Clearance mechanism of a mannosylated antibody-enzyme fusion protein used in experimental cancer therapy

MFECP1 is a mannosylated antibody-enzyme fusion protein used in antibody-directed enzyme prodrug therapy (ADEPT). The antibody selectively targets tumor cells and the targeted enzyme converts a prodrug into a toxic drug. MFECP1 is obtained from expression in the yeast Pichia pastoris and produced to clinical grade. The P. pastoris-derived mannosylation of the fusion protein aids rapid normal tissue clearance required for successful ADEPT. The work presented provides evidence that MFECP1 is cleared by the endocytic and phagocytic mannose receptor (MR), which is known to bind to mannose-terminating glycans. MR-transfected fibroblast cells internalize MFECP1 as revealed by flow cytometry and confocal microscopy. Immunofluorescence microscopy shows that in vivo clearance in mice occurs predominantly by MR on liver sinusoidal endothelial cells, although MR is also expressed on adjacent Kupffer cells. In the spleen, MFECP1 is taken up by MR-expressing macrophages residing in the red pulp and not by dendritic cells which are found in the marginal zone and white pulp. Clearance can be inhibited in vivo by the MR inhibitor mannan as shown by increased enzyme activities in blood. The work improves understanding of interactions of MFECP1 with normal tissue, shows that glycosylation can be exploited in the design of recombinant anticancer therapeutics and opens the ways for optimizing pharmacokinetics of mannosylated recombinant therapeutics.     

The role of SIGNR1 and the beta-glucan receptor (dectin-1) in the nonopsonic recognition of yeast by specific macrophages

We recently demonstrated that the beta-glucan receptor Dectin-1 (betaGR) was the major nonopsonic beta-glucan receptor on macrophages (Mphi) for the yeast-derived particle zymosan. However, on resident peritoneal Mphi, we identified an additional mannan-inhibitable receptor for zymosan that was distinct from the Mphi mannose receptor (MR). In this study, we have studied the mannose-binding potential of murine Mphi and identified the dendritic cell-specific ICAM-3-grabbing nonintegrin homolog, SIGN-related 1 (SIGNR1), as a major MR on murine resident peritoneal Mphi. Both SIGNR1 and betaGR cooperated in the nonopsonic recognition of zymosan by these Mphi. When SIGNR1 was introduced into NIH3T3 fibroblasts or RAW 264.7 Mphi, it conferred marked zymosan-binding potential on these cells. However, in the nonprofessional phagocytes (NIH3T3), SIGNR1 was found to be poorly phagocytic, suggesting that other receptors such as betaGR may play a more dominant role in particle internalization on professional phagocytes. Binding of zymosan to RAW 264.7 Mphi expressing SIGNR1 resulted in TNF-alpha production. Treatment of RAW 264.7 Mphi expressing SIGNR1, which express low levels of betaGR, with beta-glucans had little effect on binding or TNF-alpha production, indicating that there was no absolute requirement for betaGR in this process. These studies have identified SIGNR1 as a major MR for fungal and other pathogens present on specific subsets of Mphi.     

Purification and molecular identification of the human hyaluronan receptor for endocytosis

The clearance of hyaluronan (HA) and chondroitin sulfates from the circulating blood and lymph in the body is mediated by the membrane-bound HA receptor for endocytosis (HARE). Previously, we found that two HARE species of approximately 175 kDa and approximately 300 kDa are abundant in the sinusoidal endothelial cells in rat liver, spleen, and lymph nodes (Zhou et al. [2000], J. Biol. Chem., 275, 37733-37741). In the present study, immunocytochemical analysis of human tissues showed a similar pattern with abundant expression of HARE in the sinusoidal endothelial cells of human liver, spleen, and lymph nodes. The two human HARE proteins were immunoaffinity-purified from human spleen. Each protein was recognized in western blots using several anti-rat HARE monoclonal antibodies and was able to bind 125I-HA specifically. In nonreducing SDS-PAGE, these two human HARE species migrated at approximately 190 kDa and approximately 315 kDa; both proteins are approximately 15 kDa larger than the corresponding rat HAREs, although the de-N-glycosylated core proteins are essentially the same mass. After reduction, the human 190-kDa HARE gave a single 196-kDa species, which was not seen in the approximately 315-kDa HARE after reduction. The reduced approximately 315-kDa HARE yielded two major proteins at approximately 250 kDa and approximately 220 kDa. We determined the sequence of the human 190-kDa HARE cDNA based on analysis of internal tryptic peptides, as well as RT-PCR and 5' RACE analyses using human spleen and lymph node cDNA libraries. The human gene that encodes HARE is on chromosome 12.     

