cell surface domains of epithelial cells; hence assembly of tight junctions could occur from both of the two plasma membrane domains. We show here that the C-terminal cytoplasmic domain of occludin, the only known transmembrane protein of tight junctions, was sufficient to mediate basolateral expression of a chimeric protein. Since this chimera was transported directly to the basolateral membrane during biosynthesis, the C-terminal domain of occludin contains a basolateral targeting signal. Additionally, the C-terminal domain of occludin was also able to mediate endocytosis. Thus, the C-terminal cytoplasmic domain appears to govern intracellular transport of occludin. To test whether the basolateral membrane is an obligatory intermediate in transport of occludin to tight junctions, we analyzed the expression of occludin molecules rendered unable to efficiently integrate into tight junctions by the introduction of N-linked glycosylation sites into the two extracellular loops. Indeed, glycosylated occludin accumulated in the basolateral membrane, supporting a model in which the biogenesis of tight junctions occurs from this cell-surface domain.

Transepithelial migration of neutrophils.

Neutrophils are a key cell type of nonadaptive immune system and are the first phagocytic cell type that reaches mucosal inflammatory sites. On the last stage of their journey from the blood stream to a mucosal surface, neutrophils cross a generally sealed epithelium by migrating along the paracellular pathway to the luminal side of the epithelial layer. This last step involves a specific receptor-mediated adhesion event of the neutrophil to the epithelium, followed by a rapid and highly coordinated reversible opening of the epithelial intercellular junctions that allows the transmigration of the neutrophils. Although we do not yet understand the molecular mechanisms that mediate this transmigration process, the last years witnessed the discovery of the first neutrophil and epithelial cell surface proteins critically involved in transepithelial migration of neutrophils.

The SH3 domain of the tight junction protein ZO-1 binds to a serine protein kinase that phosphorylates a region C-terminal to this domain.

ZO-1 is a tight junction phosphoprotein partially homologous to a tumor suppressor in Drosophila. The homologous region contains an SH3 domain with an unidentified function. Using fusion proteins containing the SH3 domain and various N- and C-terminal sequences, we tested for association of a kinase with this protein domain in extracts of MDCK cells. We show that the SH3 domain of ZO-1 binds a serine protein kinase that phosphorylates a region immediately C-terminal to the SH3 domain. This kinase associates specifically with the SH3 domain of ZO-1 and appears to be also associated with junctional complexes extracted from MDCK cells.

Copper/zinc and manganese superoxide dismutases in alcoholic liver disease: immunohistochemical quantitation.

Alcohol damage to the liver can, among other factors, be mediated through the action of toxic oxygen radicals generated by ethanol. Major antioxidants in the liver are copper/zinc and manganese superoxide dismutases (Cu/Zn- and Mn-SODs). In order to test whether SODs may be differentially expressed in alcoholic liver disease (ALD), biopsies from 45 patients with ALD were analyzed for qualitative and quantitative immunoreactivity of Cu/Zn- and Mn-SOD in hepatocytes. The overall amount of Cu/Zn-SOD reactivity was significantly lower in ALD than in control biopsies, whereas no difference was found for Mn-SOD. Staining for both enzymes was decreased in ballooned hepatocytes. Low Cu/Zn-SOD was correlated with advanced lattice-like perisinusoidal fibrosis. In hepatocytes forming cirrhotic nodules, SOD reactivity was similar to that of control cells. The results suggest that SODs may be differentially regulated in ALD, and that Mn-SOD, an inducible enzyme, may be involved in recovery and cell protection in ALD.

Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein.

