Safety and efficacy of VB-111, an anticancer gene therapy, in patients with recurrent glioblastoma: results of a phase I/II study.

BACKGROUND: VB-111 is a non-replicating adenovirus carrying a Fas-chimera transgene, leading to targeted apoptosis of tumor vascular endothelium and induction of a tumor-specific immune response. This phase I/II study evaluated the safety, tolerability, and efficacy of VB-111 with and without bevacizumab in recurrent glioblastoma (rGBM). METHODS: Patients with rGBM (n = 72) received VB-111 in 4 treatment groups: subtherapeutic (VB-111 dose escalation), limited exposure (LE; VB-111 monotherapy until progression), primed combination (VB-111 monotherapy continued upon progression with combination of bevacizumab), and unprimed combination (upfront combination of VB-111 and bevacizumab). The primary endpoint was median overall survival (OS). Secondary endpoints were safety, overall response rate, and progression-free survival (PFS). RESULTS: VB-111 was well tolerated. The most common adverse event was transient mild-moderate fever. Median OS time was significantly longer in the primed combination group compared with both LE (414 vs 223 days; hazard ratio [HR], 0.48; P = 0.043) and unprimed combination (414 vs 141.5 days; HR, 0.24; P = 0.0056). Patients in the combination phase of the primed combination group had a median PFS time of 90 days compared with 60 in the LE group (HR, 0.36; P = 0.032), and 63 in the unprimed combination group (P = 0.72). Radiographic responders to VB-111 exhibited characteristic, expansive areas of necrosis in the areas of initial enhancing disease. CONCLUSIONS: Patients with rGBM who were primed with VB-111 monotherapy that continued after progression with the addition of bevacizumab showed significant survival and PFS advantage, as well as specific imaging characteristics related to VB-111 mechanism of action. These results warrant further assessment in a randomized controlled study.

Treatment with lecinoxoids attenuates focal and segmental glomerulosclerosis development in nephrectomized rats.

Focal segmental glomerulosclerosis (FSGS) is a scarring process associated with chronic low-grade inflammation ascribed to toll-like receptor (TLR) activation and monocyte migration. We developed synthetic, small-molecule lecinoxoids, VB-201 and VB-703, that differentially inhibit TLR-2- and TLR-4-mediated activation and monocyte migration. The efficacy of anti-inflammatory lecinoxoid treatment on FSGS development was explored using a 5/6 nephrectomy rat model. Five-sixths of nephrectomized rats were treated with lecinoxoids VB-201, VB-703 or PBS, for 7 weeks. Upon sacrifice, albumin/creatinine ratio, glomerulosclerosis, fibrosis-related gene expression and the number of glomerular and interstitial monocyte were evaluated. Treatment of nephrectomized rats with lecinoxoids ameliorated glomerulosclerosis. The percentage of damaged glomeruli, glomerular sclerosis and glomeruli fibrotic score was significantly reduced following VB-201 and VB-703 treatment. VB-703 attenuated the expression of fibrosis hallmark genes collagen, fibronectin (FN) and transforming growth factor ß (TGF-ß) in kidneys and improved albumin/creatinine ratio with higher efficacy than did VB-201, but only VB-201 significantly reduced the number of glomerular and interstitial monocytes. These results indicate that treatment with TLR-2, and more prominently, TLR-4 antagonizing lecinoxioids, is sufficient to significantly inhibit FSGS. Moreover, inhibiting monocyte migration can also contribute to treatment of FSGS. Our data demonstrate that targeting TLR-2-TLR-4 and/or monocyte migration directly affects the priming phase of fibrosis and may consequently perturb disease parthogenesis.

Newly characterized motile sperm domain-containing protein 2 promotes human breast cancer metastasis.

