The antiviral role of zinc and metallothioneins in hepatitis C infection.

Metallothioneins (MTs) are small, cysteine-rich proteins characterized by a high affinity for monovalent and divalent cations, such as copper and zinc. Of the four known MT isoforms, only, members of the MT 1 and 2 subfamilies are widely expressed, acting as metal chaperones whose primary role is to mediate intracellular zinc homoeostasis. Metallothioneins are potently induced by heavy metals and other sources of oxidative stress where they facilitate metal binding and detoxification as well as free radical scavenging. Metallothionein expression is well documented in the context of viral infection; however, it remains uncertain whether MTs possess specific antiviral roles or whether induction is merely a consequence of cellular stress. To better understand the role of MTs following hepatitis C virus (HCV) infection, we examined MT expression and localization in vitro and in vivo and used a siRNA knockdown approach to ascertain their antiviral efficacy. We confirmed HCV-driven MT induction in vitro and demonstrated MT accumulation in the nucleus of HCV-infected hepatocytes by immunofluorescence. Using a pan-MT siRNA to knock down all members of the MT1 and MT2 subfamilies, we demonstrate that they are mildly antiviral against the JFH1 strain of HCV in vitro (~1.4 fold increase in viral RNA, P < .05). Furthermore, the antiviral effect of zinc treatment against HCV in vitro was mediated through MT induction (P < .05). Our data suggest a potential benefit of using zinc as a low-cost adjunct to current HCV antiviral therapies and suggest that zinc may facilitate the antiviral role of MTs against other viruses.

The autoimmune risk gene ZMIZ1 is a vitamin D responsive marker of a molecular phenotype of multiple sclerosis.

Multiple Sclerosis (MS) is a neurological condition driven in part by immune cells from the peripheral circulation, the targets for current successful therapies. The autoimmune and MS risk gene ZMIZ1 is underexpressed in blood in people with MS. We show that, from three independent sets of transcriptomic data, expression of ZMIZ1 is tightly correlated with that of hundreds of other genes. Further we show expression is partially heritable (heritability 0.26), relatively stable over time, predominantly in plasmacytoid dendritic cells and non-classical monocytes, and that levels of ZMIZ1 protein expression are reduced in MS. ZMIZ1 gene expression is increased in response to calcipotriol (1,25 Vitamin D3) (p < 0.0003) and associated with Epstein Barr Virus (EBV) EBNA-1 antibody titre (p < 0.004). MS therapies fingolimod and dimethyl fumarate altered blood ZMIZ1 gene expression compared to untreated MS. The phenotype indicates susceptibility to MS, and may correspond with clinical response and represent a novel clinical target.

Cistromic and genetic evidence that the vitamin D receptor mediates susceptibility to latitude-dependent autoimmune diseases.

The vitamin D receptor (VDR) is a ligand-activated transcription factor that regulates gene expression in many cell types, including immune cells. It requires binding of 1,25 dihydroxy vitamin D3 (1,25D3) for activation. Many autoimmune diseases show latitude-dependent prevalence and/or association with vitamin D deficiency, and vitamin D supplementation is commonly used in their clinical management. 1,25D3 is regulated by genes associated with the risk of autoimmune diseases and predominantly expressed in myeloid cells. We determined the VDR cistrome in monocytes and monocyte-derived inflammatory (DC1) and tolerogenic dendritic cells (DC2). VDR motifs were highly overrepresented in ChIP-Seq peaks in stimulated monocyte (40%), DC1 (21%) and DC2 (47%), P

Hepatic metallothionein expression in chronic hepatitis C virus infection is IFNL3 genotype-dependent.

