Chemosaturation with percutaneous hepatic perfusions of melphalan for hepatic metastases: experience from two European centers.

PURPOSE: Chemosaturation with percutaneous hepatic perfusion (PHP; Hepatic CHEMOSAT(®) Delivery System; Delcath Systems Inc, USA) is a minimally invasive, repeatable regional therapy for unresectable hepatic metastases. It uses a system of catheters and filters to isolate hepatic venous blood from the systemic circulation, allowing delivery of high-dose chemotherapy to the hepatic artery. Effluent hepatic venous blood is filtered before being returned to the systemic circulation, thereby reducing exposure to chemotherapy. We describe our experiences with chemosaturation-PHP at 2 European centers. MATERIALS AND METHODS: 14 patients presented unresectable hepatic metastases from solid tumors; 13 received 1 - 3 sessions of chemosaturation-PHP. Melphalan 2.0 (n = 1) or 3.0 (n = 12) mg/kg was given as a 30-minute infusion into the hepatic artery. 12 patients were evaluable for tumor response. RESULTS: One complete (cholangiocarcinoma, n = 1) and 6 partial responses (ocular, n = 3 or cutaneous melanoma, n = 3) were observed, 5 patients had stable disease (ocular melanoma, n = 3; breast cancer, n = 1; gastric cancer, n = 1). Mild to moderate filter-related toxicity (i. e. thrombocytopenia, anemia) was observed immediately post-procedure. Grade 3/4 melphalan-related pancytopenia developed after 1 - 2 weeks. All hematological events were managed effectively with transfusions and/or other supportive measures. The new high-efficiency filter showed milder toxicity and faster recovery. In one case, chemosaturation-PHP was abandoned prematurely due to heparin-induced vaginal bleeding, and one patient died due to retroperitoneal hemorrhage from heparin anti-coagulation. CONCLUSION: Chemosaturation-PHP for non-resectable liver metastases is a feasible treatment option when performed by an experienced multi-disciplinary team. It may be a promising regional therapy for patients with no effective treatment options.

Aquaculture fouling: Efficacy of potassium monopersulphonate triple salt based disinfectant (Virkon® Aquatic) against Ciona intestinalis.

With the increasing spread of invasive marine species and their detrimental effects on aquaculture operations globally, mitigation strategies need to be optimized to mitigate economic impacts. The efficacy of a potassium monopersulphonate triple salt based disinfectant used in the aquaculture industry (Virkon® Aquatic at 0.5-5%) was evaluated against the solitary tunicate Ciona intestinalis, as well as the susceptibility of three different age groups of C. intestinalis to the treatment and the effect of the disinfectant on mussel mortality. Younger C. intestinalis were most affected by all treatments, and almost all immersion applications significantly decreased the biomass of C. intestinalis compared to untreated plates. Disinfectant solutions of ≥ 1% reduced biomass below pre-treatment levels. Mussel mortality was low, especially for solutions <3%. C. intestinalis should be treated 4 weeks post-settlement to maximize antifouling treatment effects. Immersion in 3% disinfectant for 30 s reduced the biomass of C. intestinalis by up to 89% and would be feasible in field applications using existing treatment equipment.

Up-regulation of hepatic ABCC2, ABCG2, CYP1A1 and GST in multixenobiotic-resistant killifish (Fundulus heteroclitus) from the Sydney Tar Ponds, Nova Scotia, Canada.

Cellular defence against accumulation of toxic xenobiotics includes metabolism by phase I and II enzymes and export of toxicants and their metabolites via ATP-binding cassette (ABC) transporters. Liver gene expression of representatives of these three protein groups was examined in a population of multixenobiotic-resistant killifish (Fundulus heteroclitus) from the Sydney Tar Ponds, Nova Scotia, Canada. The Tar Ponds are heavily polluted with polycyclic aromatic hydrocarbons, polychlorinated biphenyls and heavy metals. The relationship among ABC transporters ABCB1, ABCB11, ABCC2, ABCG2, phase I enzyme cytochrome P4501A1 (CYP1A1) and phase II enzyme glutathione-S-transferase (GST-mu) was investigated by quantifying hepatic transcript abundance. In Tar Pond killifish, hepatic mRNA expression levels of ABCC2, ABCG2, CYP1A1 and GST-mu were elevated compared to reference sites, suggesting that hydrophobic contaminants undergo phase I and II metabolism and are then excreted into the bile of these fish. Hepatic ABCB1 and ABCB11 mRNA were not up-regulated in Tar Pond fish compared to two reference sites, indicating that these two proteins are not involved in conferring multixenobiotic resistance to Tar Pond killifish. The results suggest instead that liver up-regulation of phase I and II enzymes and complementary ABC transporters ABCC2 and ABCG2 may confer contaminant resistance to Tar Pond fish.

