Immunohistochemistry of adenosine deaminase: implications for adenosine neurotransmission.

Immunohistochemical analysis of adenosine deaminase in rat brain revealed an extensive plexus of adenosine deaminase-containing neurons in the basal hypothalamus. These neurons converged on and were most numerous in three major centers, namely, the tuberal, caudal, and postmammillary caudal magnocellular nuclei. Most other brain regions were devoid of cells containing adenosine deaminase. Some adenosine deaminase-containing neurons were retrogradely labeled with the fluorescent dye fast blue when the dye was injected into the frontal cortex and striatum. Specific populations of neurons having high levels of adenosine deaminase may release adenosine as a neurotransmitter.

Intracellular restriction factors in mammalian cells--An ancient defense system finds a modern foe.

Cross-species transmission of retroviruses poses a threat to mammalian species. Zoonoses have given rise to devastating diseases because the host organism is not prepared to resist a new pathogen. Mammals have developed several layers of defense against viruses, including an intracellular antiretroviral defense, a part of innate immunity. Retroviral restrictions had been studied for decades using murine leukemia virus in mice, however it has become clear that primates too have intrinsic mechanisms to ward off infections by retroviruses. Several of these antiretroviral restriction mechanisms have recently been identified, with two particularly well described factors being members of the tripartite motif (Trim) and APOBEC families. Both systems provide a strong barrier against lentiviral infections. The viruses have developed countermeasures that allow them to replicate despite the host factors. This review discusses our current knowledge of this ancient battle between mammalian hosts and their retroviral opponents.

APOBEC family proteins: novel antiviral innate immunity.

APOBEC3G has been identified as an anti-human immunodeficiency virus type 1 (HIV-1) host factor that belongs to the APOBEC superfamily of cytidine deaminases. It deaminates cytidine to uridine in nascent minus-strand viral DNA, inducing G-to-A hypermutation in the plus-strand DNA of HIV-1. The accumulating evidence demonstrates that APOBEC family proteins also have an antiviral activity against a wide variety of viruses, including not only retroviruses but also other types of viruses, and that each virus seems to have its own strategy for escaping from APOBEC proteins. These results suggest that the APOBEC3 family plays an important role in antiviral host defense as an innate immunity. Recent progress in research on APOBEC family proteins is reviewed.

Lentiviral Vif: viral hijacker of the ubiquitin-proteasome system.

Since the beginning of time, microorganisms have been devising ways to bypass detection and destruction by our immune system. Therefore, it is no surprise that along with the identification of the cellular antiviral protein APOBEC3G (A3G) has come the recognition of the viral solution to this assault. Here, we review the research that led up to the identification of A3G and the mechanism that the human immunodeficiency virus protein Vif developed to evade A3G's antiviral activities.

Tumors expressing the cytosine deaminase suicide gene can be eliminated in vivo with 5-fluorocytosine and induce protective immunity to wild type tumor.

Successful expression of the cytosine deaminase (CD) suicide gene in vivo is demonstrated in three weakly immunogenic murine tumor models: the 102 and 205 fibrosarcomas and the 38 adenocarcinoma. Normal mammalian cells do not contain cytosine deaminase, but tumor cells transduced with retroviral vectors containing the CD gene metabolize the relatively nontoxic prodrug 5-fluorocytosine to the highly toxic 5-fluorouracil. In vitro cells expressing the CD gene are killed by 5-fluorocytosine while unmodified cells are not. When injected into syngeneic mice, CD+ tumors can also be eliminated in vivo by systemic treatment with 5-fluorocytosine without significant toxicity to the host. Animals whose CD+ tumors were eliminated with prodrug treatment resist subsequent rechallenge with unmodified wild type tumor. This posttreatment immunity appears to be tumor specific. Applications of the CD system in gene therapy models are discussed.

Detection of cytosine deaminase in genetically modified tumor cells by specific antibodies.

