Phase Ib Safety, Two-Dose Study of MultiGeneAngio in Patients with Chronic Critical Limb Ischemia.

Critical limb ischemia (CLI) is the most severe presentation of peripheral arterial disease. We developed cell-based therapy entailing intra-arterial injection of autologous venous endothelial cells (ECs) modified to express angiopoietin 1, combined with autologous venous smooth muscle cells (SMCs) modified to express vascular endothelial growth factor. This combination promoted arteriogenesis in animal models and was safe in patients with limiting claudication. In an open-label, phase Ib study, we assessed the safety and efficacy of this therapy in CLI patients who failed or were unsuitable for surgery or intravascular intervention. Of 23 patients enrolled, 18 with rest pain or non-healing ulcers (Rutherford categories 4 and 5) were treated according to protocol, and 5 with significant tissue loss (Rutherford 6) were treated under compassionate treatment. Patients were assigned randomly to receive 1 × 107 or 5 × 107 (EC-to-SMC ratio, 1:1) of the cell combination. One-year amputation-free survival rate was 72% (13/18) for Rutherford 4 and 5 patients; all 5 patients with Rutherford 6 underwent amputation. Of the 12 with unhealing ulcers at dosing, 6 had complete healing and 2 others had >66% reduction in ulcer size. Outcomes did not differ between the dose groups. No severe adverse events were observed related to the therapy.

Phase I study of multi-gene cell therapy in patients with peripheral artery disease.

UNLABELLED: Alternative treatment strategies for claudication are needed and cell-based therapies designed to induce angiogenesis are promising. The purpose of this report was to conduct a Phase I safety, dose-escalating, non-randomized, open-label study of autologous, fully differentiated venous endothelial and smooth muscle cells called MultiGeneAngio (MGA) for claudication due to peripheral artery disease. Twelve subjects, at two centers, received a single intra-arterial infusion of a suspension of equal amounts of transduced autologous venous smooth muscle cells expressing vascular endothelial growth factor (VEGF165) and endothelial cells expressing angiopoietin-1 (Ang-1) (Cohort 1: 1 × 10(7), Cohort 2: 2 × 10(7), Cohort 3: 5 × 10(7), Cohort 4: 7 × 10(7)). The treatment was given unblinded and in the more symptomatic lower extremity. Transduced cells were tested for in vitro doubling time, telomerase activity, and gene expression. The main outcomes were clinical safety and tolerability. Other safety measures included ankle-brachial index (ABI) and walking time on a treadmill. All subjects were male (mean age 60 ± 5 years) including 25% with diabetes mellitus. At 1-year follow-up, there was one serious adverse event possibly related to MGA. Safety endpoints including VEGF and Ang-1 plasma protein levels were within normal ranges in all subjects. The mean maximal walking time increased from baseline to 1 year and the index limb ABI was unchanged, indicating no safety concerns. MGA, an autologous, transduced, cell-based therapy was well tolerated and safe in this Phase I study. Further evaluation is warranted in randomized human studies. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT00390767.

Sodium dodecyl sulphate-treated proteins as ligands in ELISA.

Insoluble proteins generally do not adsorb to microtitre wells and, therefore, cannot be used as antigens in enzyme-linked immunosorbent assay (ELISA). However, denaturation and solubilization with 2% sodium dodecyl sulphate (SDS) renders these proteins suitable ligands for ELISA. In quantitative ELISA using polyclonal antibodies as primary antibody, comparable results were obtained with native and SDS-denatured protein ligands. The binding of the antibodies to the SDS-treated ligands was completely inhibited by premixing the primary antibody with the corresponding native antigen. Nonspecific binding of primary and secondary antibodies to SDS-treated ligands was not observed. SDS-treated proteins are able to attach to ELISA microwells, retain their antigenic epitopes and do not engender an elevated background. The concentration of SDS-treated proteins required for coating is the same as that of the native proteins.

Protection of cultured Cyprinus carpio against a lethal viral disease by an attenuated virus vaccine.

