Dirk Spitzer

C-terminal truncations in human 3'-5' DNA exonuclease TREX1 cause autosomal dominant retinal vasculopathy with cerebral leukodystrophy

Autosomal dominant retinal vasculopathy with cerebral leukodystrophy is a microvascular endotheliopathy with middle-age onset. In nine families, we identified heterozygous C-terminal frameshift mutations in TREX1, which encodes a 3′-5′ exonuclease. These truncated proteins retain exonuclease activity but lose normal perinuclear localization. These data have implications for the maintenance of vascular integrity in the degenerative cerebral microangiopathies leading to stroke and dementias.

Properdin Can Initiate Complement Activation by Binding Specific Target Surfaces and Providing a Platform for De Novo Convertase Assembly

Complement promotes the rapid recognition and elimination of pathogens, infected cells, and immune complexes. The biochemical basis for its target specificity is incompletely understood. In this report, we demonstrate that properdin can directly bind to microbial targets and provide a platform for the in situ assembly and function of the alternative pathway C3 convertases. This mechanism differs from the standard model wherein nascent C3b generated in the fluid phase attaches nonspecifically to its targets. Properdin-directed complement activation occurred on yeast cell walls (zymosan) and Neisseria gonorrhoeae. Properdin did not bind wild-type Escherichia coli, but it readily bound E. coli LPS mutants, and the properdin-binding capacity of each strain correlated with its respective serum-dependent AP activation rate. Moreover, properdin:single-chain Ab constructs were used to direct serum-dependent complement activation to novel targets. We conclude properdin participates in two distinct complement activation pathways: one that occurs by the standard model and one that proceeds by the properdin-directed model. The properdin-directed model is consistent with a proposal made by Pillemer and his colleagues >50 years ago.

Identification of the PGRMC1 protein complex as the putative sigma-2 receptor binding site

The sigma-2 receptor, whose gene remains to be cloned, has been validated as a biomarker for tumor cell proliferation. Here we report the use of a novel photoaffinity probe, WC-21, to identify the sigma-2 receptor binding site. WC-21, a sigma-2 ligand containing both a photoactive moiety azide and a fluorescein isothiocyanate group, irreversibly labels sigma-2 receptors in rat liver; the membrane-bound protein was then identified as PGRMC1 (progesterone receptor membrane component-1). Immunocytochemistry reveals that both PGRMC1 and SW120, a fluorescent sigma-2 receptor ligand, colocalizes with molecular markers of the endoplasmic reticulum and mitochondria in HeLa cells. Overexpression and knockdown of the PGRMC1 protein results in an increase and a decrease in binding of a sigma-2 selective radioligand, respectively. The identification of the putative sigma-2 receptor binding site as PGRMC1 should stimulate the development of unique imaging agents and cancer therapeutics that target the sigma-2 receptor/PGRMC1 complex.

New roles for the major human 3′-5′ exonuclease TREX1 in human disease

Aicardi-Goutières syndrome (AGS), Systemic Lupus Erythematosus (SLE), Familial Chilblain Lupus (FCL), and Retinal Vasculopathy and Cerebral Leukodystrophy (RVCL) {a new term encompassing three independently described conditions with a common etiology - Cerebroretinal Vasculopathy (CRV), Hereditary Vascular Retinopathy (HVR), and Hereditary Endotheliopathy, Retinopathy and Nephropathy (HERNS)} - have previously been regarded as distinct entities. However, recent genetic analysis has demonstrated that each of these diseases maps to chromosome 3p21 and can be caused by mutations in TREX1, the major human 3′-5′exonuclease. In this review, we discuss the putative functions of TREX1 in relationship to the clinical, genetic and functional characteristics of each of these conditions.

Interleukin-6 is a direct mediator of T cell migration.

