Position of the sulfhydryl group and the disulfide bonds of human glucocerebrosidase

Purified human glucocerebrosidase isolated from placenta was modified with [¹⁴C]-iodoacetic acid without reduction and digested with both protease-V8 at pH 4.0 followed by-chymotrypsin at pH 7.5. The majority of radioactivity was found in a peptide that contained the [¹⁴C]-carboxymethylated-cysteine identified as CM-Cys₁₈. Direct sequencing of the N-terminus of the intact labeled protein confirmed the modification of Cys₁₈. For identification of disulfide bond-containing peptides, another portion of glucocerebrosidase was alkylated with nonlabeled iodoacetic acid and then digested with protease V8 and-chymotrypsin as before. Twenty-eight HPLC fragments were collected. These purified peaks were then reduced with-mercaptoethanol followed by S-carboxymethylation with [¹⁴C]-iodoacetic acid. Three peptides among these 28 peptides generated two radioactive daughter peptides. These peptides were sequenced and the position of the radioactive CM-cysteines identified. The locations of these disulfides are Cys₄-Cys₁₆, Cys₂₃-Cys₃₄₂, and Cys₁₂₆-Cys₂₄₈. Attempts to reproduce the free sulfhydryl labeling experiments using the glucocerebrosidase isolated from Ceredase proved unsuccessful. No label was incorporated by this enzyme prior to reduction. This result suggests that the form of the protein used in the clinic differs from the native protein.     

Expression and secretion of human glucocerebrosidase mediated by recombinant lentivirus vectors in vitro and in vivo: implications for gene therapy of Gaucher disease

Gaucher disease is a lysosomal storage disorder resulting from a deficiency of glucocerebrosidase (GC). In this study, we showed that vascular and hepatic delivery of a HIV-1-based lentivirus vector encoding human GC cDNA produced therapeutic levels of GC protein. A high level of expression of GC was produced in cultured fibroblasts derived from patients with Gaucher disease by transducing the cells with recombinant lentivirus vectors. GC secreted by transduced fibroblasts was taken up by adjacent GC-deficient cells by endocytosis. Intraportal administration of lenti-EF-GC viral vector resulted in efficient transduction and expression of the GC. Vascular delivery of vector resulted in high levels of GC expression in mice that persisted in most organs over the four months. No significant abnormalities were found attributable to recombinant lentivirus vectors in any of the tissues examined. This study represents an initial step toward gene transfer using recombinant lentivirus vectors for treatment of Gaucher disease.     

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.     

Multicenter phase 1/2 application of adenovirus-specific T cells in high-risk pediatric patients after allogeneic stem cell transplantation

Background

Adenovirus (ADV) reactivation can cause significant morbidity and mortality in children after allogeneic stem cell transplantation. Antiviral drugs can control viremia, but viral clearance requires recovery of cell-mediated immunity.

Therapeutic potential of low-dose IL-2 in a chronic GVHD patient by in vivo expansion of regulatory T cells

Chronic graft-versus-host disease (cGVHD) is a common complication following allogeneic hematopoietic stem cell transplantation (HSCT), which is characterized by autoimmune like inflammatory responses and reduced levels of regulatory T cells (Tregs). Recently, the use of low-dose IL-2 has been reported to selectively increase Tregs and therefore facilitate immune regulation and promote clinical improvements in cGVHD patients. In this report, we describe the case of a cGVHD patient who was treated with daily low-dose IL-2 therapy. Our observations demonstrate that low-dose IL-2 could induce significant expansion of Tregs in vivo leading to improved Treg/Th17 ratios. The patient showed moderate clinical benefits suggesting that multiple factors may be involved in the immunological responses. Therefore, while the therapeutic potential of low-dose IL-2 is promising, strategic approaches may be needed to induce a clinically significant and sustained Treg effect.     

Ex vivo immunological evaluation of stable mixed chimeric patients after matched related donor allogeneic transplantation in sickle cell disease