Bacterial cytosine deaminase (CD) converts the non-toxic prodrug 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU), which is toxic for mammalian cells. Therefore, the CD gene is used in cancer gene therapy to achieve high local concentration of a toxic metabolite without significant systemic toxicity. To allow the detection of CD expression at the protein level, we raised both polyclonal rabbit antisera and a monoclonal antibody (mAb) against a histidine-tagged CD fusion protein. The specificity of the polyclonal antisera and the mAb was confirmed by immunohistochemistry, immunoblot analysis, and immunoprecipitation using CD-expressing tumor cell lines. Furthermore, the antibodies can be used for ELISA assays and flow cytometry. Finally, the CD protein could be demonstrated in frozen tissue sections of CD-modified tumors in a rat tumor model using the anti-CD serum. With these antibodies, CD expression can now be monitored throughout in vitro and in vivo gene transfer studies, including clinical protocols relying on the CD suicide gene strategy.

Solid-phase adsorption of antigens for efficient production of antibodies reactive with native and fixed tissue antigens.

A study has been made of the efficacy of different immunization protocols using low antigen levels for the generation of monoclonal antibodies capable of detecting antigens (ADCP) in processed tissues. Protocols using unprocessed native antigen immobilized on nitrocellulose were more efficient than soluble antigen in generating serum antibodies reactive with both native antigen and processed tissues. The derived monoclonal antibodies reacted with native but not processed antigen. The use of antigen immobilized on polyvinylidene (PVDF) and subsequently processed as for histochemistry was successful in generating monoclonal antibodies reactive with processed antigen.

Murine monoclonal antibodies which recognize adenosine deaminase from calf, mouse, rat and man.

Three monoclonal antibodies against calf adenosine deaminase were obtained from mice. All three strongly cross-react with the rat and human forms of adenosine deaminase, and two of them with the mouse enzyme. We show that these reagents can be useful for the preparation of adenosine deaminase-free cell culture media and consider their potential interest for the early immunofluorescence detection of T-cell acute lymphoblastic leukemias.

Development of adenosine deaminase-immunoreactive neurons in the rat brain.

It has previously been demonstrated that neurons immunoreactive for the enzyme adenosine deaminase (ADA) have a highly restricted distribution pattern in the adult rat brain. In order to determine whether the pattern of ADA expression is equally limited during the period of brain development, the localization of ADA was investigated immunohistochemically in brains of embryonic, early postnatal and young adult rats. No immunostaining for ADA was detected on the 12th embryonic day. On embryonic day 15, ADA-immunoreactive cells were first observed in the hypoglossal motor nucleus, and on day 18 in cingulate, retrosplenial and visual cortex, in the posterior basal hypothalamus, and in the facial motor nucleus. On the 20th embryonic day ADA-immunoreactive neurons appeared in various olfactory and related systems and in the superior colliculus. On the 1st postnatal day, immunoreactivity was intensified in all structures in which it was observed at preceeding ages and, in addition, appeared in several brainstem regions. On postnatal day 10 and 15, immunostained neurons appeared in several subcortical structures whereas the number of these decreased in the anterior olfactory nucleus and some related cortical areas. In animals 25 days of age the intensity of immunostaining continued to increase, essentially producing the adult pattern in all except olfactory areas where there was a dramatic loss of ADA-immunoreactive cells. These results show that the restricted pattern of ADA-immunostaining observed in adult rat brain is generated over a protracted period of development, various stages of which are characterized predominantly by the expression of ADA in greater abundance, at least to the extent this can be gleaned immunohistochemically, in greater numbers of neurons and to a minor degree by a decreased capacity to express this enzyme.

Development of class-switched, affinity-matured monoclonal antibodies following a 7-day immunization schedule.

Previously we reported making high-affinity monoclonal antibodies (MAbs) 13 days after the onset of Repetitive Immunizations Multiple Sites (RIMMS) strategy. The Ig subclass variety and affinity of these antibodies suggested that maturational processes had already begun within draining lymph nodes. We now demonstrate that this diversity can in fact be captured as early as Day 7. In the work reported here, somatic fusion of immune lymphocytes isolated from peripheral lymph nodes resulted in the isolation of affinity-matured MAbs reactive with cytosine deaminase. This model further demonstrates and substantiates at a cellular level the rapid development and maturation of T-cell-dependent B-cell responses occurring within draining lymph nodes following antigen challenge.

Trichoderma harzianum ETS 323-mediated resistance in Brassica oleracea var. capitata to Rhizoctonia solani involves the novel expression of a glutathione S-transferase and a deoxycytidine deaminase.