The IFNL3 genotype predicts the clearance of hepatitis C virus (HCV), spontaneously and with interferon (IFN)-based therapy. The responder genotype is associated with lower expression of interferon stimulated genes (ISGs) in liver biopsies from chronic hepatitis C patients. However, ISGs represent many interacting molecular pathways, and we hypothesised that the IFNL3 genotype may produce a characteristic pattern of ISG expression explaining the effect of genotype on viral clearance. For the first time, we identified an association between a cluster of ISGs, the metallothioneins (MTs) and IFNL3 genotype. Importantly, MTs were significantly upregulated (in contrast to most other ISGs) in HCV-infected liver biopsies of rs8099917 responders. An association between lower fibrosis scores and higher MT levels was demonstrated underlying clinical relevance of this association. As expected, overall ISGs were significantly downregulated in biopsies from subjects with the IFNL3 rs8099917 responder genotype (P=2.38 × 10(-7)). Peripheral blood analysis revealed paradoxical and not previously described findings with upregulation of ISGs seen in the responder genotype (P=1.00 × 10(-4)). The higher MT expression in responders may contribute to their improved viral clearance and MT-inducing agents may be useful adjuncts to therapy for HCV. Upregulation of immune cell ISGs in responders may also contribute to the IFNL3 genotype effect.

Data characterizing the ZMIZ1 molecular phenotype of multiple sclerosis.

The data presented in this article are related to the research article entitled "The autoimmune risk gene ZMIZ1 is a vitamin D responsived marker of a molecular phenotype of multiple sclerosis" Fewings et al. (2017) [1]. Here we identify the set of genes correlated with ZMIZ1 in multiple cohorts, provide phenotypic details on those cohorts, and identify the genes negatively correlated with ZMIZ1 and the cells predominantly expressing those genes. We identify the metabolic pathways in which the molecular phenotype genes are over-represented. Finally, we present the flow cytometry gating strategy we have used to identify the immune cells from blood which are producing ZMIZ1 and RPS6.

Pharmacokinetics in the rat of a panel of immunotoxins made with abrin A chain, ricin A chain, gelonin, and momordin.

A panel of immunotoxins was constructed by chemically attaching the ribosome-inactivating proteins abrin A chain, ricin A chain, gelonin, and momordin to the monoclonal mouse IgG2a antibody Fib75 by means of a disulfide linkage. All the immunotoxins were toxic in tissue culture to the EJ human bladder carcinoma cell line expressing the antigen recognized by Fib75, inhibiting the incorporation of [3H]leucine by 50% at concentrations between 1 x 10(-10) M and 8 x 10(-10) M. The pharmacokinetics of the immunotoxins in the normal Wistar rat was determined following i.v. administration. The concentrations of intact immunotoxin in serum samples taken at various intervals after injection for up to 24 h were measured by solid-phase enzyme-linked immunosorbent assays specific for each of the four different ribosome-inactivating proteins. The Fib75 immunotoxins were cleared from the circulation with comparable, but not identical, biphasic kinetics best described by a two compartment open pharmacokinetic model. The alpha-phase half-lives of the panel, between 0.35 and 0.71 h, were similar. The beta-phase half-life of Fib75-abrin A chain, 13.3 h, was significantly longer than the beta-phase half-lives of Fib75-ricin A chain, Fib75-gelonin, and Fib75-momordin, between 7.5 and 8.6 h. Fib75-abrin A chain was found to be about 3- to 4-fold more resistant than the other immunotoxins to breakdown by reduction of the disulfide linkage between the A chain and the antibody with glutathione in vitro. This suggests that the longer serum half-life of Fib75-abrin A chain may have been due to greater stability against reduction in vivo. Analysis of serum samples obtained up to 24 h after injection of Fib75-abrin A chain revealed that the chemically intact immunotoxin present in the circulation retained full cytotoxic activity. An abrin A chain immunotoxin made with a different monoclonal mouse IgG2a antibody was also found to be more stable against reduction by glutathione in vitro than an analogous ricin A chain immunotoxin. Thus, abrin A chain may posses unique molecular properties that endow immunotoxins made with this A chain with greater stability in vivo than immunotoxins made with ricin A chain or other ribosome-inactivating proteins.

Blood clearance in the rat of a recombinant mouse monoclonal antibody lacking the N-linked oligosaccharide side chains of the CH2 domains.

The serum half-lives of a wild-type recombinant mouse monoclonal antibody of the IgG2b isotype and a mutant antibody differing from the wild-type antibody by a single amino acid substitution introduced into the CH2 domain, the replacement of Asn 297 by Ala to delete the conserved site of heavy chain glycosylation, were determined in the rat. The biological half-life of the aglycosyl Asn 297-Ala mutant recombinant antibody (4.8 days) was significantly shorter than that of the normally glycosylated wild-type antibody (7.4 days) by enzyme immunoassay. A similar difference between the biological half-lives of 125I-labelled aglycosyl and wild-type antibodies (2.9 and 4.0 days, respectively) was determined by gamma counting. Analysis of serum samples demonstrated that both recombinant antibodies were present in the circulation predominantly as intact monomeric IgG and revealed no differences that could account for the more rapid elimination of the aglycosyl antibody. The results of this investigation indicate that the carbohydrate residues contribute only in part to the survival of IgG in vivo and suggest that the diminished half-life of the aglycosyl antibody is due to increased catabolism in the extravascular tissues.