Tight junctions, the most apical of the intercellular junctions that connect individual cells in a epithelial sheet, are thought to form a seal that restricts paracellular and intramembrane diffusion. To analyze the functioning of tight junctions, we generated stable MDCK strain 2 cell lines expressing either full-length or COOH-terminally truncated chicken occludin, the only known transmembrane component of tight junctions. Confocal immunofluorescence and immunoelectron microscopy demonstrated that mutant occludin was incorporated into tight junctions but, in contrast to full-length chicken occludin, exhibited a discontinuous junctional staining pattern and also disrupted the continuous junctional ring formed by endogenous occludin. This rearrangement of occludin was not paralleled by apparent changes in the junctional morphology as seen by thin section electron microscopy nor apparent discontinuities of the junctional strands observed by freeze-fracture. Nevertheless, expression of both wild-type and mutant occludin induced increased transepithelial electrical resistance (TER). In contrast to TER, particularly the expression of COOH-terminally truncated occludin led to a severalfold increase in paracellular flux of small molecular weight tracers. Since the selectivity for size or different types of cations was unchanged, expression of wild-type and mutant occludin appears to have activated an existing mechanism that allows selective paracellular flux in the presence of electrically sealed tight junctions. Occludin is also involved in the formation of the apical/basolateral intramembrane diffusion barrier, since expression of the COOH-terminally truncated occludin was found to render MDCK cells incapable of maintaining a fluorescent lipid in a specifically labeled cell surface domain.

Copper/zinc and manganese superoxide dismutase immunoreactivity in hepatic iron overload diseases.

Iron overload to the liver induces hepatic injury, eventually ending up with liver fibrosis or cirrhosis. Pathogenic mechanisms involved in liver damage are only partially known, but there is evidence for an important role of iron-induced reactive oxygen species. We have, therefore, analyzed the immunohistochemical reactivity for two major free radical scavengers, copper/zinc and manganese superoxide dismutase (Cu/Zn- and Mn-SOD's) in three situations of hepatic iron overload, and compared enzyme patterns with grades of iron deposition, grades of fibrosis, and levels of microphotometrically measured type IV collagen immunoreactivity. Cu/Zn- and Mn-SOD reactivity was detectable in hepatocytes with a heavy and a low iron burden, but Cu/Zn-SOD staining was more intense than that of Mn-SOD in the three groups analysed. There was trend for microphotometrically measured type IV collagen levels to increase with the amount of iron, and increased collagen IV was correlated with higher grades of Cu/Zn-SOD, but not of Mn-SOD, reactivity. The findings suggest that the two SOD's may be differentially expressed in states of hepatic iron overload, and that low expression of the inducible radical scavenger, Mn-SOD, may play a role in chronic iron toxicity.

Altered hepatic localization and expression of occludin after common bile duct ligation.

Epithelial tight junctions form a regulated barrier that seals the paracellular space and prevents mixing of luminal contents with the interstitium. This barrier is composed of a group of proteins including the putative "sealing" protein occludin that appears to bind directly to a cytoplasmic junction protein, ZO-1. To study the interaction and regulation of these two components when paracellular integrity is altered, we assessed protein expression and immunofluorescent (IF) localization of ZO-1 and occludin in a rat model of hepatocyte tight junction damage induced by common bile duct ligation (CBDL). Protein levels were detected in liver by immunoblotting and IF localization by 3-dimensional reconstruction of serial 0.5-micron confocal microscopic optical sections. As previously described, ZO-1 protein levels progressively increased to threefold control levels 9 days after CBDL. In contrast, occludin protein levels decreased by 50% within 2 days after CBDL and returned to control values by 9 days. In control IF sections, ZO-1 and occludin colocalized, forming thin continuous staining outlining canaliculi. After CBDL, ZO-1 staining appeared discontinuous, and a punctate pericanalicular accumulation of signal developed around junctional areas. Occludin staining was also discontinuous after CBDL, but, in contrast to ZO-1, was not punctate and remained localized either in a linear fashion along canalicular margins or in a homogeneous fashion in immediately surrounding areas. CBDL results in changes in the expression and localization of the putative tight junction sealing protein occludin in hepatocytes that are distinct from those observed for the peripheral membrane tight junction protein ZO-1.(ABSTRACT TRUNCATED AT 250 WORDS)