Background aimsAllogeneic hematopoietic stem cell transplantation is curative for sickle cell disease, and the use of matched related donors, non-myeloablative conditioning and sirolimus immunosuppression results in stable mixed chimerism without graft-versus-host disease (GVHD). However, the time to terminate sirolimus while maintaining mixed chimerism is unclear.MethodsIn this study, we developed a two-way mixed lymphocyte reaction (MLR) to evaluate ex vivo immunoreaction in mixed chimeric patients.ResultsIn co-culture of peripheral blood mononuclear cells (PBMCs) from two healthy controls (without irradiation), we detected proliferation at various ratios of PBMC mixtures (1:9 to 9:1) as well as various concentrations of sirolimus, suggesting that two-way MLR is applicable to patients (having >10% chimerism) undergoing sirolimus treatment. In two-way MLR using PBMCs (including donor and recipient cells) from mixed chimeric patients (n = 28), greater ex vivo proliferation was observed <6 months compared with >6 months post-transplant and healthy control PBMC monoculture. Robust ex vivo proliferation was observed in a patient with acute GVHD, and persistent ex vivo proliferation (until 2 years) was observed in a patient with decreasing donor chimerism.ConclusionsIn summary, we demonstrated that in two-way MLR, ex vivo immunoreaction decreases to low levels ~6 months post-transplant. These findings suggest a rationale to continue immunosuppression for 6 months.     

Production of glucocerebrosidase with terminal mannose glycans for enzyme replacement therapy of Gaucher's disease using a plant cell system

Gaucher's disease, a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCD), is currently treated by enzyme replacement therapy using recombinant GCD (Cerezyme^®) expressed in Chinese hamster ovary (CHO) cells. As complex glycans in mammalian cells do not terminate in mannose residues, which are essential for the biological uptake of GCD via macrophage mannose receptors in human patients with Gaucher's disease, an in vitro glycan modification is required in order to expose the mannose residues on the glycans of Cerezyme^®. In this report, the production of a recombinant human GCD in a carrot cell suspension culture is described. The recombinant plant-derived GCD (prGCD) is targeted to the storage vacuoles, using a plant-specific C-terminal sorting signal. Notably, the recombinant human GCD expressed in the carrot cells naturally contains terminal mannose residues on its complex glycans, apparently as a result of the activity of a special vacuolar enzyme that modifies complex glycans. Hence, the plant-produced recombinant human GCD does not require exposure of mannose residues in vitro , which is a requirement for the production of Cerezyme^®. prGCD also displays a level of biological activity similar to that of Cerezyme^® produced in CHO cells, as well as a highly homologous high-resolution three-dimensional structure, determined by X-ray crystallography. A single-dose toxicity study with prGCD in mice demonstrated the absence of treatment-related adverse reactions or clinical findings, indicating the potential safety of prGCD. prGCD is currently undergoing clinical studies, and may offer a new and alternative therapeutic option for Gaucher's disease.     

儿童获得性再生障碍性贫血造血干细胞移植后Epstein-Barr病毒血症危险因素及预后分析

目的分析儿童获得性再生障碍性贫血(AA)异基因造血干细胞移植(allo-HSCT)后Epstein-Barr病毒(EBV)血症的危险因素、利妥昔单抗的干预效果及EBV相关疾病的临床转归。方法回顾性分析2010年4月至2018年3月于上海儿童医学中心完成allo-HSCT的257例AA患儿,根据是否发生EBV血症分为:EBV血症组(141例,单纯EBV血症组125例和EBV相关疾病组16例)和非EBV血症组(116例)。采用Cox回归分析危险因素,采用Kaplan-Meier法分析累积生存率,定性资料的组间比较采用卡方检验。结果(1)257例患儿行allo-HSCT后发生EBV血症为141例,发生率为54.86﹪(141/257),其中原发感染为5.67﹪(8/257),再激活为94.33﹪(133/141)。EBV血症的中位发生时间为移植后44 d(13~568 d),单纯EBV血症的移植相关死亡率为6.40﹪(8/125);EBV相关疾病的病死率为56.25﹪(9/16)。(2)利妥昔单抗抢先治疗EBV血症的有效率为88.73﹪(63/71)。(3)与单纯EBV血症组比较,EBV相关疾病组患儿生存率降低,差异有统计学意义(Log-rank P<0.001)。(4)多因素分析结果显示,使用全身照射治疗(TBI)预处理的患儿,发生EBV感染的风险是不使用TBI预处理的患儿1.717倍(95﹪的CI:1.160~2.542);患儿移植物中CD34阳性细胞≥3×10^6个/kg是<3×10^6个/kg患儿的1.775倍(95﹪的CI:1.089~2.894)。结论移植后EBV血症的发生和TBI的应用、输注CD34阳性细胞数大于3×10^6个/kg密切相关,EBV血症进展为EBV疾病后移植相关死亡率升高,对高危患儿应积极予以利妥昔单抗抢先治疗改善预后。     

Effect and mechanism of acute graft versus host disease on early diffuse murine lung injury following allogeneic stem cell transplantation