Recombinant mouse monoclonal antibodies with single amino acid substitutions affecting Clq and high affinity Fc receptor binding have identical serum half-lives in the BALB/c mouse.

The serum half-lives of three recombinant mouse monoclonal antibodies, differing radically in their ability to bind to Clq or FcRI but only minimally in structure, were determined in the BALB/c mouse following intravenous administration. The wild-type antibody, a chimaeric antibody comprising variable domains binding 3-iodo-4-hydroxy-5-nitrophenylacetate and constant domains of the mouse IgG2b isotype, was eliminated from the bloodstream with biphasic kinetics: alpha-phase, 0.5 days; beta-phase, 7.0 days. The alpha- and beta-phase half-lives of mutant recombinant antibodies with single amino acid substitutions, either Glu 235-Leu allowing binding to the mouse FcRI, or Lys 322-Ala reducing Clq binding 30-fold, were indistinguishable from those of the wild-type antibody demonstrating that the biological half-life of intact mouse IgG is independent of the ability to bind Clq or FcRI. The major implication of the present study is that IgG molecules which have been genetically engineered to eliminate interaction with other components of the immune system should retain the long half-life typical of natural antibodies.

Ricin B chain converts a non-cytotoxic antibody-ricin A chain conjugate into a potent and specific cytotoxic agent.

We report the conversion of a non-cytotoxic antibody-ricin A chain conjugate to one displaying specific cytotoxic effects comparable with that of native ricin, by the addition of ricin B chain as a second stage reagent. The results suggest that this conversion is achieved by the association of the added B chain with the A chain of the conjugate, and not through a primary binding of B chain at the cell surface.

Purification of immunotoxins containing the ribosome-inactivating proteins gelonin and momordin using high performance liquid immunoaffinity chromatography compared with blue sepharose CL-6B affinity chromatography.

Comparable amounts of the ribosome-inactivating proteins (RIPs) ricin A chain, gelonin and momordin were allowed to bind to Blue Sepharose CL-6B (immobilised Cibacron Blue F3GA) in phosphate buffer, pH 7.5, and were then eluted quantitatively with buffer containing 0.5 M NaCl. Differences in the elution profiles indicated that the RIPs possessed different affinities for the Cibacron Blue F3GA dye. Conjugation of the RIPs to the monoclonal antibody Fib75 resulted in decreased affinity for Blue Sepharose. Under conditions allowing the complete separation of the Fib75-ricin A chain immunotoxin from unconjugated Fib75, the Fib75 immunotoxins made with gelonin and momordin failed to bind completely to the Blue Sepharose column. The Fib75-gelonin and Fib75-momordin fractions that eluted from the column with 0.5 M NaCl were free of unconjugated Fib75 but were enriched in multiply substituted conjugate molecules. A high performance liquid immunoaffinity chromatography procedure based on the selective binding of conjugated RIP to immobilised affinity-purified anti-RIP antibody permitted the complete separation of the gelonin and momordin immunotoxins from unconjugated Fib75 without altering the composition, molecular integrity or cytotoxic activity of the immunotoxins.

The low uptake of an abrin A-chain immunotoxin by rat hepatic cells in vivo and in vitro.

