ALBERT

Beschreibung: We report the generation and use of pseudotyped adeno-associated viral (AAV) vectors for the liver-specific expression of human blood coagulation factor IX (hFIX). Therefore, an AAV-2 genome encoding the hfIX gene was cross-packaged into capsids of AAV types 1 to 6 using efficient, large-scale technology for particle production and purification. In immunocompetent mice, the resultant vector particles expressed high hFIX levels ranging from 36% (AAV-4) to more than 2000% of normal (AAV-1, -2, and -6), which would exceed curative levels in patients with hemophilia. Expression was dose- and time-dependent, with AAV-6 directing the fastest and strongest onset of hFIX expression at all doses. Interestingly, systemic administration of 2 × 1012 vector particles of AAV-1, -4, or -6 resulted in hFIX levels similar to those achieved by portal vein delivery. For all other serotypes and particle doses, hepatic vector administration yielded up to 84-fold more hFIX protein than tail vein delivery, corroborated by similarly increased vector DNA copy numbers in the liver, and elicited a reduced immune response against the viral capsids. Finally, neutralization assays showed variable immunologic cross-reactions between most of the AAV serotypes. Our technology and findings should facilitate the development of AAV pseudotype-based gene therapies for hemophilia B and other liver-related diseases. (Blood. 2003;102:2412-2419)

Beschreibung: Intravenous injection of a lipopolysaccharide (LPS) into mice induces a rapid accumulation of platelets in the lung and liver. When degradation of the accumulated platelets occurs, anaphylactoid shock follows rapidly, the severity of the shock paralleling the quantity of platelets accumulated in the lung. Here we examined the contributions made by LPS structure and the complement system to the platelet response to LPS. BALB/c mice were injected with an LPS fromEscherichia coli O8, O9, O111, or K-12, or from recombinant mutants of K-12. The O-regions of the O8 and O9 LPSs consist of a mannose homopolysaccharide (MHP), while that of O111 consists of a heteropolysaccharide (not including mannose), and K-12 LPS lacks an O-region. O111 LPS was devoid of the ability to induce the platelet response or shock, while the ability of K-12 LPS was weak. The 2 recombinant LPSs—each having an O-region (from O8 or O9) linked to K-12 LPS—exhibited activities similar to or stronger than those of their original LPSs. Mannose-binding lectin (MBL) complexed with MBL-associated serine proteases (MASPs) bound strongly to LPSs containing MHP and caused C4 activation. Moreover, the abilities of these LPSs to activate the complement system corresponded well with their abilities to induce the platelet response and rapid shock. These results suggest that the structure of the O-antigen region is important for the platelet response to LPS, and that activation of the lectin pathway of the complement system is involved in this response.

Beschreibung: Antithrombin (AT) deficiency is an autosomal disorder associated with venous thromboembolism. However, a diagnosis of homozygous AT deficiency is seldom made. Most patients are heterozygous and have approximately 50% AT activities, and they are at higher risk for the development of thromboembolism. Through gene targeting we generated AT-deficient mice and previously reported that completely AT-deficient mice could not survive the prenatal period because of extensive thrombosis in the myocardium and liver sinusoids. In contrast, heterozygous AT-deficient mice with 50% AT activities have not shown spontaneous thromboembolic episodes. To demonstrate a thrombotic tendency in heterozygous AT deficiency, we challenged heterozygous AT-deficient mice (AT+/− mice) with the administration of lipopolysaccharide (LPS) or with restraint stress by immobilization. LPS injection markedly induced fibrin deposition in the kidney glomeruli, myocardium, and liver sinusoids in AT+/− mice compared with wild-type mice (AT+/+ mice). Restraint stress tests were performed by placing mice in 50-mL conical centrifuge tubes for 20 hours. Fibrin deposition was observed in the kidney ofAT+/+ and AT+/− mice, but AT+/−mice exhibited more extensive fibrin deposition thanAT+/+ mice. After prophylactic administration of human AT concentrates to increase plasma AT activities of AT+/−mice, LPS-induced fibrin deposition was effectively prevented. These results suggest that heterozygous AT deficiency is significantly associated with a tendency toward thrombosis formation in the kidney. The AT+/− mouse thus is a useful model for studying the effect of environmental or genetic risk factors on thrombogenesis.

