Analysis of CD4(+) T-cell responses to human papillomavirus (HPV) type 11 L1 in healthy adults reveals a high degree of responsiveness and cross-reactivity with other HPV types

Human papillomavirus type 11 (HPV-11) infection causes genital warts and recurrent respiratory papillomatosis. While there is compelling evidence that CD4(+) T cells play an important role in immune surveillance of HPV-associated diseases, little is known about human CD4(+) T-cell recognition of HPV-11. We have investigated the CD4(+) T-cell responses of 25 unrelated healthy donors to HPV-11 L1 virus-like particles (VLP). CD4(+) T-cell lines from 21 of 25 donors were established. Cell sorting experiments carried out on cells from six donors demonstrated that the response was located in the CD45RA(low) CD45RO(high) memory T-cell population. To determine the peptide specificity of these responses, epitope selection was analyzed by using 95 15-mer peptides spanning the entire HPV-11 L1 protein. No single region of L1 was immunodominant; responders recognized between 1 and 10 peptides, located throughout the protein, and peptide responses fell into clear HLA class II restricted patterns. Panels of L1 peptides specific for skin and genital HPV were used to show that the L1 CD4(+) T-cell responses were cross-reactive. The degree of cross-reactivity was inversely related to the degree of L1 sequence diversity between these viruses. Finally, responses to HPV-11 L1 peptides were elicited from ex vivo CD45RO(+) peripheral blood mononuclear cells, demonstrating that recognition of HPV-11 was a specific memory response and not due to in vitro selection during tissue culture. This is the first study of CD4(+) T-cell responses to HPV-11 in healthy subjects and demonstrates marked cross-reactivity with other skin and genital HPV types. This cross-reactivity may be of significance for vaccine strategies against HPV-associated clinical diseases.     

Identification of tyrosine residues in constitutively activated fibroblast growth factor receptor 3 involved in mitogenesis, Stat activation, and phosphatidylinositol 3-kinase activation

Fibroblast growth factor receptor 3 (FGFR3) mutations are frequently involved in human developmental disorders and cancer. Activation of FGFR3, through mutation or ligand stimulation, results in autophosphorylation of multiple tyrosine residues within the intracellular domain. To assess the importance of the six conserved tyrosine residues within the intracellular domain of FGFR3 for signaling, derivatives were constructed containing an N-terminal myristylation signal for plasma membrane localization and a point mutation (K650E) that confers constitutive kinase activation. A derivative containing all conserved tyrosine residues stimulates cellular transformation and activation of several FGFR3 signaling pathways. Substitution of all nonactivation loop tyrosine residues with phenylalanine rendered this FGFR3 construct inactive, despite the presence of the activating K650E mutation. Addition of a single tyrosine residue, Y724, restored its ability to stimulate cellular transformation, phosphatidylinositol 3-kinase activation, and phosphorylation of Shp2, MAPK, Stat1, and Stat3. These results demonstrate a critical role for Y724 in the activation of multiple signaling pathways by constitutively activated mutants of FGFR3.     

The influence of prey size and abundance, and individual phenotype on prey choice by the three-spined stickleback, Gasterosteus aculeatm L.

Prey selection behaviour of three-spined sticklebacks, Gasterosteus aculeatus L., was studied in two experiments. Where possible, the experimental apparatus satisfied the assumptions of the simplest optimal diet model (the basic prey model); prey were presented sequentially, the fish could not search for and handle prey at the same time, and net energy gain, handling time and encounter rate were fixed. Experiment 1 presented fish with a range of Asellus sizes so that pursuit ( p ) and handling ( h ) time could be related to prey size. Published energy values of Asellus together with pursuit and handling times were used to calculate E /( p+h ) for Asellus measuring 3,4,5,6,7 and 9 mm. Pursuit times did not differ with prey size but handling times did. E /( p+h ) was very variable particularly at the larger prey sizes. Experiment 2 presented fish with two sequences of prey differing in the encounter rate with the most profitable prey sizes. Fish did not select the diet predicted by the basic prey model tending to always ignore the largest prey even when net energy gain would have been maximized by including them in the diet. Further analysis showed that the probability of a prey size being taken was a function of prey size, fish stomach fullness and encounter rate. It is concluded that the basic prey model is too simple to capture the behaviour of the fish. One of its main faults is that the changing state of the fish through the feeding bout is ignored.     

Isolation and identification of Triticum aestivum L. em. Thell. cv Chinese Spring-T. peregrinum Hackel disomic chromosome addition lines

Analyses of RFLPs, isozymes, morphological markers and chromosome pairing were used to isolate 12 Triticum aestivum cv Chinese Spring (genomes A, B, and D)-T. peregrinum (genomes S^v and U^v) disomic chromosome addition lines. The evidence obtained indicates that each of the 12 lines contains an intact pair of T. peregrinum chromosomes. One monosomic addition line, believed to contain an intact 6S^v chromosome, was also isolated. A CS-7U^v chromosome addition line was not obtained. Syntenic relationships in common with the standard Triticeae arrangement were found for five of the seven S^v genome chromosomes. The exceptions were 4S^v and 7S^v. A reciprocal translocation exists between 4S¹ and 7S¹ in T. longissimum and evidence was obtained that the same translocation exists in T. peregrinum. In contrast, evidence for syntenic relationships in common with the standard Triticeae arrangements were found for only one U^v chromosome of T. peregrinum.; namely, chromosome 2U^v. All other U^v genome chromosomes are involved in at least one translocation, and the same translocations were found in the U genome of T. umbellulatum. Evidence was also obtained indicating that the centromeric regions of 4U and 4U^v are homoeologous to the centromeric regions of Triticeae homoeologous group-6 chromosomes, that the centromeric regions of 6U and 6U^v are homoeologous to the centromeric regions of group-4 chromosomes, and that 4U and 4U^v are more closely related overall to Triticeae homoeologous group-6 chromosomes than they are to group-4 chromosomes.     