Wound healing in the PU.1 null mouse--tissue repair is not dependent on inflammatory cells

Damage to neonatal and adult tissues always incites an influx of inflammatory neutrophils and macrophages. Besides clearing the wound of invading microbes, these cells are believed to be crucial coordinators of the repair process, acting both as professional phagocytes to clear wound debris and as a major source of wound growth factor signals. Here we report wound healing studies in the PU.1 null mouse, which is genetically incapable of raising the standard inflammatory response because it lacks macrophages and functioning neutrophils. Contrary to dogma, we show that these "macrophageless" mice are able to repair skin wounds with similar time course to wild-type siblings, and that repair appears scar-free as in the embryo, which also heals wounds without raising an inflammatory response. The growth factor and cytokine profile at the wound site is changed, cell death is reduced, and dying cells are instead engulfed by stand-in phagocytic fibroblasts. We also show that hyperinnervation of the wound site, previously believed to be a consequence of inflammation, is present in the PU.1 null wound, too.     

Unique cell type-specific junctional complexes in vascular endothelium of human and rat liver sinusoids

Liver sinusoidal endothelium is strategically positioned to control access of fluids, macromolecules and cells to the liver parenchyma and to serve clearance functions upstream of the hepatocytes. While clearance of macromolecular debris from the peripheral blood is performed by liver sinusoidal endothelial cells (LSECs) using a delicate endocytic receptor system featuring stabilin-1 and -2, the mannose receptor and CD32b, vascular permeability and cell trafficking are controlled by transcellular pores, i.e. the fenestrae, and by intercellular junctional complexes. In contrast to blood vascular and lymphatic endothelial cells in other organs, the junctional complexes of LSECs have not yet been consistently characterized in molecular terms. In a comprehensive analysis, we here show that LSECs express the typical proteins found in endothelial adherens junctions (AJ), i.e. VE-cadherin as well as α-, β-, p120-catenin and plakoglobin. Tight junction (TJ) transmembrane proteins typical of endothelial cells, i.e. claudin-5 and occludin, were not expressed by rat LSECs while heterogenous immunreactivity for claudin-5 was detected in human LSECs. In contrast, junctional molecules preferentially associating with TJ such as JAM-A, B and C and zonula occludens proteins ZO-1 and ZO-2 were readily detected in LSECs. Remarkably, among the JAMs JAM-C was considerably over-expressed in LSECs as compared to lung microvascular endothelial cells. In conclusion, we show here that LSECs form a special kind of mixed-type intercellular junctions characterized by co-occurrence of endothelial AJ proteins, and of ZO-1 and -2, and JAMs. The distinct molecular architecture of the intercellular junctional complexes of LSECs corroborates previous ultrastructural findings and provides the molecular basis for further analyses of the endothelial barrier function of liver sinusoids under pathologic conditions ranging from hepatic inflammation to formation of liver metastasis.     

Distribution of murine mannose receptor expression from early embryogenesis through to adulthood

The mannose receptor is a 175-kDa transmembrane glycoprotein that appears to be expressed on the surface of terminally differentiated macrophages and Langerhans cells. The ectodomain of the mannose receptor has eight carbohydrate recognition domains. The receptor recognizes the patterns of sugars that adorn a wide array of bacteria, parasites, yeast, fungi, and mannosylated ligands. Clearance studies in whole animals have localized radiolabeled ligands, such as mannosylated bovine serum albumen, not only to macrophages, but also to liver sinusoidal endothelial cells. Hitherto, there has been no comprehensive analysis of expression of the mannose receptor in embryonic and adult mouse tissues. In this study, we have undertaken a systematic survey of the expression of the mannose receptor from early embryogenesis through to adulthood. The mannose receptor is expressed on tissue macrophages throughout the adult mouse as expected. However, the mannose receptor is first observed on embryonic day 9 on cells that line blood island vessel walls in the yolk sac. The mannose receptor is localized on sinusoidal endothelial cells in embryonic liver by embryonic day 11 and in bone marrow at embryonic day 17. This pattern persists in these organs throughout embryogenesis into adulthood when sinusoidal endothelial cells of lymph nodes also express the mannose receptor. The receptor is also found on lymphatic endothelial cells of small intestine. In contrast, sinusoids of spleen and thymus do not express mannose receptor antigen. This study demonstrates that the mannose receptor is expressed on tissue macrophages and on subsets of vascular and lymphatic endothelial cells. Thus, the mannose receptor maybe a marker of the so-called reticuloendothelial system.     