Beschreibung: X-linked thrombocytopenia with thalassemia (XLTT; Online Mendelian Inheritance in Man [OMIM] accession number 314050) is a rare disorder characterized by thrombocytopenia, platelet dysfunction, splenomegaly, reticulocytosis, and unbalanced hemoglobin chain synthesis. In a 4-generation family, the gene responsible for XLTT was mapped to the X chromosome, short arm, bands 11-12 (band Xp11-12). The maximum lod score possible in this family, 2.39, was obtained for markers DXS8054 and DXS1003, at a recombination fraction of 0. Recombination events observed for XLTT and markers DXS8080 and DXS8023 or DXS991 define a critical region that is less than or equal to 7.65 KcM and contains the gene responsible for the Wiskott-Aldrich syndrome (WAS; OMIM accession number 301000) and its allelic variant X-linked thrombocytopenia (XLT; OMIM accession number 313900). Manifestations of WAS include thrombocytopenia, eczema, and immunodeficiency. In WAS/XLT the platelets are usually small, and bleeding is proportional to the degree of thrombocytopenia. In contrast, in XLTT the platelet morphology is normal, and the bleeding time is disproportionately prolonged. In this study no alteration in the WAS gene was detected by Northern blot or Western blot analysis, flow cytometry, or complimentary DNA dideoxynucleotide fingerprinting or sequencing. As has been reported for WAS and some cases of XLT, almost total inactivation of the XLTTgene-bearing X chromosome was observed in granulocytes and peripheral blood mononuclear cells from 1 asymptomatic obligate carrier. The XLTT carrier previously found to have an elevated :β hemoglobin chain ratio had a skewed, but not clonal, X-inactivation pattern favoring activity of the abnormal allele. Clinical differences and results of the mutation analyses make it very unlikely that XLTT is another allelic variant of WAS/XLT and strongly suggest that X-linked thrombocytopenia mapping to band Xp11-12 is a genetically heterogeneous disorder.

Beschreibung: X-linked thrombocytopenia with thalassemia (XLTT; Online Mendelian Inheritance in Man [OMIM] accession number 314050) is a rare disorder characterized by thrombocytopenia, platelet dysfunction, splenomegaly, reticulocytosis, and unbalanced hemoglobin chain synthesis. In a 4-generation family, the gene responsible for XLTT was mapped to the X chromosome, short arm, bands 11-12 (band Xp11-12). The maximum lod score possible in this family, 2.39, was obtained for markers DXS8054 and DXS1003, at a recombination fraction of 0. Recombination events observed for XLTT and markers DXS8080 and DXS8023 or DXS991 define a critical region that is less than or equal to 7.65 KcM and contains the gene responsible for the Wiskott-Aldrich syndrome (WAS; OMIM accession number 301000) and its allelic variant X-linked thrombocytopenia (XLT; OMIM accession number 313900). Manifestations of WAS include thrombocytopenia, eczema, and immunodeficiency. In WAS/XLT the platelets are usually small, and bleeding is proportional to the degree of thrombocytopenia. In contrast, in XLTT the platelet morphology is normal, and the bleeding time is disproportionately prolonged. In this study no alteration in the WAS gene was detected by Northern blot or Western blot analysis, flow cytometry, or complimentary DNA dideoxynucleotide fingerprinting or sequencing. As has been reported for WAS and some cases of XLT, almost total inactivation of the XLTTgene-bearing X chromosome was observed in granulocytes and peripheral blood mononuclear cells from 1 asymptomatic obligate carrier. The XLTT carrier previously found to have an elevated :β hemoglobin chain ratio had a skewed, but not clonal, X-inactivation pattern favoring activity of the abnormal allele. Clinical differences and results of the mutation analyses make it very unlikely that XLTT is another allelic variant of WAS/XLT and strongly suggest that X-linked thrombocytopenia mapping to band Xp11-12 is a genetically heterogeneous disorder.

Beschreibung: After a considerable dispute, it was shown that cells with hematopoietic activity in muscles are derived from hematopoietic organ. Transdifferentiation is not a frequent event, if any, and such a phenomenon cannot be applied to tissue regeneration easily. To achieve a breakthrough, we explored the possibility that genetic manipulation may enhance the efficiency of transdifferentiation. In this regard, there is a notable report that transient overexpression of a homeobox-containing transcriptional repressor Msx1 in muscles generated abundant mononuclear cells (MNCs) capable of differentiating into myotubes, chondrocytes, adipocytes and osteocytes. That is, enforced Msx1 expression caused dedifferentiation of myotubes, and subsequent Msx1 suppression induced redifferentiation (Odelberg SJ et al, Cell103:1099). Recently, we found that similar dedifferentiation-redifferentiation events also occur in vivo after transient Msx1 expression in muscles using adeno-associated virus (AAV) vectors (Endo T et al, manuscript in preparation). Since virtually all of AAV vector-mediated transgenes exist as episomes, they gradually disappear as the host cells divide. In the present study, we took advantage of this feature of AAV vectors; muscle-derived MNCs would lose Msx1 transgene through cell divisions after dedifferentiation, and a proper differentiation cue might redirect these undifferentiated cells into the hematopoietic lineage as well. AAV vector expressing Msx1 (AAV/msx1) was injected into tibialis anterior muscles of C57BL/6 mice. Flow cytometric analysis revealed that MNCs from AAV/msx1-treated muscles contained a considerable number of cells expressing hematopoietic stem cell markers. CD34−/c-Kit+ cells (1.3±0.9% of MNCs in control muscles) were increased in AAV/msx1-treated muscles (13.6±6.0% at 4 weeks). CD45+/Sca-1+ cells (2.4±0.4% in control muscles) were also increased in the AAV/msx1-treated muscle, peaking at 2 weeks (36.0±8.1%; P =.01). To evaluate hematopoietic activity, MNCs were cultured in methylcellulose medium containing stem cell factor, IL-3, IL-6 and erythropoietin. After AAV/msx1 injection, colony-forming cells in the muscles were gradually increased, reaching a peak at 3 weeks (48.9±24.1/muscle), in contrast to very few progenitors detected in control muscles (1.4±3.0/muscle; P