Plant interactions with microbial biocontrol agents are used as experimental models to understand resistance-related molecular adaptations of plants. In a hydroponic three-way interaction study, a novel Trichoderma harzianum ETS 323 mediated mechanism was found to induce resistance to Rhizoctonia solani infection in Brassica oleracea var. capitata plantlets. The R. solani challenge on leaves initiate an increase in lipoxygenase activity and associated hypersensitive tissue damage with characteristic "programmed cell death" that facilitate the infection. However, B. oleracea plantlets whose roots were briefly (6 h) colonized by T. harzianum ETS 323 developed resistance to R. solani infection through a significant reduction of the host hypersensitive tissue damage. The resistance developed in the distal leaf tissue was associated with the expression of a H(2)O(2)-inducible glutathione S-transferase (BoGST), which scavenges cytotoxic reactive electrophiles, and of a deoxycytidine deaminase (BoDCD), which modulates the host molecular expression and potentially neutralizes the DNA adducts and maintains DNA integrity. The cDNAs of BoGST and BoDCD were cloned and sequenced; their expressions were verified by reverse-transcription polymerase chain reaction analysis and were found to be transcriptionally activated during the three-way interaction.

RNA and DNA binding properties of HIV-1 Vif protein: a fluorescence study.

The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Some "non-permissive" cell lines cannot sustain replication of Vif(-) HIV-1 virions. In these cells, Vif counteracts the natural antiretroviral activity of the DNA-editing enzymes APOBEC3G/3F. Moreover, Vif is packaged into viral particles through a strong interaction with genomic RNA in viral nucleoprotein complexes. To gain insights into determinants of this binding process, we performed the first characterization of Vif/nucleic acid interactions using Vif intrinsic fluorescence. We determined the affinity of Vif for RNA fragments corresponding to various regions of the HIV-1 genome. Our results demonstrated preferential and moderately cooperative binding for RNAs corresponding to the 5'-untranslated region of HIV-1 (5'-untranslated region) and gag (cooperativity parameter omega approximately 65-80, and K(d) = 45-55 nM). In addition, fluorescence spectroscopy allowed us to point out the TAR apical loop and a short region in gag as primary strong affinity binding sites (K(d) = 9.5-14 nM). Interestingly, beside its RNA binding properties, the Vif protein can also bind the corresponding DNA oligonucleotides and their complementary counterparts with an affinity similar to the one observed for the RNA sequences, while other DNA sequences displayed reduced affinity. Taken together, our results suggest that Vif binding to RNA and DNA offers several non-exclusive ways to counteract APOBEC3G/3F factors, in addition to the well documented Vif-induced degradation by the proteasome and to the Vif-mediated repression of translation of these antiviral factors.

Multifaceted antiviral actions of APOBEC3 cytidine deaminases.

To defend against external pathogens, metazoan organisms have evolved numerous defenses that generally fall within the innate and adaptive immune responses. Considerable effort continues to focus on developing a vaccine to manipulate the adaptive immune system to protect against or control HIV-1. However, recent advances in our understanding of the innate immune system have revealed that cells have a potent intrinsic antiretroviral defense in the form of APOBEC3G, which is a member of a larger family of cytidine deaminases that are active against HIV-1 and other retroviruses. Insights into how the action of A3G is circumvented by HIV-1 through the action of its Vif protein, and the surprising mechanisms by which A3G is regulated within the cell, offer exciting new opportunities for developing novel anti-HIV-1 therapies that exploit this intrinsic antiretroviral system.

STAT1-independent cell type-specific regulation of antiviral APOBEC3G by IFN-alpha.

APOBEC3G (A3G) has broad antiviral activity against retroviruses and hepatitis B virus. However, the role of IFNs in regulating A3G during innate immunity has not been established. In this study, we show that the A3G gene is uniquely regulated by IFNs in a cell type-dependent manner. A3G was up-regulated by IFN-alpha in liver cells and macrophages, but not in T lymphoid cells or epithelial 293T cells. In contrast, other IFN-alpha-stimulated genes such as dsRNA-activated protein kinase were induced in all these cells, suggesting additional cellular factors may regulate IFN-alpha-induced A3G expression. Consistent with this idea, IFN-alpha-mediated induction of A3G, but not other IFN-alpha-stimulated genes, was potently inhibited by the drug Rottlerin, through a mechanism independent of STAT1 activation. The canonical IFN-alpha-mediated pathway of gene transcription requires both STAT1 and STAT2. Surprisingly, induction of A3G was STAT1 independent, but STAT2 dependent in liver cells. However, STAT1 signaling was functional and required for IFN-gamma induction of A3G in these cells. Our results indicate that A3G may participate in antiviral cellular defenses through a novel IFN-mediated signaling pathway.