The therapeutic value of antibody-ricin A-chain conjugates (immunotoxins) as antineoplastic agents is limited by their rapid removal from the circulation, primarily by cells of the liver which take up the immunotoxin through receptor mediated recognition of mannose-containing oligosaccharides in the toxin A-chain. We have therefore examined the uptake by rat hepatic cells of a monoclonal antibody (LICR-LOND Fib 75) conjugate assembled with the ricin related, but carbohydrate free, A-chain of the plant toxin abrin. The abrin A-chain immunotoxin was very poorly taken up in vivo and in vitro by both hepatic parenchymal and non parenchymal cells whereas a comparable conjugate assembled with ricin A-chain was actively accumulated by liver cells particularly the hepatic non-parenchymal cells. Furthermore, the abrin A-chain immunotoxin uptake by non-parenchymal cells in vitro was unaffected by mannose and the immunotoxin bound less readily to liver cells than did the ricin A-chain conjugate, consistent with a proposal that its accumulation by hepatic cells is brought about by endocytosis following non-specific binding or by fluid phase pinocytosis. These results suggest abrin A-chain immunotoxins might be further explored as anti-cancer agents since in some cases they could have an improved therapeutic efficacy over immunotoxins constructed with ricin A-chains.

Fate of an antibody-ricin A chain conjugate administered to normal rats.

The pharmacokinetics and catabolism of ricin A chain, a mouse monoclonal antibody (LICR-LOND-Fib 75) and a disulphide linked conjugate of the two have been studied following their intravenous administration to normal rats. Results indicate that the conjugate was removed from the circulation much more rapidly than the antibody but less quickly than the free ricin A chain. Disappearance of the conjugate from the circulation appeared to be biphasic with an early rapid initial phase followed by a much more rapidly than the antibody but less quickly than the free ricin A chain. Disappearance of the conjugate from the circulation appeared to be biphasic with an early rapid initial phase followed by a much slower phase. The fate of a conjugate with a 125I iodide label in the antibody component was compared with that of a conjugate similarly labelled but in the ricin A chain component. The results indicate that breakdown of the conjugate involves both cleavage of the disulphide linkage and complete catabolism of the whole conjugate molecule with the release of 125I iodide. Rapid cleavage of the disulphide bond in the vasculature does not appear to be responsible for the initial rapid disappearance of the conjugate from the circulation.

Comparative stabilities in vitro and in vivo of a recombinant mouse antibody FvCys fragment and a bisFvCys conjugate.

A murine antibody FvCys fragment with a single additional cysteine residue at the C terminus of the VH domain was expressed in Escherichia coli from a modified expression plasmid containing the structural genes for the VH and VL domains derived from the anti-lysozyme hybridoma D1.3. Chemical cross-linking between the introduced sulfhydryl groups of two FvCys fragments by means of bis-maleimidohexane was used to generate a bisFvCys conjugate. The stability of the bisFvCys conjugate and an FvCys analogue that had been reacted with N-ethyl-maleimide to block the free sulfhydryl group, FvCys(BL), were compared after 125I-labeling. The bisFvCys conjugate was completely stable to incubation in solution at 37 degrees C for 24 h whereas only 60% of the FvCys(BL) fragment remained soluble. After i.v. administration to normal Wistar rats, both Fv proteins were rapidly cleared from the circulation with biphasic kinetics that were best fitted to a two-compartment open pharmacokinetic model. The alpha-phase half-life of the bisFvCys conjugate, 0.32 h, was significantly longer than that of the FvCys(BL) fragment, 0.15 h (p less than 0.001) whereas there was no significant difference between the beta-phase half-lives, 1.4 to 1.6h. No chain cleavage or covalent attachment to serum protein was detected by SDS-PAGE analysis of serum samples. However, gel permeation HPLC revealed that both Fv proteins associated with serum proteins in vivo and in vitro.

Comparative biochemical, cytotoxic and pharmacokinetic properties of immunotoxins made with native ricin A chain, ricin A1 chain and recombinant ricin A chain.

Immunotoxins were constructed by attaching native ricin A chain, ricin A1 chain and recombinant ricin A chain to the mouse monoclonal IgG2a antibody Fib75 by means of a disulphide linkage using the hetero-bifunctional cross-linker SPDP. The Fib75 immunotoxins were of similar composition and exerted identical cytotoxic effects against the EJ human bladder carcinoma cell line in tissue culture. All 3 immunotoxins broke down to the same extent upon incubation with glutathione in vitro. The clearance of the immunotoxins from the circulation of normal Wistar rats was determined following i.v. administration. The concentration of intact immunotoxin in serum samples taken at various intervals up to 48hr after injection was measured by a ricin A chain-specific ELISA. The Fib75 immunotoxin made with native ricin A chain was removed from the circulation most rapidly. Fib75-recombinant ricin A chain persisted in the circulation at a higher level than Fib75-ricin A1 chain. A higher proportion of the ricin A1 chain immunotoxin was lost from the bloodstream during the alpha-phase. The beta-phase half-lives of Fib75-recombinant ricin A chain and Fib75-ricin A1 chain were similar, consistent with the identical susceptibility of the immunotoxins to cleavage by glutathione. The presence of the complex-type oligosaccharide side-chain on the A1 chain may have accelerated the clearance of the A1 chain immunotoxin in relation to that of the immunotoxin made with the aglycosyl recombinant A chain.