Beschreibung: Cardiovascular/thrombotic diseases are frequently induced by a variety of stressors. Obese patients are susceptible to thrombotic diseases associated with stress, but the underlying mechanisms are unknown. We previously reported that restraint stress to mice caused a significant induction of a primary inhibitor of fibrinolysis, plasminogen activator inhibitor-1 (PAI-1), in association with tissue microthrombosis. We have investigated the effect of obesity on the stress-induced PAI-1 expression using genetically obese mice. Male obese mice (C57BL/6J ob/ob) of 6 weeks old and their lean counterparts (C57BL/6J +/?) were placed into 50-100 ml conical centrifuge tubes fitted with multiple punctures so as to allow ventilation. The tubes were placed in horizontal holders and the animals thus maintained for a continuous period of restraint. After 2 or 20 hours of restraint, the mice (n=8) were sacrificed, and the plasma and tissues were harvested. PAI-1 antigen in plasma was measured by ELISA and PAI-1 mRNA in tissues was quantitated by competitive RT-PCR assay. Obese mice were hyperresponsive to short- and long-duration of restraint stress in the induction of PAI-1 antigen in plasma and PAI-1 mRNA in tissues, especially in their hearts and adipose tissues (Fig. 1). Figure Figure In situ hybridization analysis of the stressed mice revealed that strong signals for PAI-1 mRNA were localized to adipocytes, cardiovascular endothelial cells, and renal glomerular cells in obese animals. Immunohistochemical analysis for fibrin revealed that renal glomerular fibrin deposition was induced by 2 hour-restraint stress in obese mice (arrow in Fig. 2B), but rare in lean mice (Fig. 2A). Quantitative evaluation of fibrin was also achieved by counting the number of fibrin-positive glomeruli in each kidney section in a blinded fashion (Fig. 2C). Figure Figure Elevation of tumor necrosis factor-alpha (TNF-alpha) level in plasma after stress was pronounced in obese mice and pre-treatment of mice with anti-TNF-alpha antibody partially attenuated the stress-mediated induction of PAI-1 gene in adipose tissues. These results suggest that larger induction of PAI-1 may be relevant to the increased risk of stress-induced thrombosis in obese subjects and that TNF-alpha may be involved. This study presents a novel finding regarding the molecular process of the stress-induced thrombosis in obesity, and suggests that PAI-1, stress, and obesity, may be closely associated with the increased risk for cardiovascular and thrombotic diseases.

Beschreibung: Transcription factor GATA-1 is essential for the development of erythroid cells and megakaryocytes. Each of its 2 zinc fingers is critical for normal function. The C-terminal finger is necessary for DNA binding. The N finger mediates interaction with FOG-1, a cofactor for GATA-1, and also modulates DNA-binding affinity, notably at complex or palindromic GATA sites. Residues of the N finger–mediating interaction with FOG-1 lie on the surface of the N finger facing away from DNA. Strong sequence conservation of residues facing DNA suggests that this other surface may also have an important role. We report here that a syndrome of X-linked thrombocytopenia with thalassemia in humans is caused by a missense mutation (Arg216Gln) in the GATA-1 N finger. To investigate the functional consequences of this substitution, we used site-directed mutagenesis to alter the corresponding residue in GATA-1. Compared with wild-type GATA-1, Arg216Gln GATA-1 shows comparable affinity to single GATA sites but decreased affinity to palindromic sites. Arg216Gln GATA-1 interacts with FOG-1 similarly with wild-type GATA-1. Arg216Gln GATA-1 supports erythroid maturation of GATA-1 erythroid cells, albeit at reduced efficiency compared with wild-type GATA-1. Together, these findings suggest that residues of the N finger of GATA-1–facing DNA contribute to GATA-1 function apart from interaction with the cofactor FOG-1. This is also the first example of β-thalassemia in humans caused by a mutation in an erythroid transcription factor.

Beschreibung: Cryoglobulin activity associated with murine immunoglobulin G3 (IgG3) has been shown to play a significant role in the development of murine lupuslike glomerulonephritis. A fraction, but not all, IgG3 monoclonal antibodies are capable of inducing a severe acute lupuslike glomerulonephritis as a result of direct localization of IgG3 cryoglobulins, suggesting the importance of qualitative features of cryoglobulins in their nephritogenic activities. Here a remarkable difference is shown in the renal pathogenicity of 2 murine IgG3 monoclonal cryoglobulins, identical in the amino acid sequences of their heavy and light chains but different in galactosylation patterns of oligosaccharide side chains because of their synthesis in different myeloma cells. The antibody lacking the capacity to induce severe glomerulonephritis displayed an increased proportion of galactosylated heavy chains. Changes in conformation, as revealed by gel filtration analysis, reduced cryoglobulin activity, and accelerated clearance could account for the lack of the renal pathogenicity of the more galactosylated variant. This observation provides a direct demonstration for the role of IgG galactosylation in the pathogenic potential of cryoglobulins.