Berith Nilsson

Generation of iC3 at the interface between blood and gas.

Earlier studies have shown that C3 can be denatured when blood comes in contact with a polystyrene surface. This study was undertaken to sec if similar denaturation of C3 occurs at the gas plasma interface which is found in all kinds of oxygenator used during cardio‐pulmonary operations. An in vitro system consisting of gas bubbling through human blood, serum or plasma was used. The generation of C3a, as an indicator of complement activation, and iC3 and iC3 fragments were monitored. Both C3a and iC3/iC3 fragments levels were increased during bubbling. In contrast to the C3a level, no reduction in iC3/iC3 fragments formation was seen in the presence of EDTA, indicating that il was independent of complement activation. The rate of iC3/iC3 fragments generation was unaffected by the composition of the gas (pure oxygen, pure nitrogen or air), suggesting that the denaturation of C3 indeed occurred at the serum gas interface. C3 and iC3/iC3 fragments were isolated from bubbled EDTA‐chelated serum by PEG precipitation and chromatography on FPLC, using a Mono S column and detected by two ELISAs, specific for native C3 and iC3/iC3 fragments. After 240 min approximately 20% of the total amount of C3 consisted of intact iC3 and it was confirmed that this population bound to human erythrocytes.

CAR/FoxP3-engineered T regulatory cells target the CNS and suppress EAE upon intranasal delivery

BackgroundMultiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). In the murine experimental autoimmune encephalomyelitis (EAE) model of MS, T regulatory (Treg) cell therapy has proved to be beneficial, but generation of stable CNS-targeting Tregs needs further development. Here, we propose gene engineering to achieve CNS-targeting Tregs from naïve CD4 cells and demonstrate their efficacy in the EAE model.MethodsCD4+ T cells were modified utilizing a lentiviral vector system to express a chimeric antigen receptor (CAR) targeting myelin oligodendrocyte glycoprotein (MOG) in trans with the murine FoxP3 gene that drives Treg differentiation. The cells were evaluated in vitro for suppressive capacity and in C57BL/6 mice to treat EAE. Cells were administered by intranasal (i.n.) cell delivery.ResultsThe engineered Tregs demonstrated suppressive capacity in vitro and could efficiently access various regions in the brain via i.n cell delivery. Clinical score 3 EAE mice were treated and the engineered Tregs suppressed ongoing encephalomyelitis as demonstrated by reduced disease symptoms as well as decreased IL-12 and IFNgamma mRNAs in brain tissue. Immunohistochemical markers for myelination (MBP) and reactive astrogliosis (GFAP) confirmed recovery in mice treated with engineered Tregs compared to controls. Symptom-free mice were rechallenged with a second EAE-inducing inoculum but remained healthy, demonstrating the sustained effect of engineered Tregs.ConclusionCNS-targeting Tregs delivered i.n. localized to the CNS and efficiently suppressed ongoing inflammation leading to diminished disease symptoms.

Modification of the complement binding properties of polystyrene: Effects of end-point heparin attachment

In recent years, conjugation of heparin to biomaterials has been shown to improve its biocompatibility. The purpose of the present work was to compare complement activation and binding of C3 to unmodified and heparin‐treated polystyrene surfaces of microtitre plates. When polystyrene was incubated with human serum, C3 was deposited on the surface by both adsorption and binding dependent on activation of the classical (CPW) and alternative (APW) pathways After end‐point attachment of heparin, significant C3 deposition, although at reduced levels, occurred only by CPW‐mediated mechanisms. while adsorption and APW ‐mediated binding were strongly reduced. Generally, the modified surface bound lower amounts of protein, e.g. serum albumin and IgG, than the unmodified. By contrast, it had increased affinity for Clq which leads to binding of Cl and activation of complement via the CPW. Nevertheless, the net effect of the surface modification on the complement reaction was an overall reduction of C3 binding due to obliteration of APW. This can be related to an enhanced factor H/I‐dependent down‐regulation of C3b and to the lowered protein‐adsorbing property of the surface, both of which have inhibitory effects on APW and on the C3 shunt‐dependent activation of the complement system.

