IntroductionCells that are going into apoptosis expose phosphatidylserine (PS) on their surface that serves as an "eat me" signal for both professional phagocytes (such as macrophages, B lymphocytes, and immature dendritic cells) and nonprofessional phagocytes (such as fibroblasts and epithelial cells). 1 Phagocytes recognize apoptotic cells directly via the PS receptor or indirectly via ␣ v -integrins that are present on the phagocyte surface. 2 In the latter process, ␣ v ␤ 3 and ␣ v ␤ 5 integrins recognize PS through an opsonin called lactadherin, also known as milk fat globule epidermal growth factor-8 (MFG-E8). 3,4 This opsonin contains a PS-binding C-domain and an RGD-motif present in the epidermal growth factor domain, which binds to ␣ v ␤ 3 and ␣ v ␤ 5 integrins. 2,[5][6][7] Rapid clearance of apoptotic cells prevents the release of potentially harmful or immunogenic intracellular materials from these cells. 8 Lactadherin-deficient mice have shown impaired phagocytosis of apoptotic cells, induction of autoimmune diseases, and autoantibody production. 9,10 ␣ v -Integrins are densely present on macrophages and dendritic cells and play an important role in phagocytosis. 11 Notably, these integrins are also overexpressed on angiogenic endothelium 12 where they play an important role in cell adhesion and migration. 13,14 Recently, Silvestre et al discovered that lactadherin is expressed in and around (angiogenic) blood vessels in an ischemic hind limb model and that the protein seems to play a crucial role in vascular endothelial growth factor (VEGF)-dependent neovascularization. 15 As angiogenesis is also a hallmark of tumor growth and tumor angiogenic endothelium expresses ␣ v -integrins, in this study we have explored the possible role of lactadherin-induced phagocytosis by angiogenic tumor endothelial cells. Methods Cell linesB16.F10 murine melanoma cells (ATCC, Manassas, VA) were cultured on regular FCS-enriched DMEM. Human umbilical vein endothelial cells (HUVECs) were cultured on EGM-2 endothelial cell growth medium-2 (Cambrex, East Rutherford, NJ) consisting of EBM-2 medium supplemented with an EGM-2 bullet kit (containing growth factors, 2% FBS, and antibiotics). HUVECs were used as an ␣ v ␤ 3 -expressing model for proliferating endothelium to a maximal passage number of 8. Lactadherin purificationBovine lactadherin was purified as previously described. 16 Purity was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and N-terminal amino acid sequencing and shown to be Submitted June 8, 2007; accepted February 13, 2008. Prepublished online as Blood First Edition paper, February 21, 2008 DOI 10.1182 DOI 10. /blood-2007 The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on May 12, 2018. by guest www.bloodjournal.org From 97% to...

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Rab3D is a small GTP-binding protein that associates with secretory granules of endocrine and exocrine cells. The physiological role of Rab3D remains unclear. While it has initially been implicated in the control of regulated exocytosis, recent deletion-mutation studies have suggested that Rab3D is involved in the biogenesis of secretory granules. Here, we report the unexpected finding that Rab3D also associates with early Golgi compartments in intestinal goblet cells and in Brunner's gland acinar cells. Expression of Rab3D in the intestine was demonstrated by SDS-PAGE and Western blot analysis of homogenates prepared from the rat duodenum and colon. Confocal laser scanning microscopy revealed Rab3D immunofluorescence in the Golgi area of goblet cells of the duodenum and colon and in Brunner's gland acinar cells. There was no colocalization between Rab3D and a trans-Golgi network marker, TGN-38. In contrast, Rab3D colocalized partially with a cis-Golgi marker, GM-130, and with a marker of cis-Golgi and coat protein complex I vesicles, beta-COP. Strong colocalization was observed between Rab3D and the lectins Griffonia simplicifolia agglutinin II and soybean agglutinin, which have been described as markers of the medial and cis-Golgi, respectively. Rabphilin, a putative effector of Rab3D, displayed an identical pattern of Golgi localization. Incubation of colon tissue with carbamylcholine or deoxycholate to stimulate exocytosis by goblet cells caused a partial redistribution of Rab3D to the cytoplasm and mucous granule field and a concomitant transformation of the Golgi architecture. Taken together, the present data suggest that Rab3D and rabphilin may regulate the secretory pathway at a much earlier stage than what has hitherto been assumed.

In domestic brown layer fowl, reactive amyloidosis of internal organs, such as liver and spleen, and of the joints is a common disorder. In a variety of amyloid types including the AA-amyloid of the chicken, in addition to amyloid fibrils, proteoglycans and glycosaminoglycans (GAGs) are found on immunohistochemistry or after extraction. The aim of the present report is to study amyloid fibrils for the ultrastructural location of GAGs by cuprolinic blue staining and immunogold labeling. Rabbit antichicken AA antiserum was used for the immunogold labeling on conventionally embedded and cryoembedded liver tissue and revealed similar results. Therefore conventional blocks could be used for further analysis. Cuprolinic blue staining was performed on blocks of joint tissue in which clearly discernable rod-shaped glycoproteins were encountered in between collagen fibrils. Moreover, it appeared to stain larger deposits which might represent amyloid. Postlabeling with the immunogold method of the cuprolinic blue-stained tissue proved that cuprolinic blue positive fibrils represented AA-amyloid fibrils. Therefore, it was concluded that the GAGs which appeared to colocalize with the fibrillar microanatomy of amyloid, represent a structural part of the amyloid fibrils and that the avian amyloid fibrils may be considered as a pathological proteoglycan.