Expression, localization, and functional activity of TL1A, a novel Th1-polarizing cytokine in inflammatory bowel disease

TL1A is a novel TNF-like factor that acts as a costimulator of IFN-gamma secretion through binding to the death domain-containing receptor, DR3. The aim of this study was to test the hypothesis that TL1A may play an important role in inflammatory bowel disease (IBD) by functioning as a Th1-polarizing cytokine. The expression, cellular localization, and functional activity of TL1A and DR3 were studied in intestinal tissue specimens as well as isolated lamina propria mononuclear cells from IBD patients and controls. TL1A mRNA and protein expression was up-regulated in IBD, particularly in involved areas of Crohn's disease (CD; p < 0.03 vs control). TL1A production was localized to the intestinal lamina propria in macrophages and CD4(+) and CD8(+) lymphocytes from CD patients as well as in plasma cells from ulcerative colitis patients. The amount of TL1A protein and the number of TL1A-positive cells correlated with the severity of inflammation, most significantly in CD. Increased numbers of immunoreactive DR3-positive T lymphocytes were detected in the intestinal lamina propria from IBD patients. Addition of recombinant human TL1A to cultures of PHA-stimulated lamina propria mononuclear from CD patients significantly augmented IFN-gamma production by 4-fold, whereas a minimal effect was observed in control patients. Our study provides evidence for the first time that the novel cytokine TL1A may play an important role in a Th1-mediated disease such as CD.     

The dendritic cell-specific chemokine,dendritic cell-derived CC chemokine 1, enhances protective cell-mediated immunity to murine malaria

Cell-mediated immunity plays a crucial role in the control of many infectious diseases, necessitating the need for adjuvants that can augment cellular immune responses elicited by vaccines. It is well established that protection against one such disease, malaria, requires strong CD8(+) T cell responses targeted against the liver stages of the causative agent, Plasmodium spp. In this report we show that the dendritic cell-specific chemokine, dendritic cell-derived CC chemokine 1 (DC-CK1), which is produced in humans and acts on naive lymphocytes, can enhance Ag-specific CD8(+) T cell responses when coadministered with either irradiated Plasmodium yoelii sporozoites or a recombinant adenovirus expressing the P. yoelii circumsporozoite protein in mice. We further show that these enhanced T cell responses result in increased protection to malaria in immunized mice challenged with live P. yoelii sporozoites, revealing an adjuvant activity for DC-CK1. DC-CK1 appears to act preferentially on naive mouse lymphocytes, and its adjuvant effect requires IL-12, but not IFN-gamma or CD40. Overall, our results show for the first time an in vivo role for DC-CK1 in the establishment of primary T cell responses and indicate the potential of this chemokine as an adjuvant for vaccines against malaria as well as other diseases in which cellular immune responses are important.     

Interaction of tocopherols and phenolic compounds with membrane lipid components: evaluation of their antioxidant activity in a liposomal model system

This paper describes the use of complex liposomes as real membrane models to evaluate the potential benefits of several antioxidants in relation to lipid peroxidation. The xanthine oxidase/Fe(3+)-ADP-EDTA and the Fe(2+)/H2O2 systems have been used to generate hydroxyl radicals and the water soluble azo-compound 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) to generate carbon centered radicals (A*) by thermal decomposition. The antioxidant behavior of the rosemary and citrus plant extracts and vitamin-E and vitamin-E acetate alpha-tocopherols have been analyzed. The order of effectiveness in avoiding radical chain reactions has been established by using the colorimetric thiobarbituric acid reaction and the fluorescent probe DPH-PA. ESR spectroscopy has been used to carry out the pursuit of the oxidation processes on the basis of the identification of the radical species resulting from the oxidant system and the ability of the antioxidants to act as scavengers for hydroxyl and AAPH-derived radicals. The modification of the main transition temperature for the lipid mixture and the splitting of the calorimetric peak in the presence of the antioxidants were demonstrated by differential scanning calorimetry. The results obtained showed that the phenols-containing plant extracts and alpha-tocopherols perturb the phase behavior of the BBE lipid bilayer and have a fluidifying effect that could favor the known antioxidant capability and scavenging characteristics of these compounds. 31P-NMR results could be interpreted as, after the incorporation of these antioxidants, those lipid molecules interacting with antioxidants give rise to lamellar phase spectral components with resonance position at lower fields or to isotropic signals in accordance with a higher motion of their phosphate groups.     

