Inhibition of the membrane repair protein annexin-A2 prevents tumor invasion and metastasis.

Cancer cells are exposed to major compressive and shearing forces during invasion and metastasis, leading to extensive plasma membrane damage. To survive this mechanical stress, they need to repair membrane injury efficiently. Targeting the membrane repair machinery is thus potentially a new way to prevent invasion and metastasis. We show here that annexin-A2 (ANXA2) is required for membrane repair in invasive breast and pancreatic cancer cells. Mechanistically, we show by fluorescence and electron microscopy that cells fail to reseal shear-stress damaged membrane when ANXA2 is silenced or the protein is inhibited with neutralizing antibody. Silencing of ANXA2 has no effect on proliferation in vitro, and may even accelerate migration in wound healing assays, but reduces tumor cell dissemination in both mice and zebrafish. We expect that inhibiting membrane repair will be particularly effective in aggressive, poor prognosis tumors because they rely on the membrane repair machinery to survive membrane damage during tumor invasion and metastasis. This could be achieved either with anti-ANXA2 antibodies, which have been shown to inhibit metastasis of breast and pancreatic cancer cells, or with small molecule drugs.

A rare complication of an Arteriovenous shunt after bimaxillary surgery.

The most common complications following bimaxillary surgery are inferior alveolar nerve damage, hemorrhage, and relapse. Severe complications are rare, but few cases of vascular arteriovenous malformation, cavernous sinus thrombosis, formation of an aneurysm or arteriovenous shunting are reported in literature. We present a case of a 20-year-old male patient who developed a right sided tinnitus and visible pulsations close to the mandibular angle on the right side after bimaxillary surgery. CT-angiography and subsequent digital subtraction angiography (DSA) six months after surgery showed an arteriovenous fistula (AVF) from the external carotid artery to the external jugular vein. The AVF was treated by endovascular coil embolization. At six months after intervention there were no residual complaints. We discuss the possible etiology and trauma mechanisms that might have caused this pathology and present recommendations to avoid this type of complications.

Defective membrane repair machinery impairs survival of invasive cancer cells.

Cancer cells are able to reach distant tissues by migration and invasion processes. Enhanced ability to cope with physical stresses leading to cell membrane damages may offer to cancer cells high survival rate during metastasis. Consequently, down-regulation of the membrane repair machinery may lead to metastasis inhibition. We show that migration of MDA-MB-231 cells on collagen I fibrils induces disruptions of plasma membrane and pullout of membrane fragments in the wake of cells. These cells are able to reseal membrane damages thanks to annexins (Anx) that are highly expressed in invasive cancer cells. In vitro membrane repair assays reveal that MDA-MB-231 cells respond heterogeneously to membrane injury and some of them possess a very efficient repair machinery. Finally, we show that silencing of AnxA5 and AnxA6 leads to the death of migrating MDA-MB-231 cells due to major defect of the membrane repair machinery. Disturbance of the membrane repair process may therefore provide a new avenue for inhibiting cancer metastasis.

Distinction between hereditary and sporadic breast cancer on the basis of clinicopathological data.

BACKGROUND: About 5% of all breast cancer cases are attributable to germline mutations in BRCA1 or BRCA2 genes. BRCA mutations in suspected carriers, however, may be missed, which hampers genetic counselling. MATERIALS AND METHODS: Different clinicopathological features were compared between 22 breast cancers from carriers of proved BRCA1 mutations and 604 cancers from sporadic controls. In addition, 5 BRCA2-related breast cancers and 66 breast cancers of untested patients at intermediate risk and 19 breast cancers of untested patients at high risk of hereditary disease on the basis of family history were evaluated. RESULTS: A "probably sporadic" class (age >or=54 years and epidermal growth factor receptor (EGFR) negative; 68% of cases) with a 0% chance of BRCA1-related breast cancer containing 79% of the sporadic cases was yielded by using a decision tree with age, Ki67 and EGFR. A 75% chance of BRCA1-related breast cancer was shown by the "probably BRCA1-related" class (age <54 years and Ki67 >or=25%; 8% of cases) with 82% of the BRCA1-related cases but only 1.4% of the sporadic cases. Most cases at intermediate or high risk of hereditary disease on the basis of family history could be classified with high probability as either probably BRCA1 related or probably sporadic. CONCLUSION: Breast carcinomas can be classified with a high level of certainty as sporadic or related to BRCA1 germline mutations by using a decision tree with age, Ki67 and EGFR. This can be clinically useful in mutation analysis in families with a borderline risk of hereditary disease.