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among women worldwide. Breast cancer metastasis results in poor prognosis and increased mortality, but the mechanisms of breast cancer metastasis are yet to be fully resolved. Identifying distinctive proteins that regulate metastasis might be targeted to improve therapy in breast cancer. We previously described MOSPD2 as a surface membrane protein that regulates monocyte migration in vitro. In this study, we demonstrate for the first time that MOSPD2 has a major role in breast cancer cell migration and metastasis. MOSPD2 expression was highly elevated in invasive and metastatic breast cancer while it was absent or residual in normal tissue and in primary in situ tumors. In vitro experiments showed that silencing MOSPD2 in different breast cancer cell lines significantly inhibited cancer cell chemotaxis migration. Mechanistically, we found that silencing MOSPD2 profoundly abated phosphorylation events that are involved in breast tumor cell chemotaxis. In vivo, MOSPD2-silenced breast cancer cells exhibited marked impaired metastasis to the lungs. These results indicate that MOSPD2 plays a key role in the migration and metastasis of breast cancer cells and may be used to prevent the spreading of breast cancer cells and to mediate their death.

Identification of Motile Sperm Domain-Containing Protein 2 as Regulator of Human Monocyte Migration.

Binding of chemokines to their cognate receptors on monocytes instigates a cascade of events that directs these cells to migrate to sites of inflammation and cancerous tissues. Although targeting of selected chemokine receptors on monocytes exhibited preclinical efficacy, attempts to translate these studies to the clinic have failed thus far, possibly due to redundancy of the target receptor. We reveal that motile sperm domain-containing protein 2 (MOSPD2), a protein with a previously unknown function, regulates monocyte migration in vitro. This protein was found to be expressed on the cytoplasmic membrane of human monocytes. Silencing or neutralizing MOSPD2 in monocytes restricted their migration when induced by different chemokines. Mechanistically, silencing MOSPD2 inhibited signaling events following chemokine receptor ligation. When tested for expression in other immune cell subsets, MOSPD2 was apparent also, though less abundantly, in neutrophils, but not in lymphocytes. Thus, in the presence of neutralizing Abs, neutrophil migration was inhibited to some extent whereas lymphocyte migration remained intact. In view of these results, we suggest MOSPD2 as a potential target protein for treating diseases in which monocyte and neutrophil accumulation is correlated with pathogenesis.

Treatment with Oxidized Phospholipids Directly Inhibits Nonalcoholic Steatohepatitis and Liver Fibrosis Without Affecting Steatosis.

BACKGROUND AND AIMS: Previous studies demonstrated that toll-like receptors 4 and 2 (TLR-4 and TLR-2), which are expressed on liver-resident Kupffer, hepatic stellate cells, and circulating monocytes, play a role in nonalcoholic fatty liver disease. Lecinoxoids are oxidized phospholipids that antagonize TLR-2- and TLR-4-mediated activation of innate immune cells and inhibit monocyte migration. In this study, we tested the effect of two functionally different lecinoxoids on the development of nonalcoholic steatohepatitis and liver fibrosis in a mouse model. METHODS: Two-day-old C57BL/6 mice were injected with streptozotocin and fed a high-fat diet from Week 4 after birth. At Week 6 post-birth, lecinoxoids VB-201 or VB-703 were given orally, once daily, for 3 weeks. Telmisartan was administered orally, once daily, for 3 weeks, as positive control. At experiment conclusion, biochemical indices were evaluated. HE stain and quantitative PCR were used to determine the extent of steatosis and steatohepatitis, and Sirius red stain was used to assess liver fibrosis. RESULTS: Treatment with lecinoxoids did not alter the concentration of blood glucose, liver triglycerides, or steatosis compared with solvent-treated mice. However, whereas VB-201 inhibited the development of fibrosis and, to some extent, liver inflammation, VB-703 significantly lessened both liver inflammation and fibrosis. CONCLUSIONS: This study indicates that using lecinoxoids to antagonize TLR-2, and more prominently TLR-4, is sufficient to significantly inhibit nonalcoholic steatohepatitis and liver fibrosis. Inhibiting monocyte migration with lecinoxoids that are relatively weak TLR-4 antagonists may alter liver fibrosis and to some extent nonalcoholic steatohepatitis.

VB-111: a novel anti-vascular therapeutic for glioblastoma multiforme.