Interleukin (IL)‐6 is a pleiotropic cytokine involved in the differentiation and proliferation of hematopoietic cells. Hepatocytes respond to IL‐6 with the synthesis and secretion of acute‐phase proteins. In addition, IL‐6 plays a role as a migration factor in vivo. In the present paper, we studied the potential of IL‐6 to mediate migration of human primary T cells and T cell‐derived cell lines. IL‐6 was found to induce migration only in the presence of extracellular matrix, suggesting a cross‐talk between the IL‐6‐ and integrin signal transduction pathways. Furthermore, an IL‐6 gradient is required for chemotactic migration. This activity is not due to the release of secondary chemotactic activities, but is a direct response to IL‐6. T cell migration could also be observed in response to IL‐11, but no migration was found after stimulation with leukemia inhibitory factor or oncostatin M, although these cytokines signal through gp130‐containing receptor complexes. Finally, we present evidence that activation of the mitogen‐activated protein kinase (MAPK) cascade, the phosphatidylinositol 3‐kinase as well as the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is crucial for IL‐6‐induced migration. Selective activation of the JAK/STAT or the MAPK cascade by mutated receptor proteins shows a crucial role of IL‐6‐initiated SH2 domain‐containing tyrosine phosphatase 2/MAPK activity for migration.

Sustained thromboprophylaxis mediated by an RBC-targeted pro-urokinase zymogen activated at the site of clot formation

Plasminogen activators (PAs) are used to treat life-threatening thrombosis, but not for thromboprophylaxis because of rapid clearance, risk of bleeding, and central nervous system (CNS) toxicity. We describe a novel strategy that may help to overcome these limitations by targeting a thrombin-activated PA pro-drug to circulating red blood cells (RBCs). We fused a single chain antibody (scFv Ter-119) that binds to mouse glycophorin A (GPA) with a variant human single-chain low molecular weight urokinase construct that can be activated selectively by thrombin (scFv/uPA-T). scFv/uPA-T bound specifically to mouse RBCs without altering their biocompatibility and retained its zymogenic properties until converted by thrombin into an active 2-chain molecule. As a result, RBC-bound scFv/uPA-T caused thrombin-induced fibrinolysis. One hour and 48 hours after intravenous (IV) injection in mice, approximately 70% and approximately 35% of scFv/uPA-T was retained in the blood, respectively, and approximately 95% of the circulating scFv/uPA-T remained bound to RBCs. A single IV injection of scFv/uPA-T provided effective prophylaxis against arterial and venous thrombosis for up to 24 hours. Thus, prophylactic delivery of RBC-targeted PA pro-drugs activated selectively at the site of clot formation represents a new approach to prevent thrombosis in clinical settings where the risk of clotting is high.

The novel sigma-2 receptor ligand SW43 stabilizes pancreas cancer progression in combination with gemcitabine

BackgroundSigma-2 receptors are over-expressed in proliferating cancer cells, making an attractive target for the targeted treatment of pancreatic cancer. In this study, we investigated the role of the novel sigma-2 receptor ligand SW43 to induce apoptosis and augment standard chemotherapy.ResultsThe binding affinity for sigma-2 ligands is high in pancreas cancer, and they induce apoptosis with a rank order of SV119 < SW43 < SRM in vitro. Combining these compounds with gemcitabine further increased apoptosis and decreased viability. Our in vivo model showed that sigma-2 ligand treatment decreased tumor volume to the same extent as gemcitabine. However, SW43 combination treatment with gemcitabine was superior to the other compounds and resulted in stabilization of tumor volume during treatment, with minimal toxicities.ConclusionsThis study shows that the sigma-2 ligand SW43 has the greatest capacity to augment gemcitabine in a pre-clinical model of pancreas cancer and has provided us with the rationale to move this compound forward with clinical investigations for patients with pancreatic cancer.

Targeting of a mutant plasminogen activator to circulating red blood cells for prophylactic fibrinolysis.

Chemical coupling to carrier red blood cells (RBCs) converts tissue type plasminogen activator (tPA) from a problematic therapeutic into a safe agent for thromboprophylaxis. The goal of this study was to develop a more clinically relevant recombinant biotherapeutic by fusing a mutant tPA with a single-chain antibody fragment (scFv) with specificity for glycophorin A (GPA) on mouse RBCs. The fusion construct (anti-GPA scFv/PA) bound specifically to mouse but not human RBCs and activated plasminogen; this led to rapid and stable attachment of up to 30,000 copies of anti-GPA scFv/PA per mouse RBC that were thereby endowed with high fibrinolytic activity. Binding of anti-GPA scFv/PA neither caused RBC aggregation, hemolysis, uptake in capillary-rich lungs or in the reticuloendothelial system nor otherwise altered the circulation of RBCs. Over 40% of labeled anti-GPA scFv/PA injected in mice bound to RBC, which markedly prolonged its intravascular circulation and fibrinolytic activity compared with its nontargeted PA counterpart, anti-GPA scFv/PA, but not its nontargeted PA analog, prevented thrombotic occlusion in FeCl3 models of vascular injury. These results provide proof-of-principle for the development of a recombinant PA variant that binds to circulating RBC and provides thromboprophylaxis by use of a clinically relevant approach.