Cutting edge: membrane lymphotoxin regulates CD8(+) T cell-mediated intestinal allograft rejection.

Blocking the CD28/B7 and/or CD154/CD40 costimulatory pathways promotes long-term allograft survival in many transplant models where CD4(+) T cells are necessary for rejection. When CD8(+) T cells are sufficient to mediate rejection, these approaches fail, resulting in costimulation blockade-resistant rejection. To address this problem we examined the role of lymphotoxin-related molecules in CD8(+) T cell-mediated rejection of murine intestinal allografts. Targeting membrane lymphotoxin by means of a fusion protein, mAb, or genetic mutation inhibited rejection of intestinal allografts by CD8(+) T cells. This effect was associated with decreased monokine induced by IFN-gamma (Mig) and secondary lymphoid chemokine (SLC) gene expression within allografts and spleens respectively. Blocking membrane lymphotoxin did not inhibit rejection mediated by CD4(+) T cells. Combining disruption of membrane lymphotoxin and treatment with CTLA4-Ig inhibited rejection in wild-type mice. These data demonstrate that membrane lymphotoxin is an important regulatory molecule for CD8(+) T cells mediating rejection and suggest a strategy to avoid costimulation blockade-resistant rejection.     

Peroxynitrite causes endothelial cell monolayer barrier dysfunction

Nitric oxide (·NO) attenuates hydrogen peroxide(H₂O₂)-mediated barrier dysfunction in culturedporcine pulmonary artery endothelial cells (PAEC) (Gupta MP, Ober MD,Patterson C, Al-Hassani M, Natarajan V, and Hart, CM. Am JPhysiol Lung Cell Mol Physiol 280: L116-L126, 2001). However,·NO rapidly combines with superoxide (O) to formthe powerful oxidant peroxynitrite (ONOO), which wehypothesized would cause PAEC monolayer barrier dysfunction. To testthis hypothesis, we treated PAEC with ONOO (500 µM) or3-morpholinosydnonimine hydrochloride (SIN-1; 1-500 µM).SIN-1-mediated ONOO formation was confirmed by monitoringthe oxidation of dihydrorhodamine 123 to rhodamine. BothONOO and SIN-1 increased albumin clearance(P < 0.05) in the absence of cytotoxicity and alteredthe architecture of the cytoskeletal proteins actin and -catenin asdetected by immunofluorescent confocal imaging.ONOO-induced barrier dysfunction was partially reversibleand was attenuated by cysteine. Both ONOO and SIN-1nitrated tyrosine residues, including those on -catenin and actin,and oxidized proteins in PAEC. The introduction of actin treated withONOO into PAEC monolayers via liposomes alsoresulted in barrier dysfunction. These results indicate thatONOO directly alters endothelial cytoskeletal proteins,leading to barrier dysfunction.

     

Salmonella infections in a marsupial, the quokka (Setonix brachyurus), in relation to seasonal changes in condition and environmental stress.

An unusual abundance of Salmonella infections was studied in an island population of a wild marsupial, the quokka (Setonix brachyurus), which experiences starvation in summer associated with significant mortality. The frequency of infections was found to vary seasonally over most parts of the island, with high infection rates (70 to 100%) in summer and low infection rates (0 to 30%) in winter. In some samples, there was an average of as many as two isolations per animal, and up to five isolations were made from a single animal. By the end of summer, virtually all animals excreted Salmonella spp., with a median rate of excretion of approximately 3,000 Salmonella organisms per g of feces. The seasonal changes occurred over intervals of only weeks. The infections are believed to be associated with disruption of the digestive physiology of the animals caused by the poor quality of feed available in summer. This conclusion was supported by a quantitative study of the infections and by a field manipulation experiment which delayed the initiation of the infections as long as a food supplement was available. The proliferation of Salmonella spp. is discussed in terms of the ecology of the quokka and of the use of Salmonella spp. as indicators of environmental stress acting on the animals.     

The Drosophila boundary element-associated factors BEAF-32A and BEAF-32B affect chromatin structure

Binding sites for the Drosophila boundary element-associated factors BEAF-32A and -32B are required for the insulator activity of the scs' insulator. BEAF binds to hundreds of sites on polytene chromosomes, indicating that BEAF-utilizing insulators are an important class in Drosophila. To gain insight into the role of BEAF in flies, we designed a transgene encoding a dominant-negative form of BEAF under GAL4 UAS control. This BID protein encompasses the BEAF self-interaction domain. Evidence is provided that BID interacts with BEAF and interferes with scs' insulator activity and that BEAF is the major target of BID in vivo. BID expression during embryogenesis is lethal, implying that BEAF is required during early development. Expression of BID in eye imaginal discs leads to a rough-eye phenotype, and this phenotype is rescued by a third copy of the BEAF gene. Expression of BID in salivary glands leads to a global disruption of polytene chromatin structure, and this disruption is largely rescued by an extra copy of BEAF. BID expression also enhances position-effect variegation (PEV) of the w(m4h) allele and a yellow transgene inserted into the pericentric heterochromatin of chromosome 2R, while a third copy of the BEAF gene suppresses PEV of both genes. These results support the hypothesis that BEAF-dependent insulators function by affecting chromatin structure or dynamics.