Induction of tumor cell apoptosis in vivo increases tumor antigen cross-presentation,cross-priming rather than cross-tolerizing host tumor-specific CD8 T cells

Cross-presentation of cell-bound Ags from established, solid tumors to CD8 cells is efficient and likely to have a role in determining host response to tumor. A number of investigators have predicted that when tumor Ags are derived from apoptotic cells either no response, due to Ag "sequestration," or CD8 cross-tolerance would ensue. Because the crucial issue of whether this happens in vivo has never been addressed, we induced apoptosis of established hemagglutinin (HA)-transfected AB1 tumors in BALB/c mice using the apoptosis-inducing reagent gemcitabine. This shrank the tumor by approximately 80%. This induction of apoptosis increased cross-presentation of HA to CD8 cells yet neither gross deletion nor functional tolerance of HA-specific CD8 cells were observed, based on tetramer analysis, proliferation of specific CD8 T cells, and in vivo CTL activity. Interestingly, apoptosis primed the host for a strong antitumor response to a second, virus-generated HA-specific signal in that administration of an HA-expressing virus after gemcitabine administration markedly decreased tumor growth compared with viral administration without gemcitabine. Thus tumor cell apoptosis in vivo neither sequesters tumor Ags nor cross-tolerizes tumor-specific CD8 cells. This observation has fundamental consequences for the development of tumor immunotherapy protocols and for understanding T cell reactivity to tumors and the in vivo immune responses to apoptotic cells.     

Identification of a new simian immunodeficiency virus lineage with a vpu gene present among different cercopithecus monkeys (C. mona, C. cephus, and C. nictitans) from Cameroon

During a large serosurvey of wild-caught primates from Cameroon, we found 2 mona monkeys (Cercopithecus mona) out of 8 and 47 mustached monkeys (Cercopithecus cephus) out of 302 with human immunodeficiency virus (HIV)-simian immunodeficiency virus (SIV) cross-reactive antibodies. In this report, we describe the full-length genome sequences of two novel SIVs, designated SIVmon-99CMCML1 and SIVmus-01CM1085, isolated from one mona (CML1) and one mustached (1085) monkey, respectively. Interestingly, these viruses displayed the same genetic organization (i.e., presence of a vpu homologue) as members of the SIVcpz-HIV type 1 lineage and SIVgsn isolated from greater spot-nosed monkeys (Cercopithecus nictitans). Phylogenetic analyses of SIVmon and SIVmus revealed that these viruses were genetically distinct from other known primate lentiviruses but were more closely related to SIVgsn all across their genomes, thus forming a monophyletic lineage within the primate lentivirus family, which we designated the SIVgsn lineage. Interestingly, mona, mustached, and greater spot-nosed monkeys are phylogenetically related species belonging to three different groups of the genus Cercopithecus, the C. mona, C. cephus, and Cercopithecus mitis groups, respectively. The presence of new viruses closely related to SIVgsn in two other species reinforces the hypothesis that a recombination event between ancestral SIVs from the family Cercopithecinae is the origin of the present SIVcpz that is widespread among the chimpanzee population.     

Inhibitors tethered near the acetylcholinesterase active site serve as molecular rulers of the peripheral and acylation sites

The acetylcholinesterase (AChE) active site consists of a narrow gorge with two separate ligand binding sites: an acylation site (or A-site) at the bottom of the gorge where substrate hydrolysis occurs and a peripheral site (or P-site) at the gorge mouth. AChE is inactivated by organophosphates as they pass through the P-site and phosphorylate the catalytic serine in the A-site. One strategy to protect against organophosphate inactivation is to design cyclic ligands that will bind specifically to the P-site and block the passage of organophosphates but not acetylcholine. To accelerate the process of identifying cyclic compounds with high affinity for the AChE P-site, we introduced a cysteine residue near the rim of the P-site by site-specific mutagenesis to generate recombinant human H287C AChE. Compounds were synthesized with a highly reactive methanethiosulfonyl substituent and linked to this cysteine through a disulfide bond. The advantages of this tethering were demonstrated with H287C AChE modified with six compounds, consisting of cationic trialkylammonium, acridinium, and tacrine ligands with tethers of varying length. Modification by ligands with short tethers had little effect on catalytic properties, but longer tethering resulted in shifts in substrate hydrolysis profiles and reduced affinity for acridinium affinity resin. Molecular modeling calculations indicated that cationic ligands with tethers of intermediate length bound to the P-site, whereas those with long tethers reached the A-site. These binding locations were confirmed experimentally by measuring competitive inhibition constants KI2 for propidium and tacrine, inhibitors specific for the P- and A-sites, respectively. Values of KI2 for propidium increased 30- to 100-fold when ligands had either intermediate or long tethers. In contrast, the value of KI2 for tacrine increased substantially only when ligands had long tethers. These relative changes in propidium and tacrine affinities thus provided a sensitive molecular ruler for assigning the binding locations of the tethered cations.     