Novel cell type-specific antiviral mechanism of interferon gamma action in macrophages.

Interferon (IFN)-gamma and macrophages (Mphi) play key roles in acute, persistent, and latent murine cytomegalovirus (MCMV) infection. IFN-gamma mechanisms were compared in embryonic fibroblasts (MEFs) and bone marrow Mphi (BMMphi). IFN-gamma inhibited MCMV replication in a signal transducer and activator of transcription (STAT)-1alpha-dependent manner much more effectively in BMMphi (approximately 100-fold) than MEF (5-10-fold). Although initial STAT-1alpha activation by IFN-gamma was equivalent in MEF and BMMphi, microarray analysis demonstrated that IFN-gamma regulates different sets of genes in BMMphi compared with MEFs. IFN-gamma inhibition of MCMV growth was independent of known mechanisms involving IFN-alpha/beta, tumor necrosis factor alpha, inducible nitric oxide synthase, protein kinase RNA activated (PKR), RNaseL, and Mx1, and did not involve IFN-gamma-induced soluble mediators. To characterize this novel mechanism, we identified the viral targets of IFN-gamma action, which differed in MEF and BMMphi. In BMMphi, IFN-gamma reduced immediate early 1 (IE1) mRNA during the first 3 h of infection, and significantly reduced IE1 protein expression for 96 h. Effects of IFN-gamma on IE1 protein expression were independent of RNaseL and PKR. In contrast, IFN-gamma had no significant effects on IE1 protein or mRNA expression in MEFs, but did decrease late gene mRNA expression. These studies in primary cells define a novel mechanism of IFN-gamma action restricted to Mphi, a cell type key for MCMV pathogenesis and latency.

Latent murine cytomegalovirus infection in macrophages.

In this study we show that macrophages (Mphi) are latently infected with murine cytomegalovirus (MCMV). After clearance of acute MCMV infection, the predominant form of chronic infection in Balb mice is latency rather than persistence. Peritoneal exudate cells (PECs) from latently infected Balb mice (3-9 months postinfection) contained MCMV genome and reactivatable virus. Adherent cells from both resident and thioglycollate-elicited PECs carried more MCMV DNA (measured by PCR) than nonadherent cells, and were selectively enriched for Mphi. FACS sorted F4/80(+) Mphi contained MCMV DNA, while other FACS sorted cell populations from PECs were never positive for MCMV DNA. MCMV reactivated from FACS sorted F4/80(+) Mphi in 32% of cocultures with murine embryonic fibroblasts (MEFs). Since Mphi carry MCMV genome and reactivatable virus, but not lytic virus, they are latently infected with MCMV. We determined the frequency of Mphi carrying MCMV genome in PECs (about 1/50,000) using a limiting dilution PCR assay. Using this frequency and estimates of the total amount of MCMV genome in populations, we estimate that latently infected Mphi carry 1-10 copies of MCMV genome. To evaluate the origin of latently infected Mphi, we compared the frequency of cells carrying MCMV genome in the resident and elicited PECs. The frequency of Mphi carrying MCMV DNA was the same in resident and thioglycollate-elicited PECs, despite the fact that there was a ninefold increase in the number of Mphi recovered after thioglycollate elicitation. This argued for recruitment of bone marrow-derived Mphi (BMMphi) carrying MCMV genome into the peritoneum during inflammatory responses. Consistent with this hypothesis, MCMV genome, but not persistent virus, was detected in bone marrow cells from latently infected mice.

Murine cytomegalovirus with a deletion of genes spanning HindIII-J and -I displays altered cell and tissue tropism.

