T cell immunity in connective tissue disease patients targets the RNA binding domain of the U1-70kDa small nuclear ribonucleoprotein

Although the T cell dependence of autoimmune responses in connective tissue diseases has been well established, limited information exists regarding the T cell targeting of self Ags in humans. To characterize the T cell response to a connective tissue disease-associated autoantigen, this study generated T cell clones from patients using a set of peptides encompassing the entire linear sequence of the 70-kDa subunit of U1 snRNP (U1-70kDa) small nuclear ribonucleoprotein. Despite the ability of U1-70kDa to undergo multiple forms of Ag modification that have been correlated with distinct clinical disease phenotypes, a remarkably limited and consistent pattern of T cell targeting of U1-70kDa was observed. All tested T cell clones generated against U1-70kDa were specific for epitopes within the RNA binding domain (RBD) of the protein. High avidity binding of the RBD with U1-RNA was preserved with the disease-associated modified forms of U1-70kDa tested. The high avidity interaction between the U1-RBD on the polypeptide and U1-RNA may be critical in immune targeting of this region in autoimmunity. The T cell autoimmune response to U1-70kDa appears to have less diversity than is seen in the humoral response; and therefore, may be a favorable target for therapeutic intervention.     

Early variations of host thyroxine and interleukin-7 favor Schistosoma mansoni development

Schistosoma mansoni induces, in the vertebrate host, cutaneous production of interleukin-7 (IL-7), which is beneficial for parasite establishment and development. Infection of mice deficient in IL-7 expression leads to parasite dwarfism. Because similar findings were previously described in hypothyroid mice, this study aimed to elucidate the potential link between IL-7 and thyroid hormones (THs), using several models including hypo- and hyperthyroid mice, modified either transiently or constitutively. Mice treated with thyroxine led to increased worm numbers and development of giant worms, whereas an iodine-deficient diet reduced parasite maturation, egg laying, and liver pathology. Conversely, mice genetically deficient for either of the nuclear TH receptors displayed normal worm development despite modifications in hormone levels, suggesting that thyroxine action is mediated through host receptors. In addition, no modification of antibody titers has been evidenced in thyroxine-treated mice, whereas antibody levels were altered in transgenic animals. These observations suggest that the immune system is not likely to be involved in the modifications of parasite development reported in this study. Interestingly, concomitant treatment with IL-7 and thyroxine had a synergistic effect, leading to recovery of very large worms, thus raising questions about the complexity of interactions between IL-7 and metabolic hormones.     

Ashworthius Patriciapilittae n. sp. (Trichostrongyloidea: Haemonchinae), an abomasal nematode in Odocoileus virginianus from Costa Rica,and a new record for species of the genus in the Western Hemisphere

A species of Ashworthius is reported for the first time in the Western Hemisphere, and A. patriciapilittae n. sp. is described on the basis of specimens in white-tailed deer Odocoileus virginianus from Costa Rica. Among 8 known species, A. patriciapilittae is morphologically similar to A. tuyenquangi in red muntjac Muntjacas muntjak from northern Vietnam. The synlophe in A. patriciapilittae is composed of 26 ridges in the cervical zone and is continuous to the caudal extremity in males and females. Males are characterized by a complex dorsal ray and narrow trifurcate spicules (351-356 microm long) lacking an "eyelet." with dissimilar ventral and dorsal processes; the gubernaculum is 45-48% of the spicule length. Females have a prominent linguiform flap at the vulva and large eggs (108-142 microm long). The presence of A. patriciapilittae in Costa Rica is examined in the context of competing hypotheses for cospeciation or contemporary host-switching in cervids: either A. patriciapilittae is a component of an endemic Central and South American fauna that has diversified through coevolution of Ashworthius and cervid hosts or it has been introduced. Among haemonchines in the Western Hemisphere, specimens of A. patriciapilittae may be confused with 3 species of Haemonchus, including H. contortus, H. placei, and H. similis, that occur in both domestic and wild ruminants. Discovery of A. patriciapilittae emphasizes the continued need for survey and inventory to define the structure and distribution of parasite faunas in wild and domestic ruminants from the Nearctic and Neotropical regions.     