Massive mortality of koi and common carp--Cyprinus carpio species--has been observed since 1998 in many countries worldwide, resulting in severe economic losses. The cause of the disease is an as yet unclassified large DNA virus, designated carp nephritis gill necrosis virus (CNGV) or koi herpes virus (KHV). Previously, we demonstrated that the wild type CNGV lost its pathogenecity following serial transfer in cell culture, and that clones isolated from the attenuated population can be used as a prophylactic vaccine. Here, we describe the basic conditions required for proper fish immunization so that a protection protocol may be devised. We demonstrated that carps are very sensitive to the pathogenic and the attenuated viruses, and short immersion of fish in water containing the viruses is sufficient for infection. The infection of fish with the pathogenic and the attenuated viruses is temperature-restricted; fish held at the non-permissive temperature, immediately following infection, were not affected by the pathogenic virus, and were not rendered resistant to the disease. Thus, propagation of the virus in the fingerlings is a pre-requisite for immunization. In order to increase the number of random mutations in the genome of the attenuated virus, and thus, reduce the possibility of the attenuated virus reverting to pathogenic, we irradiated it and selected additional clones appropriate for vaccination. The results of our study suggest that a safe and efficient prophylactic vaccine can be developed by selecting an appropriate attenuated virus.

Description of an as yet unclassified DNA virus from diseased Cyprinus carpio species.

Numerous deaths of koi and common carp (Cyprinus carpio) were observed on many farms throughout Israel, resulting in severe financial losses. The lethal viral disease observed is highly contagious and extremely virulent, but morbidity and mortality are restricted to koi and common carp populations. Diseased fish exhibit fatigue and gasping movements in shallow water. Infected fish had interstitial nephritis and gill necrosis as well as petechial hemorrhages in the liver and other symptoms that were not consistent with viral disease, suggesting a secondary infection. Here we report the isolation of carp nephritis and gill necrosis virus (CNGV), which is the etiologic agent of this disease. The virus propagates and induces severe cytopathic effects by 5 days postinfection in fresh koi or carp fin cell cultures (KFC and CFC, respectively), but not in epithelioma papillosum cyprini cells. The virus harvested from KFC cultures induced the same clinical signs, with a mortality of 75 to 95%, upon inoculation into naive koi and common carp. Using PCR, we provide final proof that the isolated virus is indeed the etiologic agent of food and ornamental carp mortalities in fish husbandry. Electron microscopy revealed viral cores with icosahedral morphology of 100 to 110 nm that resembled herpesviruses. Electron micrographs of purified pelleted CNGV sections, together with viral sensitivities to ether and Triton X-100, suggested that it is an enveloped virus. However, the genome of the isolated virus is a double-stranded DNA (dsDNA) molecule of 270 to 290 kbp, which is larger than known herpesviruses. The viral DNA seems highly divergent and bears only small fragments (16 to 45 bp) that are similar to the genomes of several DNA viruses. Nevertheless, amino acid sequences encoded by CNGV DNA fragments bear similarities primarily to members of the Poxviridae and Herpesviridae and to other large dsDNA viruses. We suggest, therefore, that the etiologic agent of this disease may represent an as yet unclassified virus species that is endemic in C. carpio (carp).

Abrogation of Vif function by peptide derived from the N-terminal region of the human immunodeficiency virus type 1 (HIV-1) protease.

The human immunodeficiency virus type 1 (HIV-1) auxiliary gene vif is essential for virus propagation in peripheral blood lymphocytes, macrophages, and in some T-cell lines. Previously, it was demonstrated that Vif inhibits the autoprocessing of truncated HIV-1 Gag-Pol polyproteins expressed in bacterial cells, and that purified recombinant Vif and Vif-derived peptides inhibit and bind HIV-1 protease (PR). Here we show that Vif interacts with the N-terminal region of HIV-1 PR, and demonstrate that peptide derived from the N-terminal region of PR abrogates Vif function in non-permissive cells. Specifically, we show that (i) Vif protein binds HIV-1 PR, but not covalently linked tethered PR-PR; (ii) the four amino acids residing at the N terminus of HIV-1 PR are essential for Vif/PR interaction; (iii) synthetic peptide derived from the N terminus of HIV-1 PR inhibits Vif/PR binding; and (iv) this peptide inhibits the propagation of HIV-1 in restrictive cells. Based on these data, we suggest that Vif interacts with the dimerization sites of the viral protease, and that peptide residing at the N terminus of PR abrogates Vif function(s).