Early detection in saliva of polypeptides associated to isoproterenol-induced mouse parotid hypertrophy

Chronic administration of isoproterenol (IPR) results in a marked hypertrophy and in the induction of a group of putative proline-rich polypeptides in the mouse parotid glands. Some of these polypeptides (pps C-G) have been considered as molecular markers of the parotid gland enlargement. Given the secretory character of polypeptides C-G, the polypeptide composition of mouse saliva was used to monitor the IPR-induced salivary gland hypertrophy. Whole saliva was collected after an oral administration of pilocarpine (PIL). Under those conditions, PIL provoked a massive salivary secretion both in normal control mice and during the whole course of the IPR-induced gland enlargement. Striking changes in the polypeptide composition of saliva obtained from chronically IPR-stimulated animals were observed. Those changes consisted basically in the appearance and progressive increase in concentration of parotid polypeptides C-G and in the progressive diminution in concentration of a couple of normal salivary polypeptides (polypeptides A-B). The appearance of new polypeptides in saliva could be established unequivocally within the 24 h following the trophic adrenergic stimulation. On the other hand, salivary polypeptides induced in response to a single administration of IPR could be demonstrated as late as 7-9 days after the stimulation. Accordingly, detection of parotid polypeptides C-G in PIL-produced saliva obtained from IPR-stimulated mice has proved to be a highly advantageous method to evaluate salivary gland hypertrophy both at very early stages after the trophic stimulation and late after the occurrence of the trophic episode.     

Phylogenetic discordance at the species boundary: comparative gene genealogies among rapidly radiating Heliconius butterflies

Recent adaptive radiations provide excellent model systems for understanding speciation, but rapid diversification can cause problems for phylogenetic inference. Here we use gene genealogies to investigate the phylogeny of recent speciation in the heliconiine butterflies. We sequenced three gene regions, intron 3 ( approximately 550 bp) of sex-linked triose-phosphate isomerase (Tpi), intron 3 ( approximately 450 bp) of autosomal mannose-phosphate isomerase (Mpi), and 1,603 bp of mitochondrial cytochrome oxidase subunits I and II (COI and COII), for 37 individuals from 25 species of Heliconius and related genera. The nuclear intron sequences evolved at rates similar to those of mitochondrial coding sequences, but the phylogenetic utility of introns was restricted to closely related geographic populations and species due to high levels of indel variation. For two sister species pairs, Heliconius erato-Heliconius himera and Heliconius melpomene-Heliconius cydno, there was highly significant discordance between the three genes. At mtDNA and Tpi, the hypotheses of reciprocal monophyly and paraphyly of at least one species with respect to its sister could not be distinguished. In contrast alleles sampled from the third locus, Mpi, showed polyphyletic relationships between both species pairs. In all cases, recent coalescence of mtDNA lineages within species suggests that polyphyly of nuclear genes is not unexpected. In addition, very similar alleles were shared between melpomene and cydno, implying recent gene flow. Our finding of discordant genealogies between genes is consistent with models of adaptive speciation with ongoing gene flow and highlights the need for multiple locus comparisons to resolve phylogeny among closely related species.     

Importance of the N-terminal region of the phage GA-1 single-stranded DNA-binding protein for its self-interaction ability and functionality

The single-stranded DNA-binding protein (SSB) of phage GA-1 displays higher efficiency than the SSBs of the related phages phi 29 and Nf. In this work, the self-interaction ability of GA-1 SSB has been analyzed by visualization of the purified protein by electron microscopy, glycerol gradient sedimentation, and in vivo cross-linking of bacterial cultures infected with phage GA-1. GA-1 SSB contains an insert at its N-terminal region that is not present in the SSBs of phi 29 and Nf. Three deletion mutant proteins have been characterized, Delta N19, Delta N26, and Delta N33, which lack the 19, 26 or 33 amino acids, respectively, that follow the initial methionine of GA-1 SSB. Mutant protein Delta N19 retains the structural and functional behavior of GA-1 SSB, whereas mutant proteins Delta N26 and Delta N33 no longer stimulate viral DNA replication or display helix-destabilizing activity. Analysis of the mutant proteins by ultracentrifugation in glycerol gradients and electron microscopy indicates that deletion of 26 or 33 but not of 19 amino acids of the N-terminal region of GA-1 SSB results in the loss of the oligomerization ability of this protein. Our data support the importance of the N-terminal region of GA-1 SSB for the differential self-interaction ability and functional behavior of this protein.     