Glioblastoma multiforme (GBM) is among the most highly vascularized of solid tumors, contributing to the infiltrative nature of the disease, and conferring poor outcome. Due to the critical dependency of GBM on growth of new endothelial vasculature, we evaluated the preclinical activity of a novel adenoviral gene therapy that targets the endothelium within newly formed blood vessels for apoptosis. VB-111, currently in phase II clinical trials, consists of a non-replicating Adenovirus 5 (El deleted) carrying a proapoptotic human Fas-chimera (transgene) under the control of a modified murine promoter (PPE-1-3×) which specifically targets endothelial cells within the tumor vasculature. Here we report that a single intravenous dose of 2.5 × 10(11) or 1 × 10(11) VPs was sufficient to extend survival in nude rats bearing U87MG-luc2 or nude mice bearing U251-luc, respectively. Bioluminescence imaging of nude rats showed that VB-111 effectively inhibited tumor growth within four weeks of treatment. This was confirmed in a select group of animals by MRI. In our mouse model we observed that 3 of 10 nude mice treated with VB-111 completely lost U251 luciferase signal and were considered long term survivors. To assess the antiangiogenic effects of VB-111, we evaluated the tumor-associated microvaculature by CD31, a common marker of neovascularization, and found a significant decrease in the microvessel density by IHC. We further assessed the neovasculature by confocal microscopy and found that VB-111 inhibits vascular density in two separate mouse models bearing U251-RFP xenografts. Collectively, this study supports the clinical development of VB-111 as a treatment for GBM.

Therapies targeting innate immunity for fighting inflammation in atherosclerosis.

Atherosclerosis is a smoldering disease of the vasculature that can lead to the occlusion of the arteries, resulting in ischemia of the heart and brain. For many years, the asserted underlying mechanism of atherosclerosis, supported by its epidemiology, was based on the "cholesterol hypothesis" that people with high blood cholesterol are at higher risk of developing cardiovascular disease. This hypothesis instigated a vigorous search for treatment that yielded the generation of statins, which specifically reduce LDL cholesterol. Since then, statins have revolutionized the way people are treated for the prevention of atherosclerosis. Nonetheless, despite this potent class of drugs, cardiovascular disease continues to be the leading cause of death in many parts of the world, suggesting that additional mechanisms are involved in disease pathogenesis. Intensive research has revealed that the atherosclerotic plaque is enriched with leukocytes, and that macrophages constitute the majority of immune cells in the lesion. Monocytes/macrophages are now recognized as the prime immune cells involved in the development of atherosclerosis and are implicated to affect the size, composition and vulnerability of the atherosclerotic plaque. While many of the macrophage-derived pro-inflammatory mechanisms associated with atherogenesis have been characterized, such as cell adhesion, cytokine production and protease secretion, there is a dearth of drugs that specifically target innate immunity for treating patients with atherosclerosis. This review presents pre-clinical studies, and in most cases following clinical trials with antagonists and agonists that have been designed to counteract inflammation in atherosclerosis and associated diseases, highlighting targets expressed predominantly in monocytes.

Inhibition of monocyte chemotaxis by VB-201, a small molecule lecinoxoid, hinders atherosclerosis development in ApoE⁻/⁻ mice.

OBJECTIVE: Monocytes are motile cells which sense inflammatory stimuli and subsequently migrate to sites of inflammation. Key players in host defense, monocytes have nevertheless been implicated as requisite mediators of several chronic inflammatory diseases. Inhibition of monocyte chemotaxis is therefore an attractive anti-inflammatory strategy. Oxidized phospholipids (OxPL) are native regulators of inflammation, yet their direct effect on monocyte chemotaxis is poorly defined. In this study, we investigated the direct effect of natural and synthetic phospholipids on monocyte chemotaxis. METHODS: Exploring various phospholipids using in vitro chemotaxis assays, we found that the natural phospholipid 1-palmitoyl-2-glutaryl phosphatidylcholine (PGPC) can decrease monocyte chemotaxis by 50%, while other tested OxPL had no effect. We generated a library of synthetic OxPL designated lecinoxoids, which was screened for anti-inflammatory properties. RESULTS AND CONCLUSIONS: VB-201, a small-molecule lecinoxoid, exhibited up to 90% inhibition of monocyte chemotaxis in vitro. Molecular analysis revealed that the effect of VB-201 was not restricted to a specific chemotactic ligand or receptor, and resulted from inhibition of signaling pathways required for monocyte chemotaxis. Interestingly, VB-201 did not inhibit monocyte adhesion or phagocytosis and had no effect on chemotaxis of CD4(+) T-cells or neutrophils. In vivo, oral treatment with VB-201 reduced monocyte migration in a peritonitis model and inhibited atheroma development in ApoE(-/-) mice, without affecting cholesterol or triglyceride levels. Our findings highlight a novel role played by native and synthetic phospholipids in regulation of monocyte chemotaxis. The data strengthen the involvement of phospholipids as key signaling molecules in inflammatory settings and demonstrate their potential therapeutic applicability.