AID to overcome the limitations of genomic information.

The limitations of genomic information forced our ancestors to adopt a strategy for introducing somatic DNA alterations with the risk of genome instability. Although activation-induced deaminase (AID) is involved in DNA cleavage in somatic hypermutation and class-switch recombination, its mechanism of action has been debated extensively, with the two main hypotheses being distinguished by the chief target of AID: RNA or DNA. The principle distinction between the two hypotheses is the requirement for translation of edited mRNA or uracil removal from DNA for DNA cleavage. Although a series of experiments has provided support for the 'RNA-editing' hypothesis and requires reevaluation of the 'DNA-deamination' hypothesis, definitive proof is yet to come.

The MRE11-RAD50-NBS1 complex accelerates somatic hypermutation and gene conversion of immunoglobulin variable regions.

Targeted diversification of immunoglobulin variable regions is induced by activation-induced deaminase and may occur by either somatic hypermutation or gene conversion. MRE11-RAD50-NBS1 (MRN) is a ubiquitous and conserved nuclease complex critical for DNA break repair and is essential in class-switch recombination. Here we show that ectopic expression of NBS1, the regulatory subunit of MRN, accelerated hypermutation in the human B cell line Ramos and accelerated gene conversion in the chicken B cell line DT40. In both cases, accelerated diversification depended on MRN complex formation. These data suggest that MRN promotes DNA cleavage and/or mutagenic repair of lesions initiated by activation-induced deaminase, acting in the shared pathway of immunoglobulin gene diversification.

Alteration of the circulating life and antigenic properties of bovine adenosine deaminase in mice by attachment of polyethylene glycol.

Polyethylene glycol was attached covalently to adenosine deaminase (ADA) using cyanuric chloride as the coupling agent. The modified adenosine deaminase (PEG-ADA) appears to lose its immunogenicity in mice following multiple intravenous injections. PEG-ADA does not react with antibodies raised against native ADA. The circulating half-life (T1/2) of PEG-ADA was increased to 28 hr. The lack of detectable antibody formation and long circulating life may make PEG-ADA suitable for treating human ADA deficiency.

Application of monoclonal antibodies against cytosine deaminase for the in vivo clearance of a cytosine deaminase immunoconjugate.

The selective delivery of anticancer drugs to tumors vs normal tissue using targeted antibody-enzyme conjugates for prodrug activation is limited by the amount of drug generated by blood-borne enzyme. Clearance of non-tumor-associated conjugate would increase the tumor/blood conjugate ratio, and enable larger amounts of prodrugs to be administered. A method for clearing the monoclonal antibody (mAb) conjugate L6-cytosine deaminase (L6-CD) was established by using an antibody raised against CD. The mAb 102-26 was obtained by immunizing BALB/C mice with CD conjugated to keyhole limpet hemocyanin. 102-26 was able to precipitate purified CD from solution as assessed by radioimmune precipitation and recognized CD in Western blot analyses. Similar studies were used to establish that 102-26 also recognized CD when conjugated to the L6 and 1F5 mAbs. Selective removal of L6-CD from the circulation of nude mice bearing H2981 human lung adenocarcinoma (L6-antigen positive) was achieved by injecting 102-26 24 h after L6-CD administration. High T/B ratios were obtained by clearance of a L6-CD (38:1 compared to 1.3:1 without clearance).

A33scFv-cytosine deaminase: a recombinant protein construct for antibody-directed enzyme-prodrug therapy.

A recombinant fusion protein of colon carcinoma binding A33 single chain antibody with cytosine deaminase displayed specific antigen binding and enzyme activity in surface plasmon resonance and is catalytic activity assay. In vitro, it selectively increased the toxicity of 5-FC to A33 antigen-positive cells by 300-fold, demonstrating the potency of this ADEPT strategy.