A blocking antibody to the hyaluronan receptor for endocytosis (HARE) inhibits hyaluronan clearance by perfused liver

Hyaluronan (HA) and chondroitin sulfate clearance from lymph and blood is mediated by the hyaluronan receptor for endocytosis (HARE). The purification and molecular cloning (Zhou, B., Weigel, J. A., Saxena, A., and Weigel, P. H. (2002) Mol. Biol. Cell 13, 2853-2868) of this cell surface receptor were finally achieved after we developed monoclonal antibodies (mAbs) against HARE. There are actually two independent isoreceptors for HA, which in rat are designated the 175-kDa HARE and 300-kDa HARE. Only one mAb (number 174) effectively and completely blocked the specific uptake of 125I-HA at 37 degrees C by rat liver sinusoidal endothelial cells. 125I-HA binding to both the 175-kDa and 300-kDa HARE proteins in a ligand blot assay was almost completely inhibited by <1 microg/ml mAb-174, whereas mouse IgG had little or no effect. MAb-174 also performed very well in Western analysis, indirect fluorescence microscopy, and a variety of immuno-procedures. Immunohistochemistry using mAb-174 localized HARE to the sinusoidal cells of rat liver, spleen, and lymph node. Western analysis using mAb-174 revealed that the sizes of both HARE glycoproteins were the same in these three tissues. 125I-HA was taken up and degraded by excised rat livers that were continuously perfused ex vivo with a recirculating medium. This HA clearance and metabolism by liver, which is a physiological function of HARE, was very effectively blocked by mAb-174 but not by mouse IgG. The results indicate that mAb-174 will be a useful tool to study the functions of HARE and the physiological significance of HA clearance.     

Efficient Uptake of Blood-Borne BK and JC Polyomavirus-Like Particles in Endothelial Cells of Liver Sinusoids and Renal Vasa Recta

Liver sinusoidal endothelial cells (LSECs) are specialized scavenger cells that mediate high-capacity clearance of soluble waste macromolecules and colloid material, including blood-borne adenovirus. To explore if LSECs function as a sink for other viruses in blood, we studied the fate of virus-like particles (VLPs) of two ubiquitous human DNA viruses, BK and JC polyomavirus, in mice. Like complete virions, VLPs specifically bind to receptors and enter cells, but unlike complete virions, they cannot replicate. ¹²⁵I-labeled VLPs were used to assess blood decay, organ-, and hepatocellular distribution of ligand, and non-labeled VLPs to examine cellular uptake by immunohisto- and -cytochemistry. BK- and JC-VLPs rapidly distributed to liver, with lesser uptake in kidney and spleen. Liver uptake was predominantly in LSECs. Blood half-life (∼1 min), and tissue distribution of JC-VLPs and two JC-VLP-mutants (L55F and S269F) that lack sialic acid binding affinity, were similar, indicating involvement of non-sialic acid receptors in cellular uptake. Liver uptake was not mediated by scavenger receptors. In spleen, the VLPs localized to the red pulp marginal zone reticuloendothelium, and in kidney to the endothelial lining of vasa recta segments, and the transitional epithelium of renal pelvis. Most VLP-positive vessels in renal medulla did not express PV-1/Meca 32, suggesting location to the non-fenestrated part of vasa recta. The endothelial cells of these vessels also efficiently endocytosed a scavenger receptor ligand, formaldehyde-denatured albumin, suggesting high endocytic activity compared to other renal endothelia. We conclude that LSECs very effectively cleared a large fraction of blood-borne BK- and JC-VLPs, indicating a central role of these cells in early removal of polyomavirus from the circulation. In addition, we report the novel finding that a subpopulation of endothelial cells in kidney, the main organ of polyomavirus persistence, showed selective and rapid uptake of VLPs, suggesting a role in viremic organ tropism.     

