Immunohistochemical Characterization and Sensitivity to Human Adenovirus Serotypes 3, 5, and 11p of New Cell Lines Derived from Human Diffuse Grade II to IV Gliomas.

Background: Oncolytic adenoviruses show promise in targeting gliomas because they do not replicate in normal brain cells. However, clinical responses occur only in a subset of patients. One explanation could be the heterogenic expression level of virus receptors.

Oncolytic alphavirus SFV-VA7 efficiently eradicates subcutaneous and orthotopic human prostate tumours in mice.

Background: Despite recent therapeutic and diagnostic advances, prostate cancer remains the second leading cause of cancer-related deaths among men in the Western world. Oncolytic viruses that replicate selectively in tumour cells represent a novel treatment candidate for these malignancies.

Methods: We analysed infectivity of avirulent Semliki Firest virus SFV-VA7 in human prostate cancer cell lines VCaP, LNCaP and 22Rv1 and in nonmalignant prostate epithelial cell line RWPE-1.

MicroRNA-Attenuated Clone of Virulent Semliki Forest Virus Overcomes Antiviral Type I Interferon in Resistant Mouse CT-2A Glioma.

Unlabelled: Glioblastoma is a terminal disease with no effective treatment currently available. Among the new therapy candidates are oncolytic viruses capable of selectively replicating in cancer cells, causing tumor lysis and inducing adaptive immune responses against the tumor. However, tumor antiviral responses, primarily mediated by type I interferon (IFN-I), remain a key problem that severely restricts viral replication and oncolysis.

Attenuated Semliki Forest virus for cancer treatment in dogs: safety assessment in two laboratory Beagles.

Background: Dogs suffer from spontaneous tumors which may be amenable to therapies developed for human cancer patients, and dogs may serve as large-animal cancer models. A non-pathogenic Semliki Forest virus vector VA7-EGFP previously showed promise in targeting human tumor xenografts in mice, but the oncolytic capacity of the virus in canine cancer cells and the safety of the virus in higher mammals such as dogs, are not known. We therefore assessed the oncolytic potency of VA7-EGFP against canine cancer cells by infectivity and viability assays in two dog solid tumor cell lines.

Antibodies to deamidated gliadin peptide in diagnosis of celiac disease in children.

Objectives: Determination of antibodies to synthetic deamidated gliadin peptides (anti-DGPs) may work as an alternative or complement the commonly used test for tissue transglutaminase antibodies (TGA) in the diagnosis of celiac disease (CD). We analyzed the performance of a time-resolved immunofluorometric anti-DGP assay (TR-IFMA) in the diagnosis of CD in children and also retrospectively analyzed the appearance of anti-DGP antibodies before TGA seroconversion.

Methods: The study included 92 children with biopsy-confirmed CD.

Tumor Restrictions to Oncolytic Virus.

Oncolytic virotherapy has advanced since the days of its conception but therapeutic efficacy in the clinics does not seem to reach the same level as in animal models. One reason is premature oncolytic virus clearance in humans, which is a reasonable assumption considering the immune-stimulating nature of the oncolytic agents. However, several studies are beginning to reveal layers of restriction to oncolytic virotherapy that are present before an adaptive neutralizing immune response.

Synthesis of cellulose nanocrystals carrying tyrosine sulfate mimetic ligands and inhibition of alphavirus infection.

We present two facile approaches for introducing multivalent displays of tyrosine sulfate mimetic ligands on the surface of cellulose nanocrystals (CNCs) for application as viral inhibitors. We tested the efficacy of cellulose nanocrystals, prepared either from cotton fibers or Whatman filter paper, to inhibit alphavirus infectivity in Vero (B) cells. Cellulose nanocrystals were produced by sulfuric acid hydrolysis leading to nanocrystal surfaces decorated with anionic sulfate groups.

Firefly luciferase inhibitor-conjugated peptide quenches bioluminescence: a versatile tool for real time monitoring cellular uptake of biomolecules.

In this paper, novel firefly luciferase-specific inhibitor compounds (FLICs) are evaluated as potential tools for cellular trafficking of transporter conjugates. As a proof-of-concept, we designed FLICs that were suitable for solid phase peptide synthesis and could be covalently conjugated to peptides via an amide bond. The spacer between inhibitor and peptide was optimized to gain efficient inhibition of recombinant firefly luciferase (FLuc) without compromising the activity of the model peptides.

Discovery of 5-benzyl-3-phenyl-4,5-dihydroisoxazoles and 5-benzyl-3-phenyl-1,4,2-dioxazoles as potent firefly luciferase inhibitors.