Modulation of PAI-1 and proMMP-9 syntheses by soluble TNFalpha and its receptors during differentiation of the human monocytic HL-60 cell line

During phorbol ester-induced differentiation of HL-60 monocytic cells, tumor necrosis factoralpha (TNFalpha) synthesis and secretion are increased, which contributes to the autocrine regulation of TNFalpha-responsive genes. We investigated how, during phorbol ester-induced differentiation of HL-60 cells, the secreted TNFalpha modulated plasminogen activator inhibitor type I (PAI-1) and gelatinase B (MMP-9) syntheses, two proteins involved in pericellular proteolysis. The differentiation-induced release of TNFalpha, was abolished by the hydroxamate-based matrix metalloproteinase (MMP) inhibitor, RU36156. RU36156 or a neutralizing anti-TNFalpha significantly down-regulated PAI-1 synthesis exclusively during the early phases of differentiation (from promyelocyte to monocytic-like cells), which underlined the activating role of autocrine TNFalpha during this time range. As cells progressed to monocyte/macrophage phenotype, they still released TNFalpha, but RU36156 or anti-TNFalpha no longer had an effect on PAI-1 synthesis. This lack of effect was not due to a default of TNFalpha signaling since PAI-1 synthesis was still stimulated in response to exogenous TNFalpha. TNFalpha receptor RI was also actively released and was shown to reduce TNFalpha activity which may account for the inability of soluble TNFalpha to up-regulate PAI-1 synthesis. In later mature stage, cells became susceptible to exogenous TNFalpha-induced apoptosis and rapidly lost their ability to respond to TNFalpha. The MMP-9 synthesis followed similar regulation as PAI-1. Isolated human blood monocytes-derived macrophages behave like HL-60-derived macrophages. In conclusion, these results show that during leukocyte differentiation, time windows exist during which the autocrine TNFalpha is active and then down-regulated by RI, which may temper a continuous up-regulation of the synthesis of proteins involved in pericellular proteolysis.     

Intracellular maturation and transport of tumor necrosis factor alpha converting enzyme

The tumor necrosis factor alpha converting enzyme (TACE) activity is required for the shedding of a variety of biologically active membrane bound precursors. The activation of TACE necessitates the proteolytic cleavage of its prodomain, a process that was suggested to be catalyzed by the proprotein convertase furin. However, the involvement of furin in this activation process has never been experimentally demonstrated. We have shown that the furinlike cleavage site (R-V-K-R(214)) localized between the prodomain and the metalloprotease domain of TACE is the sole site that can be in vitro cleaved by furin. In Cos7 cells, the release of TACE-processed substrates was reduced by the overexpression of the furin-specific proprotein convertase inhibitor Portland alpha1-antitrypsin inhibitor, but the release of TACE-processed substrates was increased by overexpression of furin in LoVo cells (deficient in furin activity) in which a mature form of TACE was identified. The immature form of TACE was detected at the surface of LoVo cells and at the surface of Cos7 and HT29 cells upon proprotein convertase inhibition. These results suggest that furin is the major proprotein convertase involved in the maturation/activation of TACE which is not a prerequisite for its cell-surface expression.     

