Pauci-immune focal necrotizing glomerulonephritis (FNGN) is a severe inflammatory disease associated with autoantibodies to neutrophil cytoplasmic antigens (ANCA). Here we characterize autoantibodies to lysosomal membrane protein-2 (LAMP-2) and show that they are a new ANCA subtype present in almost all individuals with FNGN. Consequently, its prevalence is nearly twice that of the classical ANCAs that recognize myeloperoxidase or proteinase-3. Furthermore, antibodies to LAMP-2 cause pauci-immune FNGN when injected into rats, and a monoclonal antibody to human LAMP-2 (H4B4) induces apoptosis of human microvascular endothelium in vitro. The autoantibodies in individuals with pauci-immune FNGN commonly recognize a human LAMP-2 epitope (designated P [41][42][43][44][45][46][47][48][49] ) with 100% homology to the bacterial adhesin FimH, with which they Correspondence should be addressed to R.K. (renate.kain@meduniwien.ac.at). 7 Present addresses: Interne, Hämato-Onkologie, Krankenhaus der Elisabethinen, Fadingerstrasse 1, 4010 Linz, Austria (R.Z.) and Vela Laboratories. Entwicklung und Laboranalytik Gesellschaft mit beschränkter Haftung, Brunnerstrasse 69/3, 1230 Wien, Austria (R.J.). 8 These authors contributed equally to this work. Here we establish that autoantibodies to human LAMP-2 are highly prevalent in pauci-immune FNGN and provide evidence of their pathogenicity by showing that they activate neutrophils and kill human blood microvascular endothelium in vitro and cause pauci-immune FNGN when administered to rodents. Unexpectedly, auto-antibodies to LAMP-2 in individuals with FNGN commonly recognize an epitope with considerable homology to the bacterial adhesin FimH and cross-react with it. We therefore determined whether exposure to FimH could induce antibodies to human LAMP-2 and initiate pauci-immune FNGN through molecular mimicry. The results lead us to propose a previously undescribed molecular mechanism both for the induction and development of injury in this human disease. RESULTS Autoantibodies to human LAMP-2 are common in FNGNWe established the prevalence of autoantibodies to hLAMP-2 in sera from 84 individuals with biopsy-proven active pauci-immune FNGN, either at presentation (n = 62) or during relapse (n = 22). ANCA were detectable by standard immunofluorescence assays in 80 of them (95%), and ELISA for the canonical ANCA were positive in 70 of them (83%); myeloperoxidasespecific ANCA were found in 38 people, and proteinase-3-specific ANCA were found in 39 people, including seven with antibodies to both antigens. Using a specific ELISA, we detected antibodies to human LAMP-2 in 78 of the 84 (93%) sera (Fig. 1a), and we validated the results by western blotting and indirect immunofluorescence on the O-glycosylation deficient CHO cell line ldlD cells stably expressing human LAMP-2 on their surface ( Supplementary Fig. 1a online). Notably, the human LAMP-2 ELISA was negative in all but six of the individuals when they were in remission after immunosuppressive therapy. Assays for human LA...

In individuals with mammary carcinoma, the most relevant prognostic predictor of distant organ metastasis and clinical outcome is the status of axillary lymph node metastasis. Metastases form initially in axillary sentinel lymph nodes and progress via connecting lymphatic vessels into postsentinel lymph nodes. However, the mechanisms of consecutive lymph node colonization are unknown. Through the analysis of human mammary carcinomas and their matching axillary lymph nodes, we show here that intrametastatic lymphatic vessels and bulk tumor cell invasion into these vessels highly correlate with formation of postsentinel metastasis. In an in vitro model of tumor bulk invasion, human mammary carcinoma cells caused circular defects in lymphatic endothelial monolayers. These circular defects were highly reminiscent of defects of the lymphovascular walls at sites of tumor invasion in vivo and were primarily generated by the tumor-derived arachidonic acid metabolite 12S-HETE following 15-lipoxygenase-1 (ALOX15) catalysis. Accordingly, pharmacological inhibition and shRNA knockdown of ALOX15 each repressed formation of circular defects in vitro. Importantly, ALOX15 knockdown antagonized formation of lymph node metastasis in xenografted tumors. Furthermore, expression of lipoxygenase in human sentinel lymph node metastases correlated inversely with metastasis-free survival. These results provide evidence that lipoxygenase serves as a mediator of tumor cell invasion into lymphatic vessels and formation of lymph node metastasis in ductal mammary carcinomas.