Lysosomal membrane permeabilization is an early event in sigma-2 receptor ligand mediated cell death in pancreatic cancer

BackgroundSigma-2 receptor ligands have been studied for treatment of pancreatic cancer because they are preferentially internalized by proliferating cells and induce apoptosis. This mechanism of apoptosis is poorly understood, with varying reports of caspase-3 dependence. We evaluated multiple sigma-2 receptor ligands in this study, each shown to decrease tumor burden in preclinical models of human pancreatic cancer.ResultsFluorescently labeled sigma-2 receptor ligands of two classes (derivatives of SW43 and PB282) localize to cell membrane components in Bxpc3 and Aspc1 pancreatic cancer cells and accumulate in lysosomes. We found that interactions in the lysosome are critical for cell death following sigma-2 ligand treatment because selective inhibition of a protective lysosomal membrane glycoprotein, LAMP1, with shRNA greatly reduced the viability of cells following treatment. Sigma-2 ligands induced lysosomal membrane permeabilization (LMP) and protease translocation triggering downstream effectors of apoptosis. Subsequently, cellular oxidative stress was greatly increased following treatment with SW43, and the hydrophilic antioxidant N-acetylcysteine (NAC) gave greater protection against this than a lipophilic antioxidant, α-tocopherol (α-toco). Conversely, PB282-mediated cytotoxicity relied less on cellular oxidation, even though α-toco did provide protection from this ligand. In addition, we found that caspase-3 induction was not as significantly inhibited by cathepsin inhibitors as by antioxidants. Both NAC and α-toco protected against caspase-3 induction following PB282 treatment, while only NAC offered protection following SW43 treatment. The caspase-3 inhibitor DEVD-FMK offered significant protection from PB282, but not SW43.ConclusionsSigma-2 ligand SW43 commits pancreatic cancer cells to death by a caspase-independent process involving LMP and oxidative stress which is protected from by NAC. PB282 however undergoes a caspase-dependent death following LMP protected by DEVD-FMK and α-toco, which is also known to stabilize the mitochondrial membrane during apoptotic stimuli. These differences in mechanism are likely dependent on the structural class of the compounds versus the inherent sigma-2 binding affinity. As resistance of pancreatic cancers to specific apoptotic stimuli from chemotherapy is better appreciated, and patient-tailored treatments become more available, ligands with high sigma-2 receptor affinity should be chosen based on sensitivities to apoptotic pathways.

Complement-Protected Amphotropic Retroviruses from Murine Packaging Cells

The application of retroviruses generated from murine cells for in vivo gene therapy is restricted primarily because of the rapid inactivation of these viruses by the human complement system. To circumvent this disadvantageous property of murine retroviruses we have generated infectious amphotropic retroviruses that exhibit strong protection against human complement attack. The membrane of these viruses contains a fusion protein, DAFF2A, that is composed of the catalytic domain of the human complement regulatory protein (CRP) decay-accelerating factor (DAF) and the envelope protein of the amphotropic murine leukemia virus (MuLV) 4070A (EnvA). The fusion of two other CRPs, MCP and CD59, to the same amphotropic Env moiety did not lead to equivalent results. The fusion protein DAFF2A was stably expressed in mouse NIH 3T3-based helper cells and independently identified with either alpha-DAF MAb or alpha-Env PAb on the cell membrane. Western blot analysis confirmed the expected molecular weight of the fusion protein. Viral titers obtained from NIH 3T3 helper cell pools were 5 x 10(5) CFU for wild-type amphotropic EnvA virus and 1 x 10(5) CFU for DAFF2A virus, respectively. By blocking the catalytic domain of DAF by pretreatment with alpha-DAF MAb DAFF2A, recombinant virions could be converted to wild-type with respect to sensitivity against human serum. Since the method for producing virions that are protected against human serum should be applicable to any cell type it offers a novel tool for human in vivo gene therapy.