Biochemical, cytotoxic and pharmacokinetic properties of an immunotoxin composed of a mouse monoclonal antibody Fib75 and the ribosome-inactivating protein alpha-sarcin from Aspergillus giganteus.

An immunotoxin was synthesized by the attachment of alpha-sarcin, the ribosome-inactivating protein derived from the mould Aspergillus giganteus, to a monoclonal mouse IgG2 antibody Fib75. The alpha-sarcin immunotoxin exerted toxic effects in tissue culture against the EJ human bladder carcinoma cell line, expressing the antigen recognised by the Fib75 antibody, inhibiting the incorporation of [3H]leucine by 50% at a concentration of 0.46 nM. The cytotoxic effects of the alpha-sarcin immunotoxin were indistinguishable from those of a Fib75 immunotoxin made with ricin A chain. Fib75-alpha-sarcin was cleared from the circulation of the rat with biphasic kinetics following intravenous administration. The alpha- and beta-phase half-lives were 0.8 h and 6 h, respectively, similar to the serum half-lives of analogous Fib75 immunotoxins made with ribosome-inactivating proteins derived from plants. alpha-Sarcin was completely stable in physiological saline buffer at 37 degrees C, whereas the ribosome-inactivating activity of ricin A chain was gradually lost under identical conditions. alpha-Sarcin may be a valuable alternative to ricin A chain for the construction of therapeutic immunotoxins because of its smaller size and greater thermostability.

Effect of linkage variation on pharmacokinetics of ricin A chain-antibody conjugates in normal rats.

We have investigated the pharmacokinetics of three ricin A chain-antibody conjugates having different bridging structures. Conjugate 1 has a disulphide linkage and was prepared with the N-succinimidyl-3-(2-pyridyldithio)propionate cross-linking reagent. Conjugate 2 has a protected disulphide linkage with a methyl group substituted on the carbon atom of the bridging structure adjacent to the disulphide linkage. Its preparation necessitated the synthesis of a new cross-linking reagent N-succinimidyl 3-(2-pyridyldithio)-butyrate. Conjugate 3 has a sulphide linkage and was prepared with the cross-linking reagent N-succinimidyl 4-(iodoacetylamino)benzoate which was synthesized by a novel route. Conjugate 1 is reducible, conjugate 2 less easily reducible and conjugate 3 nonreducible. On administration to animals all three conjugates displayed biphasic kinetics. The reducibility of the bond had no significant effect on the early disappearance of the conjugate from the circulation. However, at the later time points ease of reduction of the bond was associated with a more rapid disappearance of conjugate.

Structural features of the antibody-A chain linkage that influence the activity and stability of ricin A chain immunotoxins.

The importance of the various structural elements constituting a ricin A chain immunotoxin to the stability of the disulfide bond between the antibody and A chain was examined using a panel of immunoconjugates prepared with the mouse monoclonal antibody Fib75. Analogues of the standard ricin A chain immunotoxin prepared with the N-succinimidyl 3-(2-pyridyldithio)propionate disulfide cross-linker included immunoconjugates made with N-succinimidyl 4-[(iodoacetyl)amino]benzoate the thioether cross-linker; with N-succinimidyl 3-(2-pyridyldithio)butyrate, the hindered disulfide cross-linker; with a peptide spacer between the antibody and cross-linker; or with the dodecapeptide corresponding to the C-terminus of ricin A chain. The cytotoxic activities of the immunoconjugates and their susceptibility to reduction by glutathione in vitro were compared. The thioether-linked immunotoxin could not be cleaved by glutathione in vitro and had low cytotoxic potency, consistent with the requirement of a reducible disulfide linkage for activity. The hindered disulfide-linked immunotoxin was 3-fold more stable to reduction than the immunotoxin containing a standard unhindered disulfide linkage, but the cytotoxic activities of the two constructs were indistinguishable. The introduction of a flexible peptide Ala-Ala-Pro-Ala-Ala-Ala-Pro-Ala-Pro-Ala between Fib75 and the disulfide linkage introduced by SPDP had no deleterious effect on cytotoxic activity and no effect on the susceptibility of the disulfide linkage to reduction. This finding suggests that the enforced proximity of the A chain to the antibody caused by the use of a short chemical cross-linker in a conventional immunotoxin has no influence on either of these properties in this system.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of ricin B chain on the intracellular trafficking of an A chain immunotoxin.