Murine cytomegalovirus (MCMV) gene products dispensable for growth in cell culture are likely to have important functions within the infected host, influencing tissue tropism, dissemination, or immunological responses against the virus. To identify such genes, our strategy was to delete large regions of the MCMV genome likely to contain genes nonessential for virus replication in NIH 3T3 cells. Mutant virus RV7 contained a deletion of 7.7 kb spanning portions of MCMV HindIII-J and -I. This virus grew comparably to wild-type (WT) virus in NIH 3T3 fibroblasts, primary embryo fibroblasts, and bone marrow macrophages. However, RV7 failed to replicate in target organs of immunocompetent BALB/c mice and severe combined immunodeficient mice, which are exquisitely sensitive to MCMV infection. This defect in vivo growth may be related to the observation that RV7 grew poorly in the peritoneal macrophage cell line IC-21, which is highly permissive for growth of WT MCMV. Two other mutant viruses with an insertion or smaller deletion in the region common to the RV7 deletion grew comparably to WT virus in the macrophage cell line and replicated in salivary gland tissue. The poor growth of RV7 in IC-21 cells was due to a block in immediate-early gene expression, as levels of RNA from immediate-early gene IE1 were reduced eightfold compared with levels for WT virus in macrophages infected with RV7. Consequently, levels of RNA from early and late genes were also reduced. The lower expression of IE1 in RV7-infected IC-21 macrophages was not due to defective entry of virus into the cells, as equal amounts of viral DNA were present in cells 3 h after infection with RV7 or WT MCMV. These studies demonstrate that deletion of sequences in HindIII-J and -I confer altered cell and tissue tropism.

Quantitative cytofluorimetric determination of cell membrane-associated large tumor antigen on SV40-transformed cells.

The aim of this study was to quantitate the number of cell membrane-located SV40 large tumor antigen (large T) molecules of SV40-transformed cell lines by cytofluorimetric analysis. Five different SV40-transformed cell lines were labelled by either a biotin- or a fluorescein-conjugated monoclonal antibody, PAb1605, which is specific for the large T carboxyterminus. The conjugated-antibody fluorescence signals of the stained large T molecules of transformed cells were measured via cytofluorimetry. Comparison of the fluorescence signals of calibrated beads bearing a known number of fluorescein molecules to the signals of conjugated PAb1605 antibodies bound on microbeads to a defined number of IgG binding sites made it possible to determine the number of antibody-accessible large T molecules per SV40-transformed cell. The numbers (x10(-4)) found per cell were 1.0 (ELONA, hamster), 3.0 (VLM, mouse), 3.5 (mKSA, mouse), 11 (C57SV, mouse), and 5.5 (SV80, human), respectively. Thus, the technique described allows a precise quantitation of surface-exposed, antibody-accessible viral antigen expression.

Covalent immunochemical membrane labeling of viable cells with K698-T708, a simian virus 40 tumor antigen-derived peptide.

The process of covalent immunochemical linking of viable cell membranes with a Simian Virus 40 (SV40) tumor antigen-derived undecapeptide, K(698)PPTPPPEPET(708) (KT), is described. The principle applied was the reaction of the lysine residue, K 698, of the undecapeptide with the succinimidyl moiety of a heterobifunctional linker molecule, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) or sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate (sulfo-SIAB). Thereby, upon release of N-hydroxy-succinimide, the rest of the linker molecule reacts covalently with the epsilon-NH2 group of lysine. Upon release of pyridyl-2-thion or hydrogen iodide, respectively, the second reactive moiety of the linker is then ready to form a covalent bond with SH-groups of cell membrane compounds. As a result, KT is covalently linked onto the cell membrane by an -SS- or an -S-bond, respectively. Binding is prevented by treatment of the candidate cells with iodoacetamide, an SH-reactive compound. This artificial cell membrane epitope can be demonstrated by surface immunofluorescence and by binding to immunomagnetic beads loaded with PAb1605, a KT-specific monoclonal antibody. Quantitation by cytofluorimetry shows some 10(4) KT molecules bound per cell, a number that is in the range of the number of SV40 tumor antigen molecules of genuine SV40-transformed mammalian cells.