Double-Detargeted Oncolytic Adenovirus Shows Replication Arrest in Liver Cells and Retains Neuroendocrine Cell Killing Ability

Background We have previously developed an oncolytic serotype 5 adenovirus (Ad5) with chromogranin-A (CgA) promoter-controlled E1A expression, Ad[CgA-E1A], with the intention to treat neuroendocrine tumors, including carcinoids. Since carcinoids tend to metastasize to the liver it is important to fully repress viral replication in hepatocytes to avoid adenovirus-related liver toxicity. Herein, we explore miRNA-based regulation of E1A expression as a complementary mechanism to promoter-based transcriptional control. Methodology/Principal Findings Ad[CgA-E1A-miR122], where E1A expression is further controlled by six tandem repeats of the target sequence for the liver-specific miR122, was constructed and compared to Ad[CgA-E1A]. We observed E1A suppression and replication arrest of the miR122-detargeted adenovirus in normal hepatocytes, while the two viruses killed carcinoid cells to the same degree. Repeated intravenous injections of Ad[CgA-E1A] induced liver toxicity in mice while Ad[CgA-E1A-miR122] injections did not. Furthermore, a miR122-detargeted adenovirus with the wild-type E1A promoter showed reduced replication in hepatic cells compared to wild-type Ad5 but not to the same extent as the miR122-detargeted adenovirus with the neuroendocrine-selective CgA promoter. Conclusions/Significance A combination of transcriptional (promoter) and post-transcriptional (miRNA target) regulation to control virus replication may allow for the use of higher doses of adenovirus for efficient tumors treatment without liver toxicity.

Conformational epitopes of C3 reflecting its mode of binding to an artificial polymer surface

The aim of the study was to investigate the incompletely understood mechanisms of complement (C) activation and binding on artificial biomaterials. Polystyrene in the form of microtitre plates was used as target for C binding, detectable by ELISA using monoclonal anti-C3 antibodies specific for conformational epitopes expressed by bound C3 and C3 fragments. C3 binding in whole blood/plasma/serum is maximal at low dilutions and occurs predominantly by C activation. At higher dilutions, C3 binding occurs at approximately 1/3 of maximal levels and is solely an effect of adsorption. C3 adsorption in the lower serum dilution range, occurs at low but clearly detectable levels. Comparative epitope analysis between C3 fragments, actively bound to polystyrene in the presence of serum, and of iC3b bound to sheep erythrocytes, clearly indicates that C3 binding/activation on polystyrene takes place as a C3 convertase-mediated reaction, which in serum/plasma is followed by a secondary factor I-dependent degradation of the bound C3b into iC3b. The neo-epitope analysis of serum-contacting polystyrene revealed that the adsorbed C3, throughout the entire serum dilution range tested, deposits in a state closely similar to that observed for purified C3 at a high packing density. Polystyrene surfaces with adsorbed purified C3 expressing this epitope profile were found to mediate APW dependent deposition of C3b in pig serum, presumably by forming a hybrid convertase with porcine Bb. These data therefore suggest that adsorbed C3 on serum-contacting polystyrene surfaces may initiate complement activation via the APW.

Structural and functional analysis of C3 using monoclonal antibodies

Monoclonal antibodies (MoAbs) have greatly facilitated the structural and functional analysis of proteins in general and of the third protein of complement (C3) in particular. Various aspects of the structure and functions of C3 have been addressed using MoAbs; these include: (a) the study of conformational changes occurring in the C3 molecule and its fragments during complement activation, (b) the analysis of the interactions of C3 with other complement components and receptors as well as with proteins of foreign origin, and (c) the detection of C3 activation products in biological fluids. The purpose of this review is to summarize the contribution that MoAbs have made in understanding the structure and functions of C3.

An ex vivo loop system models the toxicity and efficacy of PEGylated and unmodified adenovirus serotype 5 in whole human blood

Polyethylene glycol coating (PEGylation) of adenovirus serotype 5 (Ad5) has been shown to effectively reduce immunogenicity and increase circulation time of intravenously administered virus in mouse models. Herein, we monitored clot formation, complement activation, cytokine release and blood cell association upon addition of uncoated or PEGylated Ad5 to human whole blood. We used a novel blood loop model where human blood from healthy donors was mixed with virus and incubated in heparin-coated PVC tubing while rotating at 37 °C for up to 8 h. Production of the complement components C3a and C5a and the cytokines IL-8, RANTES and MCP-1 was significantly lower with 20K-PEGylated Ad5 than with uncoated Ad5. PEGylation prevented clotting and reduced Ad5 binding to blood cells in blood with low ability to neutralize Ad5. The effect was particularly pronounced in monocytes, granulocytes, B-cells and T-cells, but could also be observed in erythrocytes and platelets. In conclusion, PEGylation of Ad5 can reduce the immune response mounted in human blood, although the protective effects are rather modest in contrast to published mouse data. Our findings underline the importance of developing reliable models and we propose the use of human whole blood models in pre-clinical screening of gene therapy vectors.