BACKGROUND: Digalloyl-resveratrol (di-GA) is a synthetic compound aimed to combine the biological effects of the plant polyhydroxy phenols gallic acid and resveratrol, which are both radical scavengers and cyclooxygenase inhibitors exhibiting anticancer activity. Their broad spectrum of activities may probably be due to adjacent free hydroxyl groups. METHODS: Protein activation and expression were analysed by western blotting, deoxyribonucleoside triphosphate levels by HPLC, ribonucleotide reductase activity by 14 C-cytidine incorporation into nascent DNA and cell-cycle distribution by FACS. Apoptosis was measured by Hoechst 33258/propidium iodide double staining of nuclear chromatin and the formation of gaps into the lymphendothelial barrier in a three-dimensional co-culture model consisting of MCF-7 tumour cell spheroids and human lymphendothelial monolayers. RESULTS: In HL-60 leukaemia cells, di-GA activated caspase 3 and dose-dependently induced apoptosis. It further inhibited cell-cycle progression in the G1 phase by four different mechanisms: rapid downregulation of cyclin D1, induction of Chk2 with simultaneous downregulation of Cdc25A, induction of the Cdk-inhibitor p21 Cip/Waf and inhibition of ribonucleotide reductase activity resulting in reduced dCTP and dTTP levels. Furthermore, di-GA inhibited the generation of lymphendothelial gaps by cancer cell spheroidsecreted lipoxygenase metabolites. Lymphendothelial gaps, adjacent to tumour bulks, can be considered as gates facilitating metastatic spread. CONCLUSION: These data show that di-GA exhibits three distinct anticancer activities: induction of apoptosis, cell-cycle arrest and disruption of cancer cell-induced lymphendothelial disintegration.

Abstract. The transmembrane component of the dystroglycan complex, a heterodimer of ␣-and ␤-dystroglycan, was recently localized at the basal cell membrane domain of podocytes, and it was speculated that it serves as a device of the podocyte for maintaining the complex podocyte foot process architecture, and for regulating the exact position of its ligands, the matrix proteins laminin and agrin, in the glomerular basement membrane (GBM). The redistribution of dystroglycan in two experimental rat models of foot process flattening and proteinuria-i.e., podocyte damage induced by polycationic protamine sulfate perfusion, and reactive oxygen species (ROS)-associated puromycin aminonucleoside nephrosiswas examined. In both experimental diseases, aggregation and reduced density of ␣-dystroglycan by endocytosis by podocytes was observed. In in vitro solid-phase binding assays, protamine and ROS competed with the binding of ␣-dystroglycan with purified laminin and a recombinant C-terminal fragment of agrin that contains the dystroglycan-binding domain. These changes were associated with disorder of the fibrillar components of the lamina rara externa of the GBM, as confirmed quantitatively by fractal analysis. These results indicate that both polycation and ROS induce similar changes in the distribution of podocyte ␣-dystroglycan that involve competitive disruption of ␣-dystroglycan/matrix protein complexes, endocytosis of the liberated receptor by podocytes, and disorganization of the matrix protein arrangement in the lamina rara externa. This links functional damage of the dystroglycan complex with structural changes in the GBM.Extensive flattening of podocyte foot processes is the major pathologic feature of minimal change nephrotic syndrome and focal segmental glomerulosclerosis (FSGS), and is thought to be obligatorily associated with increased glomerular permeability and proteinuria (1-3). Experimentally, these pathologic podocyte changes are induced within minutes by kidney perfusion of polycations, such as protamine sulfate, in a complex energy-and actin-dependent fashion (4,5). Injection of puromycin aminonucleoside into rats induces foot process flattening within days that depends on the overproduction of reactive oxygen species (ROS) within the glomerulus (6 -8). Both experimental approaches also induce increased glomerular permeability and proteinuria (6,8,9).Podocyte proteins of potential importance for maintaining the complex podocyte architecture and their interactions and functional hierarchies are currently under intense investigation, and it appears, primarily from genetic knockout experiments, that several podocyte membrane and cytoskeletal proteins are critical for the development of mature foot process organization (reviewed in 10,11). These comprise membrane proteins of the luminal domain of podocytes, such as the sialoglycoprotein podocalyxin (12,13), several membrane proteins of the slit diaphragm domain (14 -17), the cytoskeletal protein ␣-actinin-4 (18), and the scaffolding protein CD2AP (19,20)...