Role of CD40 ligand and CD28 in induction and maintenance of antiviral CD8+ effector T cell responses

The primary aim of this report was to evaluate the immune responses of CD40 ligand-deficient (CD40L-/-) mice infected with two viruses known to differ markedly in their capacity to replicate in the host. Lymphocytic choriomeningitis virus (LCMV) is a natural mouse pathogen that replicates widely and extensively, whereas vesicular stomatitis virus (VSV) spreads poorly. We found that the primary response of CD40L-/- mice toward VSV is significantly impaired; proliferation of both CD4+ and CD8+ cells is reduced 2- to 3-fold, few CD8+ cells acquire an activated phenotype, and little functional activity is induced. Very similar results were obtained in VSV-infected, CD28-deficient mice. In contrast, neither CD40L nor CD28 was required for induction of a primary CD8+ response toward LCMV. Surprisingly, lack of CD4+ T cells had no impact on the primary immune response toward any of the viruses, even though the CD40 ligand dependence demonstrated for VSV would be expected to be associated with CD4 dependence. Upon coinfection of VSV-infected mice with LCMV, the requirement for CD40 ligand (but not CD28) could be partially bypassed, as evidenced by a 3-fold increase in the frequency of VSV-specific CD8+ T cells on day 6 postinfection. Finally, despite the fact that the primary LCMV-specific CD8+ response is virtually unimpaired in CD40L-/- mice, their capacity to maintain CD8+ effector activity and to permanently control the infection is significantly reduced. Thus, our results demonstrate that the importance of CD40/CD40L interaction for activation of CD8+ T cells varies between viruses and over time.     

Metabolic network analysis of Penicillium chrysogenum using (13)C-labeled glucose

Using (13)C-labeled glucose fed to a penicillin-overproducing strain of Penicillium chrysogenum, the intracellular fluxes were quantified, and the presence of two new pathways, not previously described in this organism, is suggested. Thus, glycine was synthesized not only by serine hydroxymethyltransferase, but also by threonine aldolase. The formation of cytosolic acetyl-CoA was found to be synthesized both via the citrate lyase-catalyzed reaction and by degradation of the penicillin side-chain precursor, phenoxyacetic acid. Furthermore, the experimental data indicate that the main activities of homocitrate synthase and alpha-isopropylmalate synthase are located in the cytosol. All experimental data on the labeling patterns were obtained using gas chromatography-mass spectrometry, which is faster and more sensitive than the nuclear magnetic resonance methods usually applied for analysis of labeling patterns.     

Analysis of pectic epitopes recognised by hybridoma and phage display monoclonal antibodies using defined oligosaccharides, polysaccharides, and enzymatic degradation

The structure of epitopes recognised by anti-pectin monoclonal antibodies (mAbs) has been investigated using a series of model lime-pectin samples with defined degrees and patterns of methyl esterification, a range of defined oligogalacturonides and enzymatic degradation of pectic polysaccharides. In immuno-dot-assays, the anti-homogalacturonan (HG) mAbs JIM5 and JIM7 both bound to samples with a wide range of degrees of methyl esterification in preference to fully de-esterified samples. In contrast, the anti-HG phage display mAb PAM1 bound most effectively to fully de-esterified pectin. In competitive inhibition ELISAs using fully methyl-esterified or fully de-esterified oligogalacturonides with 3-9 galacturonic acid residues, JIM5 bound weakly to a fully de-esterified nonagalacturonide but JIM7 did not bind to any of the oligogalacturonides tested. Therefore, optimal JIM5 and JIM7 binding occurs where specific but undefined methyl-esterification patterns are present on HG domains, although fully de-esterified HG samples contain sub-optimal JIM5 epitopes. The persistence of mAb binding to epitopes in pectic antigens, with 41% blockwise esterification (P41) and 43% random esterification (F43) subject to fragmentation by endo-polygalacturonase II (PG II) and endo-pectin lyase (PL), was also studied. Time course analysis of PG II digestion of P41 revealed that JIM5 epitopes were rapidly degraded, but a low level of PAM1 and JIM7 epitopes existed even after extensive digestion, indicating that some HG domains were more resistant to cleavage by PG II. The chromatographic separation of fragments produced by the complete digestion of P41 by pectin lyase indicated that a very restricted population of fragments contained the PAM1 epitope while a (1-->4)-beta-D-galactan epitope occurring on the side chains of pectic polysaccharides was recovered in a broad range of fractions.     

Evidence for the role of megalin in renal uptake of transthyretin

The kidney is a major organ for uptake of the thyroid hormone thyroxine (T(4)) and its conversion to the active form, triiodothyronine. In the plasma, one of the T(4) carriers is transthyretin (TTR). In the present study we observed that TTR, the transporter of both T(4) and retinol-binding protein, binds to megalin, the multiligand receptor expressed on the luminal surface of various epithelia including the renal proximal tubules. In the kidney, megalin plays an important role in tubular uptake of macromolecules filtered through the glomerulus. To evaluate the importance of megalin for renal uptake of TTR, we performed binding/uptake assays using immortalized rat yolk sac cells with high expression levels of megalin. Radiolabeled TTR, free as well as in complex with thyroxine or retinol-binding protein, was rapidly taken up by the cells, and the uptake was strongly inhibited by a polyclonal megalin antibody and by the receptor-associated protein, a chaperone-like protein inhibiting ligand binding to megalin. In cell culture, different TTR mutations presented different levels of cell association and degradation, suggesting that the structure of TTR is important for megalin recognition. Both the apo form and the T(4)-bound form were taken up by the cells. Analysis of urine from patients with Dent's disease, a renal tubular disorder that alters receptor-mediated endocytic reabsorption of proteins, identified TTR as an abundant excreted protein. Furthermore, analysis of kidney sections of megalin-deficient mice revealed no immunohistochemical TTR labeling in intracellular vesicles in the proximal tubule cells when compared with wild type control littermates. Taken together, the present data indicate that TTR represents a novel megalin ligand of importance in the thyroid hormone homeostasis.     