Cyanobacterial toxicity and migration in a mesotrophic lake in western Washington, USA

In fall 1997, the toxic cyanobacterium Microcystis aeruginosa was documented in Lake Sammamish (western Washington, U.S.A.) for the first time. Cyanobacterial activity and environmental conditions that may promote toxic cyanobacteria were investigated during summer and fall 1999. Development of toxic Microcystis was hypothesized to be due to runoff of nutrients from the watershed (external loading hypothesis) or from vertical migration of dormant cyanobacteria from the nutrient-rich sediments into the water column (cyanobacterial migration hypothesis). Microcystins were detected using an enzyme-linked immunosorbent assay during late August and early September 1999 despite low cyanobacterial abundance. Microcystin concentrations ranged between 0.19–3.8 g l^–1 throughout the lake and at all depths with the exception of the boat launch where concentrations reached 43 g l^–1. Comparison of the conditions associated with the toxic episodes in 1997 and 1999 indicate that Microcystis is associated with a stable water column, increased surface total phosphorus concentrations (> 10 g l^–1), surface temperatures greater than 22°C, high total nitrogen to phosphorus ratios (> 30), and increased water column transparency (up to 5.5 m). Migration of the cyanobacteria, Microcystis and Anabaena, occurred in both the deep and shallow portions of the lake. Microcystis dominated (89–99%) the migrating cyanobacteria with greater migration from the shallow station. External loading of nutrients due to the large rainfall preceding the 1997 toxic episode may have provided the nutrients needed to fuel that bloom. However, toxic Microcystis occurred in 1999 despite the lack of rain and subsequent external runoff. The migration of Microcystis from the nutrient-rich sediments may have been the inoculum for the toxic population detected in 1999.     

Influences of base excision repair defects on the lethality and mutagenicity induced by Me-lex,a sequence-selective N3-adenine methylating agent

Due to its minor groove selectivity, Me-lex preferentially generates N3-methyladenine (3-MeA) adducts in double-stranded DNA. We undertook a genetic approach in yeast to establish the influence of base excision repair (BER) defects on the processing of Me-lex lesions on plasmid DNA that harbors the p53 cDNA as target. We constructed a panel of isogenic strains containing a reporter gene to test p53 function and the following gene deletions: deltamag1, deltaapn1apn2, and deltaapn1apn2mag1. When compared with the wild-type strain, a decrease in survival was observed in deltamag1, deltaapn1apn2, and deltaapn1apn2mag1. The Me-lex-induced mutation frequency increased in the following order: wild type < deltamag1< deltaapn1apn2 = deltaapn1apn2mag1. A total of 77 mutants (23 in wild type, 31 in deltamag1, and 23 in deltaapn1apn2) were sequenced. Eighty-one independent mutations (24 in wild type, 34 in deltamag1, and 23 in deltaapn1apn2) were detected. The majority of base pair substitutions were AT-targeted in all strains (14/23, 61% in wild type; 20/34, 59%, in deltamag1; and 14/23, 61%, in deltaapn1apn2). The Mag1 deletion was associated with a significant decrease of GC > AT transitions when compared with both the wild-type and the AP endonuclease mutants. This is the first time that the impact of Mag1 and/or AP endonuclease defects on the mutational spectra caused by 3-MeA has been determined. The results suggest that 3-MeA is critical for Me-lex cytotoxicity and that its mutagenicity is slightly elevated in the absence of Mag1 glycosylase activity but significantly higher in the absence of AP endonuclease activity.     

Disruption of dynein/dynactin inhibits axonal transport in motor neurons causing late-onset progressive degeneration

To test the hypothesis that inhibition of axonal transport is sufficient to cause motor neuron degeneration such as that observed in amyotrophic lateral sclerosis (ALS), we engineered a targeted disruption of the dynein-dynactin complex in postnatal motor neurons of transgenic mice. Dynamitin overexpression was found to disassemble dynactin, a required activator of cytoplasmic dynein, resulting in an inhibition of retrograde axonal transport. Mice overexpressing dynamitin demonstrate a late-onset progressive motor neuron degenerative disease characterized by decreased strength and endurance, motor neuron degeneration and loss, and denervation of muscle. Previous transgenic mouse models of ALS have shown abnormalities in microtubule-based axonal transport. In this report, we describe a mouse model that confirms the critical role of disrupted axonal transport in the pathogenesis of motor neuron degenerative disease.