An oncolytic conditionally replicating adenovirus for hormone-dependent and hormone-independent prostate cancer

The use of conditionally replicating adenoviruses offers an attractive complementary treatment strategy for localized prostate cancer. We have produced a replicating adenovirus, Ad[I/PPT-E1A], where E1A gene expression is controlled by a recombinant regulatory sequence designated PPT. The PPT sequence comprises a PSA enhancer, a PSMA enhancer and a T-cell receptor γ-chain alternate reading frame protein promoter, and it is shielded from transcriptional interference from adenoviral backbone sequences by an H19 insulator. Ad[I/PPT-E1A] yields prostate-specific E1A protein expression, viral replication and cytolysis in vitro. Furthermore, Ad[I/PPT-E1A] considerably regresses the growth of subcutaneous LNCaP prostate cancer tumors in nude mice. Importantly, the viral replication and cytolytic effect of Ad[I/PPT-E1A] are independent of the testosterone levels in the prostate cancer cells. This may be beneficial in a clinical setting since many prostate cancer patients are treated with androgen withdrawal. In conclusion, Ad[I/PPT-E1A] may prove to be useful in the treatment of localized prostate cancer.

Increased therapeutic efficacy of the prostate-specific oncolytic adenovirus Ad[I/PPT-E1A] by reduction of the insulator size and introduction of the full-length E3 region

Conditionally replicating adenoviruses are developing as a complement to traditional cancer therapies. Ad[I/PPT-E1A] is an E1B/E3-deleted virus that replicates exclusively in prostate cells, since the expression of E1A is controlled by the recombinant 1.4 kb prostate-specific PPT promoter. The transcriptional integrity of PPT is maintained by the 3.0 kb mouse H19 insulator that was introduced directly upstream of the PPT sequence. In order to increase the cloning capacity to be able to reintroduce E3 sequences in the 35.7 kb Ad[I/PPT-E1A] genome, various shorter insulators were examined in a luciferase reporter gene assay. It was found that the 1.6 kb core H19 insulator (i) improves the activity of PPT, compared to the 3.0 kb full-length insulator, while still maintaining prostate cell specificity and releasing 1.4 kb of space for insertion of additional sequences. To improve the ability of the virus to efficiently lyse infected cells and persist in vivo, we inserted the adenovirus death protein (ADP) or the full-length adenovirus E3 region. The oncolytic activity of PPT-E1A-based viruses was studied using MTS, crystal violet and replication assays. The virus with the reintroduced full-length E3-region (Ad[i/PPT-E1A, E3]) showed the highest cytopathic effects in vitro. Furthermore, this virus suppressed the growth of aggressively growing prostate tumors in vivo. Therefore, we conclude that Ad[i/PPT-E1A, E3] is a prostate-specific oncolytic adenovirus with a high potential for treating localized prostate cancer.

Adenovirus with Hexon Tat-Protein Transduction Domain Modification Exhibits Increased Therapeutic Effect in Experimental Neuroblastoma and Neuroendocrine Tumors

ABSTRACT Adenovirus serotype 5 (Ad5) is widely used as an oncolytic agent for cancer therapy. However, its infectivity is highly dependent on the expression level of coxsackievirus-adenovirus receptor (CAR) on the surfaces of tumor cells. Furthermore, infected cells overproduce adenovirus fiber proteins, which are released prior to cell lysis. The released fibers block CAR on noninfected neighboring cells, thereby preventing progeny virus entry. Our aim was to add a CAR-independent infection route to Ad5 to increase the infectivity of tumor cells with low CAR expression and prevent the fiber-masking problem. We constructed Ad5 viruses that encode the protein transduction domain (PTD) of the HIV-1 Tat protein (Tat-PTD) in hypervariable region 5 (HVR5) of the hexon protein. Tat-PTD functions as a cell-penetrating peptide, and Tat-PTD-modified Ad5 showed a dramatic increased transduction of CAR-negative cell lines compared to unmodified vector. Moreover, while tumor cell infectivity was severely reduced for Ad5 in the presence of fiber proteins, it was only marginally reduced for Tat-PTD-modified Ad5. Furthermore, because of the sequence alteration in the hexon HVR, coagulation factor X-mediated virus uptake was significantly reduced. Mice harboring human neuroblastoma and neuroendocrine tumors show suppressed tumor growths and prolonged survival when treated with Tat-PTD-modified oncolytic viruses. Our data suggest that modification of Ad5 with Tat-PTD in HVR5 expands its utility as an oncolytic agent.