Review: Annexin-A5 and cell membrane repair.

Annexins are soluble proteins that bind to biological membranes containing negatively charged phospholipids, principally phosphatidylserine, in a Ca(2+)-dependent manner. Annexin-A5 (AnxA5), the smallest member of the annexin family, presents unique properties of membrane binding and self-assembly into ordered two-dimensional (2D) arrays on membrane surfaces. We have previously reported that AnxA5 plays a central role in the machinery of membrane repair by enabling rapid resealing of plasma membrane disruption in murine perivascular cells. AnxA5 promotes membrane repair via the formation of a protective 2D bandage at membrane damaged site. Here, we review current knowledge on cell membrane repair and present recent findings on the role of AnxA5 in membrane resealing of human trophoblasts.

Antibiotics enhance binding of lipid A-specific murine monoclonal antibody E5 to Gram-negative bacteria.

Cross-protection by anti-lipid A antibodies may be mediated by contribution to host defence mechanisms such as enhancement of bactericidal activity and phagocytosis. The lipid A-specific murine monoclonal antibody (mAb) E5 was evaluated in vitro for its ability to enhance antibacterial effects in concert with antibiotics in order to investigate underlying mechanisms for the proposed in vivo efficacy. The effect of antibiotic exposure of several E. coli strains on binding by mAb E5 was examined in solid phase enzyme-linked immunoassay (ELISA) and in complement activation and phagocytosis assays. MAb E5 binds scarcely to live E. coli, both encapsulated and unencapsulated, but exposure to subinhibitory concentrations of aztreonam and ceftriaxone led to enhanced binding of mAb E5 compared to mock treated strains. Enhanced binding of mAb E5 to aztreonam-treated E. coli O111:B4 or O7K1 resulted in a modest increase in complement consumption but complement-mediated phagocytosis by human polymorphonuclear leukocytes (PMN) of these complexes was not affected. It was concluded that exposure of E. coli to antibiotics induced specific alterations in the bacteria, resulting in accessbility of epitopes recognized by mAb E5. Enhanced binding was found to support complement-mediated host defense mechanisms and might contribute to better protection in a joint action of antibiotics and antibodies.

[Fusobacterium nucleatum isolated from a patient presenting lachrymal canaliculitis]. / Fusobacterium nucleatum isolé chez un patient présentant une canaliculite.

A 68-year-old woman presented with a painless inflammation of the right superior eyelid that had started several weeks before. The clinical diagnosis concluded in canaliculitis and the solid concretions were surgically extracted from the superior canalicula. The anaerobic bacteria Fusobacterium nucleatum sp. nucleatum was isolated. Signs dramatically regressed two weeks after surgery followed by one course of oral amoxicillin and clavulanic acid associated with topical tobramycin. The clinical signs had disappeared two months later.

Interplay of flexibility and stability in the control of estrogen receptor activity.

Previously, we have identified an imperfect estrogen response element (rtERE) in the promoter of the rainbow trout vitellogenin gene. Although this ERE leads to a lower transcriptional activation, a better estradiol stimulation in vivo as compared to consensus ERE (EREcs) was observed. Here we examine the ability of recombinant human estrogen receptor alpha (rhERalpha) to bind DNA containing the EREcs or the natural imperfect rtERE, which contains three mismatches. At low salt concentration, whatever the ERE sequence, dissociation equilibrium constants of the specific rhERalpha-ERE complexes are similar (K(D) = 2 nM) with the same stoichiometry. As salt concentration increases from 80 to 200 mM KCl, the affinity of the rhERalpha-rtERE complex largely diminishes whereas that of rhERalpha-EREcs seems less affected. Hence the nature of the interactions stabilizing these complexes is different: more ionic in rhERalpha-rtERE as compared to rhERalpha-EREcs. Moreover, kinetic measurements showed that specific rhERalpha-ERE complexes exhibit shorter half-lives (few seconds) and that the rhERalpha-EREcs complex is more stable (33 s) than the complex that formed with rtERE (19.8 s), in accordance with equilibrium binding results. Finally, dynamic studies of rhERalpha have shown that the protein fluctuations are damped when the salt concentration increases or when bound to ERE and all the more with rtERE. The interplay of affinity, complex half-lives, and protein dynamics in the transcriptional regulation of estrogen receptor is discussed.

Cross-reactivity of monoclonal antibodies against lipopolysaccharides of gram-negative bacteria.