Luciferase reporter assays are commonly used in high-throughput screening methods. Here, we report new firefly luciferase (FLuc) inhibitors based on 5-benzyl-3-phenyl-4,5-dihydroisoxazoles and 5-benzyl-3-phenyl-1,4,2-dioxazoles, which showed up as "false positives" in a luciferase reporter gene-based assay for nuclear receptor antagonists. The inhibition was shown to be noncompetitive for both natural enzyme substrates (d-luciferin and ATP) and selective to FLuc and proven to arise from a direct interaction between the enzyme and the inhibitor.

Resistance to two heterologous neurotropic oncolytic viruses, Semliki Forest virus and vaccinia virus, in experimental glioma.

Attenuated Semliki Forest virus (SFV) may be suitable for targeting malignant glioma due to its natural neurotropism, but its replication in brain tumor cells may be restricted by innate antiviral defenses. We attempted to facilitate SFV replication in glioma cells by combining it with vaccinia virus, which is capable of antagonizing such defenses. Surprisingly, we found parenchymal mouse brain tumors to be refractory to both viruses.

Attenuation of Semliki Forest virus neurovirulence by microRNA-mediated detargeting.

Artificial target sequences for tissue-specific miRNAs have recently been introduced as a new means for altering the tissue tropism of viral replication. This approach can be used to improve the safety of oncolytic viruses for cancer virotherapy by restricting their replication in unwanted tissues, such as the liver. Semliki Forest virus (SFV) is a positive-strand RNA virus and, similar to the related alphaviruses, like Sindbis virus, has potential as a gene therapy vector and an oncolytic virotherapy agent, but this potential is limited by the neurovirulence of these alphaviruses.

Interferon-β sensitivity of tumor cells correlates with poor response to VA7 virotherapy in mouse glioma models.

In our recent study, replicative alphaviral vector VA7 was found to be effective against orthotopic human U87-glioma xenografts in an athymic mouse model eradicating the tumors with single intravenous (i.v.) injection.

SH3 domain-mediated recruitment of host cell amphiphysins by alphavirus nsP3 promotes viral RNA replication.

Among the four non-structural proteins of alphaviruses the function of nsP3 is the least well understood. NsP3 is a component of the viral replication complex, and composed of a conserved aminoterminal macro domain implicated in viral RNA synthesis, and a poorly conserved carboxyterminal region. Despite the lack of overall homology we noted a carboxyterminal proline-rich sequence motif shared by many alphaviral nsP3 proteins, and found it to serve as a preferred target site for the Src-homology 3 (SH3) domains of amphiphysin-1 and -2.

Intravenously administered alphavirus vector VA7 eradicates orthotopic human glioma xenografts in nude mice.

Background: VA7 is a neurotropic alphavirus vector based on an attenuated strain of Semliki Forest virus. We have previously shown that VA7 exhibits oncolytic activity against human melanoma xenografts in immunodeficient mice. The purpose of this study was to determine if intravenously administered VA7 would be effective against human glioma.

Human embryonic stem cell-derived mesenchymal stromal cell transplantation in a rat hind limb injury model.

Background Aims: Mesenchymal stromal cells (MSC) have been used in a wide variety of pre-clinical experiments and in an increasing number of human clinical trials. Although many of these studies have shown different levels of engraftment, the exact fate of MSC after transplantation and the tissue response to their engraftment have not been investigated in detail. In the present work we studied the distribution of human MSC in a rat hind limb ischemic injury model immediately after transplantation and also analyzed the recipient tissue response to transplanted cells.

Sphingosine kinase as a regulator of calcium entry through autocrine sphingosine 1-phosphate signaling in thyroid FRTL-5 cells.

Calcium entry is one of the main regulators of intracellular signaling. Here, we have described the importance of sphingosine, sphingosine kinase 1 (SK1), and sphingosine 1-phosphate (S1P) in regulating calcium entry in thyroid FRTL-5 cells. In cells incubated with the phosphatase inhibitor calyculin A, which evokes calcium entry without mobilizing sequestered intracellular calcium, sphingosine inhibited calcium entry in a concentration-dependent manner.

Oncolytic Semliki forest virus vector as a novel candidate against unresectable osteosarcoma.

Oncolytic viruses are a promising tool for treatment of cancer. We studied an oncolytic Semliki Forest virus (SFV) vector, VA7, carrying the enhanced green fluorescent protein gene (EGFP), as a novel virotherapy candidate against unresectable osteosarcoma. The efficiency and characteristics of the VA7-EGFP treatment were compared with a widely studied oncolytic adenovirus, Ad5Delta24, both in vitro and in vivo.

Replication competent Semliki Forest virus prolongs survival in experimental lung cancer.

We evaluated the therapeutic potential of the replication competent vector VA7-EGFP, which is based on the avirulent Semliki Forest virus (SFV) strain A7 (74) carrying the EGFP marker gene in an orthotopic lung cancer tumor model in nude mice. We have previously shown that this oncolytic vector destroys tumor cells efficiently in vitro and in vivo (in subcutaneous tumor model). Tumor growth in animals with orthotopically implanted adenocarcinoma cells (A549) were monitored during the study with small animal CT.