Characterization of Ejl,the cell-wall amidase coded by the pneumococcal bacteriophage Ej-1

The Ejl amidase is coded by Ej-1, a temperate phage isolated from the atypical pneumococcus strain 101/87. Like all the pneumococcal cell-wall lysins, Ejl has a bimodular organization; the catalytic region is located in the N-terminal module, and the C-terminal module attaches the enzyme to the choline residues of the pneumococcal cell wall. The structural features of the Ejl amidase, its interaction with choline, and the structural changes accompanying the ligand binding have been characterized by CD and IR spectroscopies, differential scanning calorimetry, analytical ultracentrifugation, and FPLC. According to prediction and spectroscopic (CD and IR) results, Ejl would be composed of short beta-strands (ca. 36%) connected by long loops (ca. 17%), presenting only two well-predicted alpha-helices (ca. 12%) in the catalytic module. Its polypeptide chain folds into two cooperative domains, corresponding to the N- and C-terminal modules, and exhibits a monomer <--> dimer self-association equilibrium. Choline binding induces small rearrangements in Ejl secondary structure but enhances the amidase self-association by preferential binding to Ejl dimers and tetramers. Comparison of LytA, the major pneumococcal amidase, with Ejl shows that the sequence differences (15% divergence) strongly influence the amidase stability, the organization of the catalytic module in cooperative domains, and the self-association state induced by choline. Moreover, the ligand affinity for the choline-binding locus involved in regulation of the amidase dimerization is reduced by a factor of 10 in Ejl. Present results evidence that sequence differences resulting from the natural variability found in the cell wall amidases coded by pneumococcus and its bacteriophages may significantly alter the protein structure and its attachment to the cell wall.     

In vitro and in vivo assays of 3,5-disubstituted-tetrahydro-2H-1,3,5-thiadiazin-2-thione derivatives against Trypanosoma cruzi

Cytotoxicity assays of 24 new 3,5-disubstituted-tetrahydro-2H-1,3,5-thiadiazin-2-thione derivatives were performed. The 17 compounds with higher anti-epimastigote activity and lower cytotoxicity were, thereafter, screened against amastigote of Trypanosoma cruzi. Out of these 17 derivatives S-2d was selected to be assayed in vivo, because of its remarkable trypanocidal properties. To determine toxicity against J774 macrophages, a method based on quantification of cell damage, after 24 h, was used. Cell respiration, an indicator of cell viability, was assessed by the reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to formazan. Anti-amastigote activity was estimated after 48 h by microscopic counts of May Grünwald-Giemsa-stained monolayers. Nifurtimox and benznidazole were used as reference drugs. For the in vivo experiences, mice were infected with 10(4) blood trypomastigotes and then treated during 15 days with S-2d or nifurtimox by oral route. All of the compounds were highly toxic at 100 micro g/ml for macrophages and a few of them maintained this cytotoxicity even at 10 microg/ml. Of the derivatives assayed against amastigotes 3k and S-2d showed an interesting activity, that was held even at 1microg/ml. It is demonstrated that the high anti-epimastigote activity previously reported is mainly due to the non-specific toxicity of these compounds. In vivo assays assessed a reduction of parasitemia after administration of S-2d to infected mice.     

Mouse novel Ly9: a new member of the expanding CD150 (SLAM) family of leukocyte cell-surface receptors

Human CS1, also known as novel Ly9, 19A24, or CRACC, is a member of the immunoglobulin gene superfamily (IgSF) expressed on natural killer cells and other leukocytes. Here we describe the cloning of the mouse homologue of this gene. The mouse novel Ly9 gene is shown to encode a transmembrane protein composed of two extracellular immunoglobulin-like domains, a transmembrane region and an 88-amino acid cytoplasmic domain. Mouse novel Ly9 is structurally similar to the extracellular domains of CD84 and CD229 (Ly9). Both mouse and human novel Ly9 genes mapped close to the CD229gene in a region where other members of the CD150 family have also been mapped, and analysis of their genomic sequences showed that they have an identical intron/exon organization. Northern blot analysis revealed that the expression of mouse and human novel Ly9 was predominantly restricted to hematopoietic tissues, with the exception of testis. Here we show that SAP (SH2D1A), an adapter protein responsible for the X-linked lymphoproliferative disease, binds to the phosphorylated cytoplasmic tail of human but not mouse novel Ly9. Taken together, these data indicate that mouse novel Ly9 is a new member of the expanding CD150 family of cell surface receptors.     

3'-azido-3'-deoxy-5'-O-isonicotinoylthymidine, a new prodrug of zidovudine. Synthesis, solid state characterization, and anti HIV-1 activity

Synthesis, solid state characterization and anti HIV-1 activity of 3'-azido-3'-deoxy-5'-O-isonicotinoylthymidine (2), a new prodrug of zidovudine (AZT, 1), are described. Two solid forms of 2 prepared by crystallization from ethyl acetate-petroleum ether (form alpha) and from a melt sample of form alpha (amorphous form) were characterized by X-ray diffractometry, infrared spectroscopy, differential scanning calorimetry (DSC) and thermogravimetry (TGA) techniques. The novel nucleoside exhibited antiviral activity against standard and resistant strain panels of HIV-1 as well as cytotoxicity similar to that of AZT.