Human immunodeficiency virus type 1 Vif binds the viral protease by interaction with its N-terminal region.

The vif gene, one of the six auxiliary genes of human immunodeficiency virus (HIV), is essential for virus propagation in peripheral blood lymphocytes and macrophages and in certain T-cell lines. Previously, it was demonstrated that Vif inhibits the autoprocessing of truncated HIV type 1 (HIV-1) Gag-Pol polyproteins expressed in bacterial cells, as well as the protease-mediated cleavage of synthetic peptides in vitro. Peptides derived from the aa 78-98 region in the Vif molecule specifically inhibit and bind the HIV-1 protease in vitro and arrest the production of infectious viruses in HIV-1-infected cells. This study demonstrates that (i) purified recombinant Vif protein and HIV-1 but not avian sarcoma leukaemia virus protease specifically bind each other and (ii) the interaction between these two proteins takes place at the N terminus of the protease (aa 1-9) and the central part of Vif (aa 78-98). The data presented in this report suggest a model in which Vif interacts with the dimerization sites of the viral protease.

Efficient vaccine against the virus causing a lethal disease in cultured Cyprinus carpio.

We have isolated a virus, which causes a mortal disease in cultured ornamental Koi and Common carps (Cyprinus carpio) in many countries worldwide. This unclassified virus, which causes nephritis and gill necrosis, and so has been given the name carp nephritis and gill necrosis virus (CNGV), has a morphology resembling the herpes virus, but bears a genomic DNA of ca 250-300 kbp. So far, both others and we have been unable to find CNGV-DNA sequences possessing a significant similarity to known DNA viruses. The virus induces a lethal disease when water temperature ranges between 18 and 25 degrees C (permissive temperature). In this report, we demonstrate that carps, exposed to the virus at 23 degrees C for 3-5 days and then transferred to the non-permissive temperature of 30 degrees C, became resistant to a challenged infection and their sera demonstrated a high level of virus-specific antibodies. We have isolated attenuated non-pathogenic viruses that render virus-vaccinated carps resistant to the disease. Furthermore, vaccinated fish developed high levels of antibodies against the virus. We suggest, therefore, that this attenuated virus could be used as a live vaccine for the eradication of the mortal disease afflicting Common and ornamental carp fisheries in many countries.

Pathogenesis of acute viral disease induced in fish by carp interstitial nephritis and gill necrosis virus.

A lethal disease of koi and common carp (species Cyprinus carpio) has afflicted many fish farms worldwide since 1998, causing severe financial losses. Morbidity and mortality are restricted to common carp and koi and appear in spring and autumn, when water temperatures are 18 to 28 degrees C. We have isolated the virus causing the disease from sick fish, propagated it in koi fin cell culture, and shown that virus from a single clone causes lethal disease in carp and koi upon infection. Intraperitoneal virus injection or bathing the fish in virus-containing water kills 85 to 100% of the fish within 7 to 21 days. This virus is similar to the previously reported koi herpesvirus; however, it has characteristics inconsistent with the herpesvirus family, and thus we have called it carp interstitial nephritis and gill necrosis virus. We examined the pathobiology of this disease in carp by using immunohistochemistry and PCR. We found large amounts of the virus in the kidneys of sick fish and smaller amounts in liver and brain. A rapid increase in the viral load in the kidneys was detected by using both immunofluorescence and semiquantitative PCR. Histological analyses of fish at various times after infection revealed signs of interstitial nephritis as early as 2 days postinfection, which increased in severity up to 10 days postinfection. There was severe gill disease evidenced by loss of villi with accompanying inflammation in the gill rakers. Minimal focal inflammation was noted in livers and brains. This report describes the etiology and pathology of a recently described viral agent in fish.