The importance of CTLA-4 polymorphism and human leukocyte antigen genotype for the induction of diabetes-associated cytokine response in healthy school children.

Background: Type 1 diabetes (T1D) is an autoimmune disease associated with the destruction of pancreatic beta cells and genetically linked to human leukocyte antigen (HLA) class II DR3-DQ2 and DR4-DQ8 haplotypes. The +49A/G polymorphism of the immunoregulatory cytotoxic T-lymphocyte antigen 4 (CTLA-4) gene is also associated with T1D. Genetic and environmental risk factors precede the onset of T1D, which is characterized by a T helper 1 cell-dominating cytokine response to diabetes-related autoantigens.

Evaluation of cancer virotherapy with attenuated replicative Semliki forest virus in different rodent tumor models.

Semliki Forest virus (SFV) is one of the latest candidates for a virotherapeutic agent against cancer, and recent studies have demonstrated its efficacy in tumor models. In the present study, we examined the antitumor efficacy of an avirulent SFV strain A7(74) and its derivative, a replication-competent SFV vector VA7-EGFP, in a partially immunodeficient mouse tumor model (subcutaneous A549 human lung adenocarcinoma in NMRI nu/nu mouse) and in an immunocompetent rat tumor model (intracranial BT4C glioma in BDIX rat). When subcutaneous mouse tumors were injected 3 times with VA7-EGFP, intratumorally treated animals showed almost complete inhibition of tumor growth, while systemically treated mice displayed only delayed tumor growth (intravenous injection) or no response at all (intraperitoneal injection).

Oncolytic viruses in cancer therapy.

Oncolytic virotherapy is a promising form of gene therapy for cancer, employing nature's own agents to find and destroy malignant cells. The purpose of this review is to provide an introduction to this very topical field of research and to point out some of the current observations, insights and ideas circulating in the literature. We have strived to acknowledge as many different oncolytic viruses as possible to give a broader picture of targeting cancer using viruses.

Semliki Forest virus vectors expressing transforming growth factor beta inhibit experimental autoimmune encephalomyelitis in Balb/c mice.

Cytokine immunomodulation of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, has remained a formidable treatment option, but access into the CNS is hampered due to the impermeability of the blood-brain barrier. In this report, we describe the construction and characterization of CNS-homing gene delivery/therapy vectors based on avirulent Semliki Forest virus (SFV) expressing either native or mutant transforming growth factor beta 1 (TGF-beta1). Biological activity of the expressed inserts was demonstrated by PAI-1 promoter driven luciferase production in mink cells and TGF-beta1 mRNA was demonstrated in the CNS of virus treated mice by in situ hybridization and RT-PCR.

Time-resolved immunofluorometric dual-label assay for simultaneous detection of autoantibodies to GAD65 and IA-2 in children with type 1 diabetes.

Background: Autoantibodies to glutamic acid decarboxylase (GADAs), specifically the 65-kDa isoform GAD65, and autoantibodies to the protein tyrosine phosphatase-like molecule IA-2 (IA-2As) predict development of diabetes. Our aim was to develop a time-resolved immunofluorometric (TR-IFMA) dual-label assay method for the simultaneous detection of these autoantibodies and to evaluate the diagnostic sensitivity of the method compared with single-label TR-IFMA and fluid-phase radiobinding assay (RBA) in screening children with type 1 diabetes.

Methods: We incubated combined biotinylated GAD65 and IA-2 proteins, glutathione S-transferase (GST)-IA-2, europium-labeled GAD65, terbium-labeled anti-GST antibody, and serum sample or calibrator and transferred aliquots to a streptavidin-coated 96-well microtiter plate for a second incubation.

Type I interferon response against viral and non-viral gene transfer in human tumor and primary cell lines.

Background: Type I interferon (IFN-alpha/beta) response is one of the major host defence mechanisms against viruses. Some recent reports suggest that IFNs may interfere with the efficacy of both non-viral and virus-vector-mediated therapeutic gene transfer.

Methods: The type I IFN response upon different gene transfer methods in human tumor and primary cell lines was studied by analysing IFN-beta mRNA expression, secretion of type I IFNs and accumulation of IFN-alpha/beta-induced MxA protein (myxovirus resistance protein A).

Oncolytic capacity of attenuated replicative semliki forest virus in human melanoma xenografts in severe combined immunodeficient mice.

Oncolytic viruses have gained attention as a novel form of cancer treatment. Many viral vectors in use today have been rendered safe by deletion of genes encoding viral structural proteins, thus making them unable to spread beyond the first infected cells. Hence, such replication-deficient constructs may lack efficacy.