Production of Factor VIII by Human Liver Sinusoidal Endothelial Cells Transplanted in Immunodeficient uPA Mice

Liver sinusoidal endothelial cells (LSECs) form a semi-permeable barrier between parenchymal hepatocytes and the blood. LSECs participate in liver metabolism, clearance of pathological agents, immunological responses, architectural maintenance of the liver and synthesis of growth factors and cytokines. LSECs also play an important role in coagulation through the synthesis of Factor VIII (FVIII). Herein, we phenotypically define human LSECs isolated from fetal liver using flow cytometry and immunofluorescence microscopy. Isolated LSECs were cultured and shown to express endothelial markers and markers specific for the LSEC lineage. LSECs were also shown to engraft the liver when human fetal liver cells were transplanted into immunodeficient mice with liver specific expression of the urokinase-type plasminogen activator (uPA) transgene (uPA-NOG mice). Engrafted cells expressed human Factor VIII at levels approaching those found in human plasma. We also demonstrate engraftment of adult LSECs, as well as hepatocytes, transplanted into uPA-NOG mice. We propose that overexpression of uPA provides beneficial conditions for LSEC engraftment due to elevated expression of the angiogenic cytokine, vascular endothelial growth factor. This work provides a detailed characterization of human midgestation LSECs, thereby providing the means for their purification and culture based on their expression of CD14 and CD32 as well as a lack of CD45 expression. The uPA-NOG mouse is shown to be a permissive host for human LSECs and adult hepatocytes, but not fetal hepatoblasts. Thus, these mice provide a useful model system to study these cell types in vivo. Demonstration of human FVIII production by transplanted LSECs encourages further pursuit of LSEC transplantation as a cellular therapy for the treatment of hemophilia A.     

Modulation of phagocytosis of apoptotic neutrophils by supernatant from dexamethasone-treated macrophages and annexin-derived peptide Ac(2-26)

Phagocytic clearance of apoptotic leukocytes plays an important role in the resolution of inflammation. The glucocorticoid-inducible protein annexin 1 and annexin 1-derived peptides show potent anti-inflammatory responses in acute and chronic inflammation. In this study, we report that the annexin 1-derived peptide (Ac(2-26)) significantly stimulates nonphlogistic phagocytosis of apoptotic polymorphonuclear leukocytes (PMNs) by human monocyte-derived macrophages (Mphi). Peptide Ac(2-26)-stimulated phagocytosis is accompanied by rearrangement of the Mphi actin cytoskeleton. To investigate the potential role of endogenous annexin on clearance of apoptotic cells, Mphi were cultured for 5 days in the presence of dexamethasone. Supernatants collected from dexamethasone-treated Mphi significantly enhanced the ability of naive Mphi to engulf apoptotic PMNs. This effect was blocked by an annexin blocking Ab, by immunodepletion of the supernatants, and by the formyl peptide receptor/lipoxin receptor antagonist Boc1. In addition, we show that bone marrow-derived Mphi from annexin 1-null mice present a 40% decreased phagocytosis of apoptotic PMNs compared with cells taken from littermate controls. In conclusion, these results emphasize the pivotal role of annexin 1 as mediator for clearance of apoptotic cells and expand its potential therapeutic role in controlling inflammatory diseases.     

Engulfment and clearance of apoptotic cells based on a GlcNAc-binding lectin-like property of surface vimentin

The clearance of apoptotic cells is important to maintain tissue homeostasis. The engulfment of apoptotic cells is performed by professional phagocytes, such as macrophages, and also by non-professional phagocytes, such as mesenchymal cells. Here, we show that vimentin, a cytoskeletal protein, functions as an engulfment receptor on neighboring phagocytes, which recognize O-linked β-N-acetylglucosamine (O-GlcNAc)-modified proteins from apoptotic cells as "eat me" ligands. Previously, we reported that vimentin possesses a GlcNAc-binding lectin-like property on cell surface. However, the physiological relevance of the surface localization and GlcNAc-binding property of vimentin remained unclear. In the present study, we observed that O-GlcNAc proteins from apoptotic cells interacted with the surface vimentin of neighboring phagocytes and that this interaction induced serine 71-phosphorylation and recruitment of vimentin to the cell surface of the neighboring phagocytes. Moreover, tetrameric vimentin that was disassembled by serine 71-phosphorylation possessed a GlcNAc-binding activity and was localized to the cell surface. We demonstrated our findings in vimentin-expressing common cell lines such as HeLa cells. Furthermore, during normal developmental processes, the phagocytic engulfment and clearance of apoptotic footplate cells in mouse embryos was mediated by the interaction of surface vimentin with O-GlcNAc proteins. Our results suggest a common mechanism for the clearance of apoptotic cells, through the interaction of surface vimentin with O-GlcNAc-modified proteins.     