Phase I dose-escalation study of VB-111, an antiangiogenic virotherapy, in patients with advanced solid tumors.

PURPOSE: VB-111 is an antiangiogenic agent consisting of a nonreplicating adenovirus vector (Ad-5) with a modified murine pre-proendothelin promoter leading to apoptosis of tumor vasculature by expressing a Fas-chimera transgene in angiogenic endothelial cells. In a phase I dose-escalation study, pharmacokinetics, pharmacodynamics, safety, and efficacy of a single dose of VB-111 in patients with advanced solid tumors were evaluated. EXPERIMENTAL DESIGN: VB-111 was administered as a single i.v. infusion at escalating doses from 1 × 10(10) (cohort 1) to 1 × 10(13) (cohort 7) viral particles (VP) in successive cohorts. Assessments included pharmacokinetic and pharmacodynamic profiles, tumor response, and overall survival. RESULTS: Thirty-three patients were enrolled. VB-111 was safe and well-tolerated; self-limited fever and chills were seen at doses above 3 × 10(11) VPs. Transgene expression was not detected in blood but was detected in an aspirate from a subcutaneous metastasis after treatment. One patient with papillary thyroid carcinoma had a partial response. CONCLUSIONS: VB-111 was safe and well tolerated in patients with advanced metastatic cancer at a single administration of up to 1 × 10(13) VPs. Evidence of transgene expression in tumor tissue and tumor response was observed.

A Lecinoxoid, an oxidized phospholipid small molecule, constrains CNS autoimmune disease.

Oxidized phospholipids (Ox-PLs) are generated in abundance at sites of inflammation. Recent studies have indicated that Ox-PLs may also exhibit anti-inflammatory activities. In this study, we investigated the beneficial effect of VB-201, a pure synthetic Ox-PL analog that we synthesized, on the development of a central nervous system (CNS) autoimmune inflammatory disease, in vivo. Oral administration of VB-201 ameliorated the severity of experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) peptide MOG35-55, and restrained the encephalogenicity of MOG35-55-specific T-cells. Our data presents a novel prospect for the role of Ox-PL analogs in CNS inflammatory diseases.

Systemic administration of radiation-potentiated anti-angiogenic gene therapy against primary and metastatic cancer based on transcriptionally controlled HSV-TK.

Transcription-targeted gene delivery directed against angiogenic endothelial cells is a new approach against advanced cancer. Moreover, the herpes simplex virus-thymidine kinase (HSV-TK) gene coupled with low dose radiotherapy is an efficient and externally controlled cytotoxic system. We have previously demonstrated enhanced endothelial-specific cell expression and killing using the modified murine pre-proendothelin-1 promoter (PPE1-3x) to direct adenoviral expression of a pro-apoptotic gene. The purpose of this study was to create an externally potentiated systemic antiangiogenic gene delivery system based on an adenoviral vector expressing HSV-TK under the regulation of PPE1-3X promoter combined with radiotherapy for eradicating metastatic cancer. Ad-PPE1-3x-TK induced endothelial-specific cell killing in-vitro upon introduction of the prodrug ganciclovir (GCV). BALB/c mice bearing a primary CT-26 colon carcinoma tumor showed tumor growth suppression and diminished tumor angiogenesis when the vector was administered intravenously, activated with GCV and potentiated with a single sub-therapeutic and non-toxic radiation dose. Moreover, intravenous administration of the vector, activated with GCV and potentiated with chest aimed radiation, to C57BL/6 mice bearing Lewis lung carcinoma metastases resulted in prolongation of mice survival. PPE1-3x-regulated HSV-TK expression was detected only in lung metastases in contrast to CMV-regulated expression. This novel system may benefit patients with metastatic disease.