Covalent linkage of the A chain of ricin to the LICR-LOND-Fib75 monoclonal antibody produced an immunotoxin, Fib75-SS-ricin A, which demonstrated immunospecific toxicity to human bladder carcinoma cells in tissue culture (Forrester et al., 1984). The present studies have shown that ricin B chain potentiates the toxicity of the immunotoxin by two orders of magnitude and also significantly increases the rate of protein synthesis inhibition. Using immunoelectron microscopy, the receptor-mediated endocytosis and intracellular routing of the immunotoxin was studied with and without ricin B chain treatment after immunolocalisation of the conjugate. Fib75-SS-ricin A was internalised by the EJ cells predominantly in uncoated pits and vesicles and directed to the endosomes. Some degradation of the complex appeared to take place in multivesicular endosomes at early timepoints and 24 h after internalisation, most of the immunotoxin was found in lysosomes. Some ricin A chain epitopes were detected in Golgi vesicles. Cells treated with immunotoxin and ricin B chain endocytosed the complex predominantly in coated pits and coated vesicles. Using pre-embedding immunoperoxidase techniques, ricin chains were found in the whole Golgi complex and most of the conjugate escaped lysosomal degradation. Internalised immunotoxin was recycled back to the plasma membrane in an active form associated with vesicles which appeared to be derived predominantly from multivesicular endosomes. A similar mode of recycling has recently been reported (McIntosh et al., 1990) for ricin holotoxin in the same cell line. These observations may explain the potentiating effect of toxin B chains in the antibody-directed targeting of toxin A chains.

Safety risk analysis of an innovative environmental technology.

The authors describe a decision and risk analysis performed for the cleanup of a large Department of Energy mixed-waste subsurface disposal area governed by the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA). In a previous study, the authors worked with the site decision makers, state regulators, and U.S. Environmental Protection Agency regional regulators to develop a CERCLA-based multiobjective decision analysis value model and used the model to perform a screening analysis of 28 remedial alternatives. The analysis results identified an innovative technology, in situ vitrification, with high effectiveness versus cost. Since this technology had not been used on this scale before, the major uncertainties were contaminant migration and pressure buildup. Pressure buildup was a safety concern due to the potential risks to worker safety. With the help of environmental technology experts remedial alternative changes were identified to mitigate the concerns about contaminant migration and pressure buildup. The analysis results showed that the probability of an event with a risk to worker safety had been significantly reduced. Based on these results, site decision makers have refocused their test program to examine in situ vitrification and have continued the use of the CERCLA-based decision analysis methodology to analyze remedial alternatives.

Delivery of ricin and abrin A-chains to human carcinoma cells in culture following covalent linkage to monoclonal antibody LICR-LOND-Fib 75.

With the object of generating specific cytotoxic agents, we have prepared covalent conjugates of the A-chains of ricin and of abrin with monoclonal antibody LICR-LOND-Fib 75 and investigated their toxicity toward a human tumor cell line in culture. Both conjugates proved to be potent cytotoxins toward cells carrying the appropriate antigen. The agent containing abrin A-chain was toxic at a significantly lower concentration and exerted its maximum effect more rapidly than the one containing ricin A-chain. Inclusion of chloroquine in the incubation medium significantly enhanced the toxic action of both conjugates without loss of immunospecificity. Because of the widespread occurrence on human tumor cell lines of the antigen recognized by Fib 75, these conjugates, particularly the one containing abrin A-chain, may find application in freeing human bone marrow ex vivo of infiltrated tumor cells prior to reinfusion as an autograft.