Immunolocalization of hepatocyte growth factor and its receptor (c-Met) during mouse liver development

Although hepatocyte growth factor (HGF) was discovered as a potent hepatotrophic factor responsible for liver regeneration and may involve some organ development in embryogenesis, it remains to be revealed what roles HGF plays in liver development. The present study was undertaken to determine which cells express HGF and its receptor c-Met and when c-Met is activated in mouse liver development by using immunoblotting and immunohistochemical techniques. HGF was detected in hepatocytes and non-parenchymal cells, including biliary epithelial cells, periportal connective tissue cells, megakaryocytes, endothelial cells, and sinusoidal cells, throughout liver development. Positive HGF immunostaining in hepatocytes increased during postnatal development, and reached the maximal level in the adult stage. c-Met protein was also expressed in hepatocytes throughout liver development, but maximal staining was obtained in 1- or 2-week-old livers. Phosphorylation of tyrosine residues in the c-Met beta chain also occurred in these stages. These results suggest that HGF signaling is implicated in hepatocyte growth during postnatal liver development, and its action could be in a paracrine mode; HGF produced by non-parenchymal cells such as sinusoidal cells acts on hepatocytes expressing c-Met receptors. Positive immunostaining in adult and postnatal hepatocytes may be derived from their blood clearance of HGF.     

Induction of LYVE-1/stabilin-2-positive liver sinusoidal endothelial-like cells from embryoid bodies by modulation of adrenomedullin-RAMP2 signaling

Embryonic stem cells (ESCs) are a useful source for various cell lineages. So far, however, progress toward reconstitution of mature liver morphology and function has been limited. We have shown that knockout mice deficient in adrenomedullin (AM), a multifunctional endogenous peptide, or its receptor-activity modifying protein (RAMP2) die in utero due to poor vascular development and hemorrhage within the liver. In this study, using embryoid bodies (EBs)-culture system, we successfully induced liver sinusoidal endothelial-like cells by modulation of AM-RAMP2. In an EB differentiation system, we found that co-administration of AM and SB431542, an inhibitor of transforming growth factor β (TGFβ) receptor type 1, markedly enhanced differentiation of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1)/stabilin-2-positive endothelial cells. These cells showed robust endocytosis of acetylated low-density lipoprotein (Ac-LDL) and upregulated expression of liver sinusoidal endothelial cells (LSECs)-specific markers, including factor 8 (F8), Fc-γ receptor 2b (Fcgr2b), and mannose receptor C type 1 (Mrc1), and also possessed fenestrae-like structure, a key morphological feature of LSECs. In RAMP2-null liver, by contrast, LYVE-1 was downregulated in LSECs, and the sinusoidal structure was disrupted. Our findings highlight the importance of AM-RAMP2 signaling for development of LSECs.     

Changes in the populations of null, NK1.1+, and Thy1lo lymphocytes in the bone marrow of tumor-bearing mice: effect of indomethacin treatment.

Mouse bone marrow produces many "null" lymphocytes which lack B and T lineage markers (B220-Thy1-). A subset of these cells expresses the natural killer (NK) cell marker, NK1.1. In addition, some rapidly renewed bone marrow lymphocytes express low intensities of Thy1 (Thy1lo). In view of their possible implication in tumor-host interactions these various cell populations have now been examined in mice injected with either the nonmetastatic Ehrlich ascites (EA) tumor or the Lewis lung carcinoma (LLc), a highly metastatic solid tumor. In each case, the number of null lymphocytes, as defined by a lack of radioautographic labeling of either B220 glycoprotein or Thy1, increased markedly in both the bone marrow and spleen. Treatment with the prostaglandin inhibitor, indomethacin, enhanced the increase in null cells in the bone marrow and spleen of LLc-bearing mice. The number of null small lymphocytes expressing NK1.1, as detected by combined radioautographic and immunoperoxidase techniques, increased almost 30-fold in LLc-bearing mice. The number of Thy1lo small lymphocytes increased in parallel with null cells during EA tumor growth. The findings accord with the hypothesis that the null lymphocyte population produced in mouse bone marrow includes newly formed NK lineage cells which sequentially express NK1.1 and Thy1lo. The present work demonstrates that the populations of null, NK1.1+, and Thy1lo lymphocytes in mouse bone marrow expand rapidly during the early growth of transplanted tumors, the initial increase in null lymphocytes apparently being curtailed by prostaglandin production. The results suggest that the production of null lymphocytes in mouse bone marrow is responsive to tumor development, possibly providing cells to be involved in tumor-host interactions.     