Monoclonal antibodies were produced against Escherichia coli O111, Escherichia coli J5, and the rough (R) mutant of Salmonella typhimurium M206, and tested by enzyme-linked immunosorbent assay against lipopolysaccharides of several gram-negative strains. The monoclonal antibodies were also identified with an immunoblotting assay. Anti-Escherichia coli O111 monoclonal antibodies reacted only with homologous O antigens. Anti-J5 monoclonal antibodies cross-reacted with core lipopolysaccharide, especially with Rc lipopolysaccharide. IgM anti-J5 monoclonal antibodies showed more extensive cross-reactivity than IgG3 monoclonal antibodies. Anti-Re monoclonal antibodies cross-reacted weakly with all rough lipopolysaccharide tested. Thus, the varying specificity of these monoclonal antibodies seems to indicate that the core regions in the lipopolysaccharides of various gram-negative bacteria are not similar.

Binding characteristics and cross-reactivity of three different antilipid A monoclonal antibodies.

A detailed characterization of binding specificity and cross-reactivity of three antilipid A murine mAb was performed. Binding characteristics of these three mAb were investigated against Ag (ReLPS, lipid A, derivatives of lipid A) in solid phase (ELISA) and in fluid phase (C consumption, inhibition studies), and upon incorporation in membranes (E: passive hemolysis assay, and liposomes: inhibition studies). Cross-reactivity with heterologous Ag was investigated in ELISA (LPS, Gram-negative bacteria) and immunoblot experiments (LPS). The binding specificity of mAb 26-5 (IgG2b), raised against synthetic lipid A, was located in the hydrophilic region of biphospholipid A and was also exposed after membrane incorporation of lipid A or after preincubation of lipid A with polymyxin B (PMX). mAb 26-20 (IgM), also raised against synthetic lipid A, showed binding specificity for the hydrophobic region of lipid A: no binding to membrane-associated lipid A could be demonstrated, and binding in ELISA could be blocked very efficiently by PMX. The reaction pattern of mAb 8-2 (IgM), raised against the heat-killed Re mutant of Salmonella typhimurium, was in part similar to that of mAb 26-20. However, inhibition of binding with PMX was less efficient and a high specificity for ReLPS, also after membrane incorporation of this Ag, was demonstrated. In contrast to mAb 26-5 and 26-20, mAb 8-2 showed extensive cross-reactivity with heterologous LPS preparations and heat-killed as well as live Gram-negative bacteria. It is concluded that each of the three mAb binds to a different antigenic epitope in lipid A and that exposure of those epitopes for antibody binding is restricted in a differential manner, depending on mode of Ag presentation. The here defined reaction patterns provide a basis for the interpretation of potential inhibitory effects on in vitro and in vivo biologic (and toxic) activities of endotoxins and Gram-negative bacteria.

Complement activating and opsonic capacity of monoclonal antibodies raised against Escherichia coli O111 and its rough mutant J5.

Six monoclonal antibodies raised against Escherichia coli O111 and against its rough mutant J5 (chemotype Rc) were studied. One IgG2A, one IgM anti-J5, and one IgG2A anti-O111 monoclonal antibody did not bind to lipopolysaccharides of the homologous strain, but cross-reacted with heterologous gram-negative rods in an enzyme-linked immunosorbent assay. These three monoclonal antibodies activated complement when incubated with homologous or heterologous strains, but were opsonic neither in the presence nor in the absence of complement. The other three monoclonal antibodies were directed against lipopolysaccharide of the homologous strain, but showed no cross-reactivity. The IgG3 and one IgM anti-J5 monoclonal antibodies activated complement and were opsonic only in the presence of complement. The IgM anti-O111 monoclonal antibody activated complement and was opsonic both in the presence and absence of complement. Thus, the outcome of the interaction between bacteria, antibodies, and complement is influenced primarily by whether antibodies are directed against lipopolysaccharides or against other cell wall components.

Monoclonal antibodies specific for the phase-variant O-acetylated K1 capsule of Escherichia coli.

Two monoclonal antibodies, one each of the immunoglobulin M and G2b types, were produced from mouse spleen cells. These monoclonal antibodies only reacted with approximately 50% of the Escherichia coli K1 strains and not against group B meningococci. No reaction was observed after the strains were boiled. E. coli K1 strains that reacted with the monoclonal antibodies could become nonreactive after subculture. Based on these findings, we conclude that the monoclonal antibodies react with the O-acetylated K1 capsule.