Consistent patterns in the development and immunodominance of human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T-cell responses following acute HIV-1 infection

Human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T-cell responses generated during acute infection play a critical role in the initial control of viremia. However, little is known about the viral T-cell epitopes targeted during acute infection or about their hierarchy in appearance and relative immunodominance over time. In this study, HIV-1-specific CD8+ T-cell responses in 18 acutely infected individuals expressing HLA-A3 and/or -B7 were characterized. Detailed analysis of CD8 responses in one such person who underwent treatment of acute infection followed by reexposure to HIV-1 through supervised treatment interruptions (STI) revealed recognition of only two cytotoxic T-lymphocyte (CTL) epitopes during symptomatic acute infection. HIV-1-specific CD8+ T-cell responses broadened significantly during subsequent exposure to the virus, ultimately targeting 27 distinct CTL epitopes, including 15 different CTL epitopes restricted by a single HLA class I allele (HLA-A3). The same few peptides were consistently targeted in an additional 17 persons expressing HLA-A3 and/or -B7 during acute infection. These studies demonstrate a consistent pattern in the development of epitope-specific responses restricted by a single HLA allele during acute HIV-1 infection, as well as persistence of the initial pattern of immunodominance during subsequent STI. In addition, they demonstrate that HIV-1-specific CD8+ T-cell responses can ultimately target a previously unexpected and unprecedented number of epitopes in a single infected individual, even though these are not detectable during the initial exposure to virus. These studies have important implications for vaccine design and evaluation.     

Allelopathic effects and root distribution of Ceratiola ericoides (Empetraceae) on seven rosemary scrub species

We studied the root distribution and the effects of leachates from the dominant shrub in rosemary scrub, Florida rosemary (Ceratiola ericoides), on germination of seven subordinate rosemary scrub species. For rosemary scrub specialists, (Eryngium cuneifolium and Hypericum cumulicola), germination was suppressed by the leaf and litter leachates. For species that are not found exclusively in rosemary scrub (Liatris ohlingerae, Polygonella basiramia, Paronychia chartacea, and Palofoxia feayi) litter and leaf leachate did not suppress germination significantly. Species limited to gaps in rosemary scrub (E. cuneifolium, H. cumulicola, and Lechea deckertii) showed reduced germination from rosemary leachates while species not limited to rosemary-free gaps (L. ohlingerae and P. feayi) were not affected by rosemary leachates. Rosemary root abundance was greatest near shrubs, at a shallow depth, and at sites not recently burned. As rosemary scrub patches age, rosemary roots are more likely to interact with herbaceous species in gaps.     

Phylogenetic relationships in the Gesnerioideae (Gesneriaceae) based on nrDNA ITS and cpDNA trnL-F and trnE-T spacer region sequences

The Gesnerioideae includes most of the New World members of the Gesneriaceae family and is currently considered to include five tribes: Beslerieae, Episcieae, Gesnerieae, Gloxinieae, and Napeantheae. This study presents maximum parsimony and maximum likelihood phylogenetic analyses of nuclear ribosomal DNA internal transcribed spacer regions (ITS), and the chloroplast DNA trnL intron, trnL-trnF intergenic spacer region, and trnE-trnT intergenic spacer region sequences. The ITS and cpDNA data sets strongly support the monophyly of a Beslerieae/Napeantheae clade; an Episcieae clade; a Gesnerieae clade; a Gloxinieae clade minus Sinningia, Sinningia relatives, and Gloxinia sarmentiana; and a Sinningia/Paliavana/Vanhouttea clade. This is the first study to provide strong statistical support for these tribes/clades. These analyses suggest that Sinningia and relatives should be considered as a separate tribe. Additionally, generic relationships are explored, including the apparent polyphyly of Gloxinia. Chromosome number changes are minimized on the proposed phylogeny, with the exception of the n = 11 taxa of the Gloxinieae. Scaly rhizomes appear to have been derived once in the Gloxinieae sensu stricto. The number of derivations of the inferior ovary is unclear: either there was one derivation with a reversal to a superior ovary in the Episcieae, or there were multiple independent derivations of the inferior ovary.     