Two widely spaced initiator binding sites create an HMG1-dependent parvovirus rolling-hairpin replication origin

Minute virus of mice (MVM) replicates via a linearized form of rolling-circle replication in which the viral nickase, NS1, initiates DNA synthesis by introducing a site-specific nick into either of two distinct origin sequences. In vitro nicking and replication assays with substrates that had deletions or mutations were used to explore the sequences and structural elements essential for activity of one of these origins, located in the right-end (5') viral telomere. This structure contains 248 nucleotides, most-favorably arranged as a simple hairpin with six unpaired bases. However, a pair of opposing NS1 binding sites, located near its outboard end, create a 33-bp palindrome that could potentially assume an alternate cruciform configuration and hence directly bind HMG1, the essential cofactor for this origin. The palindromic nature of this sequence, and thus its ability to fold into a cruciform, was dispensable for origin function, as was the NS1 binding site occupying the inboard arm of the palindrome. In contrast, the NS1 site in the outboard arm was essential for initiation, even though positioned 120 bp from the nick site. The specific sequence of the nick site and an additional NS1 binding site which directly orients NS1 over the initiation site were also essential and delimited the inboard border of the minimal right-end origin. DNase I and hydroxyl radical footprints defined sequences protected by NS1 and suggest that HMG1 allows the NS1 molecules positioned at each end of the origin to interact, creating a distortion characteristic of a double helical loop.     

DNA vaccination prevents and/or delays carcinoma development of papillomavirus-induced skin papillomas on rabbits

Malignant progression is a life-threatening consequence of human papillomavirus-associated lesions. In this study, we tested the efficacy of papillomavirus early-gene-based vaccines for prevention of carcinoma development of papillomavirus-induced skin papillomas on rabbits. Rabbit skin papillomas were initiated by infection with cottontail rabbit papillomavirus (CRPV). The papillomas were allowed to grow for 3 months without any treatment intervention. Rabbits were then immunized by gene gun-mediated intracutaneous administration of four DNA plasmids encoding CRPV E1, E2, E6, and E7 genes, respectively. All eight control rabbits receiving vector alone developed invasive carcinoma within 8 to 13 months. In contrast, only two of eight vaccinated rabbits developed carcinoma at 12 and 15 months, respectively. Papilloma growth was suppressed in the majority of vaccinated rabbits but not completely eradicated. These results indicate that gene gun-mediated immunization with papillomavirus early genes may be a promising strategy for prevention of malignant progression of human papillomavirus-associated lesions in humans.     

The EXT1/EXT2 tumor suppressors: catalytic activities and role in heparan sulfate biosynthesis

The D-glucuronyltransferase and N-acetyl-D-glucosaminyltransferase reactions in heparan sulfate biosynthesis have been associated with two genes, EXT1 and EXT2, which are also implicated in the inherited bone disorder, multiple exostoses. Since the cell systems used to express recombinant EXT proteins synthesize endogenous heparan sulfate, and the EXT proteins tend to associate, it has not been possible to define the functional roles of the individual protein species. We therefore expressed EXT1 and EXT2 in yeast, which does not synthesize heparan sulfate. The recombinant EXT1 and EXT2 were both found to catalyze both glycosyltransferase reactions in vitro. Coexpression of the two proteins, but not mixing of separately expressed recombinant EXT1 and EXT2, yields hetero-oligomeric complexes in yeast and mammalian cells, with augmented glycosyltransferase activities. This stimulation does not depend on the membrane-bound state of the proteins.     

Observation of yeast cell movement and aggregation in a small-scale MHz-ultrasonic standing wave field

Aggregation of suspended yeast cells in a small-scale ultrasonic standing wave field has been monitored and quantified. The aggregation effect is based on the acoustic radiation force, which concentrates the cells in clumps. The ultrasonic chamber employed (1.9 MHz, one wavelength pathlength) had a sonication volume of 60 l. The aggregation process was observed from above the transducer through a transparent glass reflector. A distinct, reproducible, pattern of clumps formed rapidly in the sound field. The sound pressure was estimated experimentally to be of the order of 1 MPa. Microscopic observations of the formation of a single clump were recorded onto a PC. The time dependent movement patterns and travelling velocities of the cells during the aggregation process were extracted by particle image velocimetry analysis. A time dependent change was seen in the particle motion pattern during approach to its completion of clump formation after 45 s. Streaming eddies were set-up during the first couple of seconds. The scale of the eddies was consistent with Rayleigh micro-streaming theory. An increase in the travelling velocity of the cells was observed after 30 s from initially about 400 m s^–1 to about 1 mm s^–1. The influence of a number of mechanisms on particle behaviour (e.g. micro-streaming, particle interactions and convective flow) is considered. The experimental set-up introduced here is a powerful tool for aggregation studies in ultrasonic standing waves and lays the foundation for future quantitative experiments on the individual contributions of the different mechanisms.