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We have designed short peptides composed of two functional domains, one a tumor blood vessel 'homing' motif and the other a programmed cell death-inducing sequence, and synthesized them by simple peptide chemistry. The 'homing' domain was designed to guide the peptide to targeted cells and allow its internalization. The pro-apoptotic domain was designed to be nontoxic outside cells, but toxic when internalized into targeted cells by the disruption of mitochondrial membranes. Although our prototypes contain only 21 and 26 residues, they were selectively toxic to angiogenic endothelial cells and showed anti-cancer activity in mice. This approach may yield new therapeutic agents.

The anti-neutrophil cytoplasmic antibody (ANCA)associated vasculitides (AAVs) are diseases characterized by inflammation of blood vessels, endothelial injury and tissue damage. The three types of small-vessel vasculitis, namely granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) and eosinophilic GPA (EGPA; previously known as Churg-Strauss syndrome), feature a loss of tolerance to neutrophil primary granule proteins, most often leukocyte proteinase 3 (PR3; also known as myeloblastin) or myeloperoxidase (MPO) (Table 1). The vessels involved in AAV are typically capillaries, arterioles and venules but small arteries and veins may also be affected. Autoimmunity is documented clinically by serum ANCAs to PR3 (PR3-ANCA) or MPO (MPO-ANCA), which are generally associated with the main syndromic AAV presentations (box 1). AAVs collectively represent one of several types of autoimmune vasculitis (Fig. 1). GPA and MPA can involve small blood vessels in any organ or tissue but commonly affect the upper and lower respiratory tract and the kidneys (box 2). Patients with AAV typically present with severe organ-threatening or life-threatening disease, although less severe presentations also occur. GPA is predominantly associated with PR3-ANCA and its clinical features typically include sinonasal disease, lower respiratory tract involvement with pulmonary haemorrhage and granulomatous inflammation, and glomerulonephritis. MPA is usually associated with MPO-ANCA and clinical features include more severe renal disease and some of the manifestations of GPA but without granulomatous inflammation. EGPA is characterized by asthma, eosinophilia and, in many (but not all) cases, vasculitis. EGPA is less common than GPA or MPA and, in some cases, is associated with ANCAs, mainly MPO-ANCA (Table 1). Although categorized as a form of AAV, EGPA has less overlap with the other AAVs than that between GPA and MPA with regard to its genetic, pathogenetic, and clinical features and its management and is typically considered a separate entity. Improvements in treatment and prognosis for patients with AAV have resulted from the translation of both preclinical and clinical research findings. Here, we provide an updated overview of the clinical and

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...

Eph receptor tyrosine kinases and their ephrin ligands have been implicated in embryonic vascular development and in in vivo models of angiogenesis. Eph proteins may also regulate tumor neovascularization, but this role has not been previously investigated. To screen for Eph proteins expressed in tumor blood vessels, we used tumor xenografts grown in nude mice from MDA-MB-435 human breast cancer cells or KS1767 human Kaposi's sarcoma cells. By immunohistochemistry, the ephrin-A1 ligand and one of its receptors, EphA2, were detected throughout tumor vasculature. Double-labeling with anti-CD34 antibodies demonstrated that both ephrin-A1 and EphA2 were expressed in xenograft endothelial cells and also tumor cells. Furthermore, EphA2 was tyrosinephosphorylated in the xenograft tumors, indicating that it was activated, presumably by interacting with ephrin-A1. Ephrin-A1 and EphA2 were also detected in both the vasculature and tumor cells of surgically removed human cancers. In an in vitro angiogenesis model, a dominant negative form of EphA2 inhibited capillary tube-like formation by human umbilical vein endothelial cells (HUVECs), demonstrating a requirement for EphA receptor signaling. These data suggest that ephrin-A1 and EphA2 play a role in human cancers, at least in part by in¯uencing tumor neovascularization. Eph proteins may represent promising new targets for antiangiogenic cancer treatments. Oncogene (2000) 19, 6043 ± 6052.

Focal segmental glomerulosclerosis (FSGS) is a frequent and severe glomerular disease characterized by destabilization of podocyte foot processes. We report that transgenic expression of the microRNA miR-193a in mice rapidly induces FSGS with extensive podocyte foot process effacement. Mechanistically, miR-193a inhibits the expression of the Wilms' tumor protein (WT1), a transcription factor and master regulator of podocyte differentiation and homeostasis. Decreased expression levels of WT1 lead to downregulation of its target genes PODXL (podocalyxin) and NPHS1 (nephrin), as well as several other genes crucial for the architecture of podocytes, initiating a catastrophic collapse of the entire podocyte-stabilizing system. We found upregulation of miR-193a in isolated glomeruli from individuals with FSGS compared to normal kidneys or individuals with other glomerular diseases. Thus, upregulation of miR-193a provides a new pathogenic mechanism for FSGS and is a potential therapeutic target.

Organ specific drug targeting was explored in mice as a possible alternative to surgery to treat prostate diseases. Peptides that specifically recognize the vasculature in the prostate were identified from phage-displayed peptide libraries by selecting for phage capable of homing into the prostate after an i.v. injection. One of the phage selected in this manner homed to the prostate 10 -15 times more than to other organs. Unselected phage did not show this preference. The phage bound also to vasculature in the human prostate. The peptide displayed by the prostate-homing phage, SMSIARL (single letter code), was synthesized and shown to inhibit the homing of the phage when co-injected into mice with the phage. Systemic treatment of mice with a chimeric peptide consisting of the SMSIARL homing peptide, linked to a proapoptotic peptide that disrupts mitochondrial membranes, caused tissue destruction in the prostate, but not in other organs. The chimeric peptide delayed the development of the cancers in prostate cancerprone transgenic mice (TRAMP mice). These results suggest that it may be possible to develop an alternative to surgical prostate resection and that such a treatment may also reduce future cancer risk. Diseases affecting the prostate have gained major significance clinically and economically, primarily because of the increasing average age of the male population in the industrialized countries. Benign prostate hyperplasia affects to some degree most elderly men. Even more serious, the prostate is a frequent site of cancer. Some autopsy studies find that most men older than 70 have occult or overt cancer in the prostate (1). The surgical therapies of prostate hypertrophy and prostate cancer are associated with serious side effects, such as incontinence and impotence.We have sought to develop a strategy that would provide a less traumatic treatment for prostate disease than is currently available. Our strategy is based on identification of peptides that home to specific sites in the vasculature by in vivo screening of intravenously injected phage libraries. These studies have revealed a surprising degree of specialization in the endothelia of various normal tissues (2, 3). Screening phage libraries for tumor homing has yielded a collection of peptides that home to tumor vasculature (4). We and others have used these tumor-homing peptides to direct therapies into tumors in mice (4, 5). We report here the identification of peptides that home to the vasculature of the prostate and the use of one of these homing peptides to deliver a proapoptotic peptide to the prostate. Materials and MethodsMaterials. Peptides were synthesized to our specifications by AnaSpec (San Jose, CA) or by our Peptide Synthesis Facility. The peptides were purified by HPLC and their identity was confirmed with mass spectrometry.Apotag Kit for TUNEL staining was purchased from Intergen (Purchase, NY). Testosterone pellets (12.5 mg) and control pellets were from Innovative Research of America (Sarasota, FL), and controlled release pumps ...

CD55 deficiency with hyperactivation of complement, angiopathic thrombosis, and protein-losing enteropathy (the CHAPLE syndrome) is caused by abnormal complement activation due to biallelic loss-of-function mutations in CD55. (Funded by the National Institute of Allergy and Infectious Diseases and others.).