Systemic administration of a conditionally replicating adenovirus, targeted to angiogenesis, reduced lung metastases burden in cotton rats.

PURPOSE: Angiogenesis is an essential process for solid tumor development. To interfere with angiogenesis, AdPPE3x-E1, an adenovirus that is transcriptionally targeted to replicate in angiogenic endothelial cells, was constructed, by replacing the E1 promoter with the modified preproendothelin-1 promoter, PPE-1-3x, previously shown to induce specific transcription in angiogenic endothelial cells. EXPERIMENTAL DESIGN: The specificity of AdPPE3x-E1 to endothelial cells was shown by quantitative PCR and immunostaining, and its antiangiogenic effect was evaluated in Matrigel models. The in vivo efficacy of AdPPE3x-E1 was also tested in a cotton rat lung metastases model. RESULTS: The replication rate of AdPPE3x-E1 in endothelial cells was similar to that of AdCMV-E1, a nonselective replicating adenovector, but the replication rate was reduced up to 60-fold in nonendothelial cells. Moreover, AdPPE3x-E1 reduced endothelial cell viability by 90% whereas nonendothelial cells were not affected. In in vitro and in vivo Matrigel models, endothelial cells infected with AdPPE3x-E1 did not develop capillary-like structures. The systemic administration of AdPPE3x-E1 reduced the lung metastases burden in a cotton rat model by 55%, compared with saline-treated rats, without significant evidence of toxicity. Quantitative PCR analysis showed that the viral copy number of AdPPE3x-E1 was increased 3-fold in the lung metastases but not in the liver, compared with a nonreplicating adenovector control. CONCLUSIONS: We have shown here for the first time an antimetastatic effect induced by an angiogenesis-transcriptionally targeted adenovirus following systemic administration. Because adenovirus replication is more efficient in humans than in cotton rats, we assume a significant effect for AdPPE3x-E1 treatment in fighting human solid tumors and metastases.

Endothelial-targeted gene transfer of hypoxia-inducible factor-1alpha augments ischemic neovascularization following systemic administration.

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a key regulator of the response to low oxygen levels and has been used for therapeutic angiogenesis. Various routes of administration have been used for delivering genes to the ischemic region including the intramuscular (IM) and intraarterial routes. When compared with these delivery methods, the intravenous (IV) route confers many advantages, including less invasiveness and lower cost. However, its use is hampered by the fact that it does not result in specific and robust tissue expression of the genes. Our aim was to determine the feasibility, safety, and therapeutic efficacy of systemic administration of adenoviral-mediated HIF-1alpha targeted to the endothelium. Using confocal microscopy and biodistribution studies we demonstrated that a modified murine preproendothelin-1 promoter (PPE1-3x) can target gene expression specifically to endothelial cells within ischemic muscle following systemic IV administration in C57BL/6 mice. Accordingly, an adenovirus expressing a PPE1-3x-regulated stabilized HIF-1alpha molecule, further activated by constitutive activation of its C-transactivation domain (C-TAD), was created. Systemic tail-vein administration of this adenovirus in a mouse hindlimb ischemia model resulted in enhanced blood perfusion, improved clinical outcome, and increased capillary density without systemic toxicity, in contrast to the profound systemic side effects and lack of therapeutic efficacy following cytomegalovirus (CMV)-regulated HIF-1alpha administration. Collectively, these data suggest that transcriptionally controlled systemic proangiogenic gene therapy is feasible, safe, and efficacious.

Activation of C-transactivation domain is essential for optimal HIF-1 alpha-mediated transcriptional and angiogenic effects.