Fluid endocytosis by rat liver and spleen. Experiments with 125I-labelled poly(vinylpyrrolidone) in vivo.

1. Rates of fluid endocytosis of rat liver, spleen, hepatocytes and sinusoidal liver cells have been determined, by using 125I-labelled poly(vinylpyrrolidone) as marker. Poly(vinylpyrrolidone) was injected intravenously into rats, and plasma clearance and uptake by liver and spleen were estimated. From these data, rates of fluid endocytosis of 1.2 and 1.8 ml of plasma/g of protein per day were calculated for liver and spleen respectively. Essentially the same results were found in nephrectomized rats. 2. Hepatocytes and sinusoidal cells were separately isolated by the collagenase/Pronase method, and sinusoidal cells were further fractionated by centrifugal elutriation. Hepatocytes, sinusoidal cells, Kupffer cells and endothelial cells showed rates of fluid endocytosis of 0.96, 9.0, 19 and 13 ml of plasma/g of cell protein per day respectively. Total-body X-irradiation did not influence uptake of poly(vinylpyrrolidone) by spleen, indicating that spleen lymphocytes are not significantly involved in fluid endocytosis. 3. For liver a rate constant of exocytosis of 5% per day was found, whereas for spleen no significant loss of accumulated label could be demonstrated during a 21-day period. 4. Distribution of label over a great number of organs and tissues was measured 9 days after the injection. Liver, skin, bone and muscle together contained about 70% of the label present in the carcass; only spleen and lymph nodes contained more label per g fresh weight of tissue than liver.     

Uptake of chemically modified low density lipoproteins in vivo is mediated by specific endothelial cells

Acetoacetylated (AcAc) and acetylated (Ac) low density lipoproteins (LDL) are rapidly cleared from the plasma (t1/2 approximately equal to 1 min). Because macrophages, Kupffer cells, and to a lesser extent, endothelial cells metabolize these modified lipoproteins in vitro, it was of interest to determine whether endothelial cells or macrophages could be responsible for the in vivo uptake of these lipoproteins. As previously reported, the liver is the predominant site of the uptake of AcAc LDL; however, we have found that the spleen, bone marrow, adrenal, and ovary also participate in this rapid clearance. A histological examination of tissue sections, undertaken after the administration of AcAc LDL or Ac LDL (labeled with either 125I or a fluorescent probe) to rats, dogs, or guinea pigs, was used to identify the specific cells binding and internalizing these lipoproteins in vivo. With both techniques, the sinusoidal endothelial cells of the liver, spleen, bone marrow, and adrenal were labeled. Less labeling was noted in the ovarian endothelia. Uptake of AcAc LDL by endothelial cells of the liver, spleen, and bone marrow was confirmed by transmission electron microscopy. These data suggest uptake through coated pits. Uptake of AcAc LDL was not observed in the endothelia of arteries (including the coronaries and aorta), veins, or capillaries of the heart, testes, kidney, brain, adipose tissue, and duodenum. Kupffer cells accounted for a maximum of 14% of the 125I-labeled AcAc LDL taken up by the liver. Isolated sinusoidal endothelial cells from the rat liver displayed saturable, high affinity binding of AcAc LDL (Kd = 2.5 X 10(-9) M at 4 degrees C), and were shown to degrade AcAc LDL 10 times more effectively than aortic endothelial cells. These data indicate that specific sinusoidal endothelial cells, not the macrophages of the reticuloendothelial system, are primarily responsible for the removal of these modified lipoproteins from the circulation in vivo.     