To explore the effect and pathogenssis of acute graft-versus-host disease (aGVHD) on early diffuse lung injury in allogeneic hematopoietic stem cell transplantation (allo-HSCT), we established an aGVHD model of C57BL/6→BALB/c mice. Chest computed tomography (CT) scans, histopathology and the levels of cytokines including tumor necrosis factor α (TNFa) and Interferon (IFNg) in lungs were dynamically detected in recipient mice after transplantation. The incidence of aGVHD was respectively 0%, 0% and 100% in simple irradiation group (A), syngeneic transplant group(B) and allogeneic transplant group (C). Chest CT scans of recipient mice were normal in 3 groups on days +3 and +7 after transplantation. CT showed that two of ten mice had bilateral lung diffuse infiltrate on day +12 (on the brink of death) in group A and 6 of 10 mice had bilateral lung diffuse infiltrate on day +14 (3 d after aGVHD occurring) in group C, and were normal on days +12 and +14 in group B after transplantation. Histopathology of lungs in the 3 groups was similar, consisting of minor interstitial pneumonitis on day +3. Group A showed edema, hyperplasia of epithelial cells and widened alveolar interval on day +7, and epithelial cell necrosis, lymphocyte infiltration, hemorrhage, protein leakage, and local consolidation on day +12. The histopathology of group B showed slight edema of epithelial cells on +7 day, which were slighter than that on day +3, and virtually normal on day +14. The histopathology in group C was characterized by the significant expansion and congestion of capillaries, and lymphocyte infiltration on day +7, the acute pneumonitis was present involving tissue edema, lymphocyte and macrophage infiltration, protein leakage and perivascular inflammation on day +14. In group A, the levels of TNFa were lower on day +7 than on day +3. In group B, the levels of TNFa attained a peak on day +3, which decreased on days +7 and +14. In group C, the levels of TNFa were highest on day +7 and there was a significant difference between those on days +7 and +14 (P=0.816). In group A, the levels of IFNg on day +7 were higher than on day +3. In group B, the levels of IFNg increased progressively, but the comparison of IFNg levels in different times had no statistical significance (P=0.521, 0.118, 0.340). In group C, the levels of IFNg attained a peak by day +7 and decreased on day +14. aGVHD is the main cause of early non-infectious lung injury. T lymphocytes and TNFa are possibly implicated in the pathogenesis of acute GVHD-induced lung injury. The decreased levels of IFNg in lung tissues following transplantation might be associated with pulmonary fibrosis in late non-infectious pulmonary complications.     

Introduction of the green fluorescent protein gene into hematopoietic stem cells results in prolonged discrepancy of in vivo transduction levels between bone marrow progenitors and peripheral blood cells in nonhuman primates

Background

The green fluorescent protein (GFP) has proven a useful marker in retroviral gene transfer studies targeting hematopoietic stem cells (HSCs) in mice. However, several investigators have reported very low in vivo peripheral blood marking levels in nonhuman primates after transplantation of HSCs transduced with the GFP gene. We retrovirally marked cynomolgus monkey HSCs with the GFP gene, and tracked in vivo marking levels within both bone marrow progenitor cells and mature peripheral blood cells following autologous transplantation after myeloablative conditioning.

Methods

Bone marrow cells were harvested from three cynomolgus macaques and enriched for the primitive fraction by CD34 selection. CD34⁺ cells were transduced with one of three retroviral vectors all expressing the GFP gene and were infused after myeloablative total body irradiation (500 cGy × 2). Following transplantation, proviral levels and fluorescence were monitored among clonogenic bone marrow progenitors and mature peripheral blood cells.

Results

Although 13–37% of transduced cells contained the GFP provirus and 11–13% fluoresced ex vivo, both provirus and fluorescence became almost undetectable in the peripheral blood within several months after transplantation regardless of the vectors used. However, on sampling of bone marrow at multiple time points, significant fractions (5–10%) of clonogenic progenitors contained the provirus and fluoresced ex vivo reflecting a significant discrepancy between GFP gene marking levels within bone marrow cells and their mature peripheral blood progeny. The discrepancy (at least one log) persisted for more than 1 year after transplantation. Since no cytotoxic T lymphocytes against GFP were detected in the animals, an immune response against GFP is an unlikely explanation for the low levels of transduced peripheral blood cells. Administration of granulocyte colony stimulating factor and stem cell factor resulted in mobilization of transduced bone marrow cells detectable as mature granulocyte progeny which expressed the GFP gene, suggesting that transduced progenitor cells in bone marrow could be mobilized into the peripheral blood and differentiated into granulocytes.