UNLABELLED: HIF-1 is a transcription factor that regulates genes involved in oxygen homeostasis. In normoxia, degradation of the HIF-1 alpha subunit is enabled by two prolyl hydroxylations at residues P402 and P564, while inactivation occurs through asparaginyl hydroxylation at residue N803 within its C-transactivation domain (C-TAD). For therapeutic angiogenesis purposes, HIF-1 alpha stabilization was previously achieved by either deleting its oxygen-dependent degradation domains, or introducing two proline point mutations at residues P402 and P564. We assessed the hypothesis that constitutive activation of HIF-1 alpha in addition to its stabilization would result in greater HIF-1 alpha transcriptional activity and angiogenic effects than mere stabilization of the molecule. For this, we constructed a Triple mutant HIF-1 alpha (TM), bearing mutations P402A and P564G N803A. Transient co-transfections with hypoxia-responsive element-luciferase construct revealed 2 to 2.5-fold increase in transcriptional activity of TM compared with P402A P564G double mutant and wild-type HIF-1 alpha. In-vitro angiogenesis assay using transfected human umbilical vein endothelial cells (HUVEC) showed that TM stimulated tube formation to a greater extent than both P402A P564G mutant and wild-type HIF-1 alpha. Accordingly, ELISA revealed that VEGF levels within the transfected HUVEC were about 10-fold greater with the TM. CONCLUSIONS: Constitutive activation of the HIF-1 alpha C-TAD, and not merely stabilization of the HIF-1 alpha molecule, is essential for optimal HIF-mediated transcriptional and angiogenic effects. This finding could have important implications for therapeutic angiogenesis using HIF-1 alpha.

Endothelial-targeted Gene Transfer of Hypoxia-inducible Factor-1α Augments Ischemic Neovascularization Following Systemic Administration.

Hypoxia-inducible factor-1α (HIF-1α) is a key regulator of the response to low oxygen levels and has been used for therapeutic angiogenesis. Various routes of administration have been used for delivering genes to the ischemic region including the intramuscular (IM) and intraarterial routes. When compared with these delivery methods, the intravenous (IV) route confers many advantages, including less invasiveness and lower cost. However, its use is hampered by the fact that it does not result in specific and robust tissue expression of the genes. Our aim was to determine the feasibility, safety, and therapeutic efficacy of systemic administration of adenoviral-mediated HIF-1α targeted to the endothelium. Using confocal microscopy and biodistribution studies we demonstrated that a modified murine preproendothelin-1 promoter (PPE1-3x) can target gene expression specifically to endothelial cells within ischemic muscle following systemic IV administration in C57BL/6 mice. Accordingly, an adenovirus expressing a PPE1-3x-regulated stabilized HIF-1α molecule, further activated by constitutive activation of its C-transactivation domain (C-TAD), was created. Systemic tail-vein administration of this adenovirus in a mouse hindlimb ischemia model resulted in enhanced blood perfusion, improved clinical outcome, and increased capillary density without systemic toxicity, in contrast to the profound systemic side effects and lack of therapeutic efficacy following cytomegalovirus (CMV)-regulated HIF-1α administration. Collectively, these data suggest that transcriptionally controlled systemic proangiogenic gene therapy is feasible, safe, and efficacious.

Vascular wall maturation and prolonged angiogenic effect by endothelial-specific platelet-derived growth factor expression.

BACKGROUND: The implementation of angiogenic gene therapy at clinics is hindered by the transience of the therapeutic effect. Recruiting vascular wall smooth muscle cells, a process termed 'maturation', can stabilize newly formed vessels. OBJECTIVE: To induce angiogenesis followed by vessel maturation in a murine ischemic limb model by endothelial cell-specific promoter regulated expression of vascular endothelial growth factor (VEGF) and platelet-derived growth factor-BB (PDGF-BB). METHODS: We constructed adenoviral vectors containing angiogenic factors VEGF and PDGF-B regulated by a modified preproendothelin-1 (PPE-1-3x) promoter and investigated their angiogenic effect in a murine ischemic limb model. RESULTS: VEGF gene therapy increased perfusion and the vessel density in the limb shortly after expression with PPE-1-3x promoter or cytomegalovirus (CMV) promoter vectors, but only PPE-1-3xVEGF treatment exhibited a sustained effect. Expression of PDGF-B by PPE-1-3x promoter resulted in morphological maturation of the vasculature and further increased the perfusion, while nonspecific expression of PDGF-B with CMV promoter had no therapeutic effect. Regulation of dual therapy with VEGF and PDGF-B by PPE-1-3x promoter resulted in an early-onset, sustained angiogenic effect accompanied by vessel maturation. CONCLUSIONS: Systemic gene therapy with the angiogenic factors VEGF and PDGF-B under angiogenic- endothelial cell-specific regulation was effective in inducing functionally and morphologically mature vasculature.