Evidence consistent with human L1 retrotransposition in maternal meiosis I

We have used a unique polymorphic 3' transduction to show that a human L1, or LINE-1 (long interspersed nucleotide element-1), retrotransposition event most likely occurred in the maternal primary oocyte during meiosis I. We characterized a truncated L1 retrotransposon with a 3' transduction that was inserted, in a Dutch male patient, into the X-linked gene CYBB, thereby causing chronic granulomatous disease. We used the unique flanking sequence to localize the precursor L1 locus, LRE3, to chromosome 2q24.1. In a cell culture assay, the retrotransposition frequency of LRE3 is greater than that for any other element that has been tested to date. The patient's mother had two LRE3 alleles that differed slightly in the 3'-flanking genomic DNA. The patient had a single LRE3 allele that was identical to one of the maternal alleles; however, the patient's insertion matched the maternal LRE3 allele that he did not inherit. Other data indicate that there is only a small chance that the father (unavailable for analysis) carries the precursor LRE3 allele. In addition, paternal origin of the insertion would have required that an LRE3 mRNA transcribed before meiosis II be carried separately from its precursor LRE3 allele in the fertilizing sperm. Since the mother carries a potential precursor allele and the insertion was on the patient's maternal X chromosome, it is highly likely that the insertion originated during maternal meiosis I.     

Evolutionary dynamics of intron size, genome size, and physiological correlates in archosaurs

It has been proposed that intron and genome sizes in birds are reduced in comparison with mammals because of the metabolic demands of flight. To test this hypothesis, we examined the sizes of 14 introns in a nonflying relative of birds, the American alligator (Alligator mississippiensis), and in 19 flighted and flightless birds in 12 taxonomic orders. Our results indicate that a substantial fraction (66%) of the reduction in intron size as well as in genome size had already occurred in nonflying archosaurs. Using phylogenetically independent contrasts, we found that the proposed inverse correlation of genome size and basal metabolic rate (BMR) is significant among amniotes and archosaurs, whereas intron and genome size variation within birds showed no significant correlation with BMR. We show statistically that the distribution of genome sizes in birds and mammals is underdispersed compared with the Brownian motion model and consistent with strong stabilizing selection; that genome size differences between vertebrate clades are overdispersed and punctuational; and that evolution of BMR and avian intron size is consistent with Brownian motion. These results suggest that the contrast between genome size/BMR and intron size/BMR correlations may be a consequence of different intensities of selection for these traits and that we should not expect changes in intron size to be significantly associated with metabolically costly behaviors such as flight.     

Immunodetection of small o-phenylenebismaleimide-labeled peptides through carrier protein display on polyvinylidene fluoride

Protein modification and peptide analysis are important techniques for the elucidation of the structure and function of enzymes. We describe a new technique for the identification of peptides covalently modified with the maleimide cross-linker o-phenylenebismaleimide (OPBM). The method can identify labeled peptides without the use of sophisticated instrumentation or radioactive markers and takes advantage of the separating power of RPLC and of the sensitivity of immunoblotting. Chloroplast ATPase F1 was labeled at a single cysteine residue by OPBM and trypsinized. Fractions collected by RPLC were bound to polyvinylidene fluoride (PVDF). Despite the small size of the OPBM-labeled peptide (1.84 kDa) it was possible to immobilize it on PVDF by using glutaraldehyde to conjugate the peptide to a larger, unlabeled protein. Polyclonal antibodies raised against the cross-linker N,N',1,5-naphthalenebismaleimide (NBM) cross-react with OPBM. These antibodies detected the presence of OPBM displayed on the PVDF and correctly identified the RPLC fraction containing the OPBM-labeled peptide as verified by both mass spectroscopy and radiolabeling of OPBM. This method could be adapted to detect the presence of linear epitopes recognized by an antibody and is a broadly applicable technique for the immunodetection of peptides.