Conclusions

Low levels of GFP‐transduced mature cells in the peripheral blood of nonhuman primates may reflect a block to differentiation associated with GFP; this block can be overcome in part by nonphysiological cytokine treatment ex vivo and in vivo. Copyright © 2002 John Wiley & Sons, Ltd.

     

Identification of the binding and activating sites of the sphingolipid activator protein, saposin C, with glucocerebrosidase.

Saposin C is a sphingolipid activator protein of 8.5 kDa that activates lysosomal glucocerebrosidase. Previously, we synthesized and characterized a synthetic full-length human saposin C protein that displays 85% of the activity of the native saposin C. In this study we use shorter synthetic peptides derived from the saposin C sequence to map binding and activation sites. By determining the activity and kinetic constant (Kact) values of these peptides, we have identified two functional domains, each comprising a binding site adjacent to or partially overlapping with an activation site. Domains 1 and 2 are located within amino acid positions 6-34 and 41-60, respectively. The activation sites span residues 27-34 and 41-49, whereas binding sites encompass residues 6-27 and 45-60. Peptides containing the sequences of either domain displayed 90% of the activity of the full-length synthetic saposin C. Domain 2, however, bound to glucocerebrosidase by at least an order of magnitude more strongly than domain 1. Binding sites within these domains contain sequences that are excellent candidates for forming amphipathic helical structures. Competition assays demonstrated that the binding of one domain to glucocerebrosidase prevents binding of the other domain, and that saposin A and saposin C bind to the same sites on glucocerebrosidase. A model predicting a saposin C:glucocerebrosidase complex with a stoichiometry of 4:2, respectively, is presented.     

Marking and gene expression by a lentivirus vector in transplanted human and nonhuman primate CD34(+) cells

Recently, gene delivery vectors based on human immunodeficiency virus (HIV) have been developed as an alternative mode of gene delivery. These vectors have a number of advantages, particularly in regard to the ability to infect cells which are not actively dividing. However, the use of vectors based on human immunodeficiency virus raises a number of issues, not the least of which is safety; therefore, further characterization of marking and gene expression in different hematopoietic lineages in primate animal model systems is desirable. We use two animal model systems for gene therapy to test the efficiency of transduction and marking, as well as the safety of these vectors. The first utilizes the rhesus animal model for cytokine-mobilized autologous peripheral blood CD34(+) cell transplantation. The second uses the SCID-human (SCID-hu) thymus/liver chimeric graft animal model useful specifically for human T-lymphoid progenitor cell reconstitution. In the rhesus macaques, detectable levels of vector were observed in granulocytes, lymphocytes, monocytes, and, in one animal with the highest levels of marking, erythrocytes and platelets. In transplanted SCID-hu mice, we directly compared marking and gene expression of the lentivirus vector and a murine leukemia virus-derived vector in thymocytes. Marking was observed at comparable levels, but the lentivirus vector bearing an internal cytomegalovirus promoter expressed less efficiently than did the murine retroviral vector expressed from its own long terminal repeats. In assays for infectious HIV type 1 (HIV-1), no replication-competent HIV-1 was detected in either animal model system. Thus, these results indicate that while lentivirus vectors have no apparent deleterious effects and may have advantages over murine retroviral vectors, further study of the requirements for optimal use are warranted.     

CD4+ T cells in aged or thymectomized recipients of allogeneic stem cell transplantations

Background

CD4+CD25highFOXP3+ regulatory T (Treg) cells, which include thymus-derived and peripherally induced cells, play a central role in immune regulation, and are therefore crucial to prevent graft-versus-host disease (GVHD). The increasing use of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for elderly patients with thymus regression, and our case of allo-HSCT shortly after total thymectomy, raised questions about the activity of thymus-derived Treg cells and peripherally induced Treg cells, which are otherwise indistinguishable.

Results

We found that despite pre-transplant thymectomy or older age, both naïve and effector Treg cells, as well as naïve and effector conventional T cells, proliferated in allo-HSCT recipients. Higher proportions of total Treg cells 1 month post allo-HSCT, and naïve Treg cells 1 year post allo-HSCT, appeared in patients achieving complete chimera without developing significant chronic GVHD, including our thymectomized patient, compared with patients who developed chronic GVHD.

Conclusions

Treg cells that modulate human allogeneic immunity may arise peripherally as well as in the thymus of allo-HSCT recipients.