Suppression of early atherosclerosis in LDL-receptor deficient mice by oral tolerance with beta 2-glycoprotein I.

BACKGROUND: Atherosclerosis is considered analogous to chronic inflammatory diseases. Beta 2-glycoprotein I (beta 2GPI) is a phospholipid binding protein shown to serve as a target for prothrombotic antiphospholipid antibodies. It has recently been demonstrated to drive an immune mediated reaction and enhance murine atherosclerosis. Oral tolerance is a method in which feeding a given antigen, downregulates the respective immune responses towards it, and attenuates concomitant organ specific disorders. Herein, we tested the hypothesis, that inhibiting cellular immunity to beta 2GPI would result in suppression of fatty streak formation in mice. METHODS AND RESULTS: LDL receptor deficient mice were fed different doses of human or bovine beta 2GPI or BSA and than switched to an atherogenic diet. To determine the effect of feeding on lymph node proliferative indices, separate groups of mice were fed beta 2GPI and then immunized with the respective antigen. Feeding either human or bovine beta 2GPI was effective in attenuating atherosclerosis as compared to control fed animals. Oral feeding with of beta 2GPI inhibited lymph node cell reactivity to beta 2GPI in mice immunized against the human protein. Oral tolerance was also capable of reducing reactivity to oxidized LDL in mice immunized against oxLDL. IL-4 and IL-10 production was upregulated in lymph node cells of beta 2GPI-tolerant mice immunized against beta 2GPI, upon priming with the respective protein. CONCLUSION: Thus, oral administration of beta 2GPI is an effective means of suppressing atherogenesis in mice and should further be investigated.

Transcription-controlled gene therapy against tumor angiogenesis.

A major drawback of current approaches to antiangiogenic gene therapy is the lack of tissue-specific targeting. The aim of this work was to trigger endothelial cell-specific apoptosis, using adenoviral vector-mediated delivery of a chimeric death receptor derived from the modified endothelium-specific pre-proendothelin-1 (PPE-1) promoter. In the present study, we constructed an adenovirus-based vector that targets tumor angiogenesis. Transcriptional control was achieved by use of a modified endothelium-specific promoter. Expression of a chimeric death receptor, composed of Fas and TNF receptor 1, resulted in specific apoptosis of endothelial cells in vitro and sensitization of cells to the proapoptotic effect of TNF-alpha. The antitumoral activity of the vectors was assayed in two mouse models. In the model of B16 melanoma, a single systemic injection of virus to the tail vein caused growth retardation of tumor and reduction of tumor mass with central tumor necrosis. When the Lewis lung carcinoma lung-metastasis model was applied, i.v. injection of vector resulted in reduction of lung-metastasis mass, via an antiangiogenic mechanism. Moreover, by application of the PPE-1-based transcriptional control, a humoral immune response against the transgene was avoided. Collectively, these data provide evidence that transcriptionally controlled, angiogenesis-targeted gene therapy is feasible.

Altered memory B-cell homeostasis in human aging.

Previous studies of age-associated immune system changes revealed alterations in expressed immunoglobulin heavy chain variable domain repertoires, and variability in the fraction of expressed heavy chain variable domain genes with mutations. To test whether the latter finding reflected a variation in memory B-cell numbers, we measured circulating memory B cells of 11 healthy elderly subjects, 173 nursing-home residents, and 34 healthy young adults. A large fraction of old adults have low values for memory cells both as a percentage of all B cells and as an absolute memory B-cell concentration. The range of both values is much wider in old adults than in young adults, and it is much wider than the range of T-cell concentrations. Memory B-cell concentration, which was positively correlated with memory T-cell concentrations but inversely related to in vitro T-cell responses to mitogens, may reflect highly individual rates of immune senescence, and it may serve as an amplified marker of underlying T-cell function.