AAV8-mediated expression of glucocerebrosidase ameliorates the storage pathology in the visceral organs of a mouse model of Gaucher disease

BACKGROUND: Gaucher disease is the most common of the lysosomal storage disorders. The primary manifestation is the accumulation of glucosylceramide (GL-1) in the macrophages of liver and spleen (Gaucher cells), due to a deficiency in the lysosomal hydrolase glucocerebrosidase (GC). A Gaucher mouse model (D409V/null) exhibiting reduced GC activity and accumulation of GL-1 was used to evaluate adeno-associated viral (AAV)-mediated gene therapy. METHODS: A recombinant AAV8 serotype vector bearing human GC (hGC) was administered intravenously to the mice. The levels of hGC in blood and tissues were determined, as were the effects of gene transfer on the levels of GL-1. Histopathological evaluation was performed on liver, spleen and lungs. RESULTS: Vector administration to pre-symptomatic Gaucher mice resulted in sustained hepatic secretion of hGC at levels that prevented GL-1 accumulation and the appearance of Gaucher cells in the liver, spleen and lungs. AAV administration to older mice with established disease resulted in normalization of GL-1 levels in the spleen and liver and partially reduced that in the lung. Analysis of the bronchoalveolar lavage fluid (BALF) from treated mice showed significant correction of the abnormal cellularity and cell differentials. No antibodies to the expressed hGC were detected following a challenge with recombinant enzyme suggesting the animals were tolerized to human enzyme. CONCLUSIONS: These data demonstrate the effectiveness of AAV-mediated gene therapy at preventing and correcting the biochemical and pathological abnormalities in a Gaucher mouse model, and thus support the continued consideration of this vector as an alternative approach to treating Gaucher disease.     

Smad3基因剔除对小鼠造血功能的影响

研究Smad3基因剔除对小鼠造血功能的影响。实验小鼠分为 5组 ,每组有Smad3基因剔除小鼠(Smad3 - - )和其同窝孪生的野生型小鼠 (Smad3 + + )各 1只。小鼠的造血功能用 14天形成的脾结节 (CFU S1 4 )、多系祖细胞 (CFU GEMM)、粒 单系祖细胞 (CFU GM)、红系祖细胞 (BFU E)测定及外周血象、骨髓象等实验血液学指标来确定。每组小鼠取尾血作白细胞、红细胞和血小板计数 ,涂片作白细胞分类计数。将一侧股骨的骨髓冲出 ,制成单细胞悬液 ,计数其中有核细胞数 ,测定CFU GM、BFU E、CFU GEMM值。将每只小鼠的 4× 10 4个骨髓有核细胞 ,经尾静脉注入 3只 8～ 10周经致死量射线照射的同系雌性小鼠体内 ,测定 14天的CFU S。取一部分胸骨、肝脏、脾脏固定做病理切片 ,其余胸骨冲出骨髓 ,涂片作分类计数。结果Smad3 - - 小鼠外周血白细胞和血小板计数明显高于Smad3 + + 小鼠 ,红细胞数无显著差异。外周血白细胞分类结果也表明粒细胞显著增高。骨髓有核细胞数无显著差异 ,CFU GM显著增高 ,BFU E无显著差异 ,CFU GEMM明显减少 ,CFU S显著减少。病理形态学观察发现骨髓增生极度活跃 ,以粒系为主 ,肝脾无显著差别。骨髓涂片分类表明粒系增多 ,粒系 :红系比例增高。因此得出结论Smad3基因剔除使小鼠造血干祖细胞数目     

Mannose-receptor-mediated clearance of lysosomal alpha-mannosidase in scavenger endothelium of cod endocardium.

Mannose-receptor-mediated clearance of circulating glycoproteins was studied in Atlantic cod (Gadus morhua). Distribution studies with radioiodinated and fluorescently labelled ligands showed that cod liver lysosomal alpha-mannosidase and yeast invertase were rapidly eliminated from blood via a mannose specific pathway in liver parenchymal cells and endocardial endothelial cells of atrium and ventricle. Asialo-orosomucoid, a galactose-terminated glycoprotein, was cleared by liver only. In vitro studies were performed with primary cultures of atrial-endocardial endothelial cells (AEC), incubated at 12 degrees C in a serum free medium. Cod AEC endocytosed mannose-terminated glycoproteins (125I-alpha-mannosidase, 125I-invertase, 125I-mannan, 125I-ovalbumin and unlabelled lysosomal alpha-mannosidase), whereas 125I-asialo-orosomucoid was not recognised. Uptake of radiolabelled mannose-terminated ligands was inhibited 80-100% in the presence of excess amounts of mannan, invertase, D-mannose, L-fucose or EGTA. Our results suggest that the cod endocardial endothelial cells express a specific Ca(2+)-dependent mannose receptor, analogous to the mannose receptor on mammalian macrophages and liver sinusoidal endothelial cells.