Analysis of Human RSV Immunity at the Molecular Level: Learning from the Past and Present

Human RSV is one of the most prevalent viral pathogens of early childhood for which no vaccine is available. Herein we provide an analysis of RSV epitope data to examine its application to vaccine design and development. Our objective was to provide an overview of antigenic coverage, identify critical antibody and T cell determinants, and then analyze the cumulative RSV epitope data from the standpoint of functional responses using a combinational approach to characterize antigenic structure and epitope location. A review of the cumulative data revealed, not surprisingly, that the vast majority of epitopes have been defined for the two major surface antigens, F and G. Antibody and T cell determinants have been reported from multiple hosts, including those from human subjects following natural infection, however human data represent a minority of the data. A structural analysis of the major surface antigen, F, showed that the majority of epitopes defined for functional antibodies (neutralizing and/or protective) were either shown to bind pre-F or to be accessible in both pre- and post-F forms. This finding may have has implications for on-going vaccine design and development. These interpretations are in agreement with previous work and can be applied in the larger context of functional epitopes on the F protein. It is our hope that this work will provide the basis for further RSV-specific epitope discovery and investigation into the nature of antigen conformation in immunogenicity.     

Retinoic acid application to chick wing buds leads to a dose-dependent reorganization of the apical ectodermal ridge that is mediated by the mesenchyme

Local application of retinoic acid to wing buds of chick embryos leads to dose- and position-dependent changes in the pattern of cellular differentiation. Early effects of retinoid treatment on the apical ectodermal ridge coordinate pattern changes and morphogenesis. The length of the apical ridge increases when additional digits will form but decreases when digits are lost. These changes in length can be understood in terms of a threshold response to the local retinoid concentration that results in either disappearance or maintenance of the ridge (Lee & Tickle, J. Embryol. exp. Morph. 90, 139-169 (1985)). Here, we have analysed the mechanisms involved in ridge disappearance by locally applying retinoic acid to the apex of stage 20 chick wing buds. With this treatment regime, low doses give duplicated digit patterns and higher doses truncations. The height of the apical ridge is progressively reduced with increasing doses of retinoid and the time course of ridge flattening indicates that the height of the ridge is correlated with bud outgrowth. With high doses of retinoic acid, the typical ridge, a pseudostratified epithelium in which the columnar cells are tightly packed, disappears and the epithelium at the tip of the bud consists of loosely packed cuboidal cells. Shortly after treatment, there is a decrease in the number of gap junctions between ridge cells. This early change in cell contacts suggests that gap junctions may be involved in maintaining epithelial morphology. When treated epithelium is recombined with untreated mesenchyme, an apical ridge is reestablished and distal structures can be generated. In contrast, when treated mesenchyme is recombined with the epithelium from normal buds, only proximal structures are formed. Therefore, retinoids can lead to a reorganization of the apical ectodermal ridge which is mediated and maintained by the mesenchyme.     

Field cage performance of two tachinid parasitoids of the tomato fruitworm on insect resistant and susceptible tomato lines

Rates of parasitism ofHelicoverpa (=Heliothis) zea (Boddie) (Lepidoptera: Noctuidae) by two tachinid parasitoid species differing in larviposition habits were measured in field cages on tomato plant lines with or without methyl-ketone (2-tridecanone and 2-undecanone)-mediated insect resistance. Hosts were placed on resistant or susceptible plants, exposed to parasitoids for 24–48 h, then held on artificial diet for parasitoid emergence. Rates of parasitism byArchytas marmoratus (Townsend) (Diptera: Tachinidae), which larviposits on its host's food plant, were significantly reduced on the resistant plants, relative to those on the susceptible plants. Parastism by another Tachinid,Eucelatoria bryani Sabrosky, which larviposits directly into its host and does not directly contact the foliage of its hosts' food plant as a small larva, was not affected by the methyl-ketone mediated resistance.     

Induced tolerance of neonate Heliothis zea to host plant allelochemicals and carbaryl following incubation of eggs on foliage of Lycopersicon hirsutum f. glabratum

Summary Incubation of Heliothis zea (Boddie) eggs on foliage of Lycopersicon hirsutum f. glabratum C.H. Mull (accession PI 134417) results in neonates with elevated levels of tolerance to the toxic effects of PI 134417 foliage attributable to 2-tridecanone found in the glandular trichomes which abound on that foliage. The neonates from such eggs are also shown to have elevated levels of tolerance to the carbamate insecticide carbaryl. Incubation of eggs in an atmosphere containing 2-tridecanone similarly produced elevated levels of tolerance to 2-tridecanone among resulting neonates, indicating that 2-tridecanone is the likely inducing agent and that exposure to 2-tridecanone vapor, which is known to emanate from PI 134417 foliage, is sufficient for induction. Analysis of the cytochrome P-450 content in gut microsomes of fifth instar larvae indicated that exposure of larvae to 2-tridecanone in artificial diet or to PI 134417 foliage resulted in significantly elevated levels of cytochrome P-450 relative to larvae fed diet without 2-tridecanone or foliage of L. esculentum which contains no 2-tridecanone. In addition, removal of the glandular trichomes from PI 134417 foliage eliminated the ability of that foliage to induce elevated levels of cytochrome P-450. These results provide circumstantial evidence that cytochrome P-450 may be involved in the induced tolerance to xenobiotics among neonates from eggs exposed to 2-tridecanone or PI 134417 foliage.Support for this research was provided by the USDA Competitive Research Grants Program in Biological Stress under Grant No. 83-CRCR-1-1241 and Grant No. 85-CRCR-1-1615, and the North Carolina Agricultural Research Service. Paper No. 10856 of Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC, USA 27650. Use of trade names does not imply endorsement of products named nor criticisms of similar ones not mentioned     

Role of c-myc and other genes in interleukin 2 regulated CT6 T lymphocytes and their malignant variants

The cloned murine cytotoxic T cell line CT6 solely requires interleukin 2 (IL 2) for viability and cell cycle progression. Treatment of G arrested cultures of CT6 cells with recombinant IL 2 induces the rapid sequential expression of the nuclear proto-oncogenes c-fos, c-myc, and c-myb but does not affect the expression of several cytosolic or membrane-associated proto-oncogenes. A comparison of early genes induced by growth factor treatment of quiescent NIH/3T3 fibroblasts and CT6 cells demonstrated that only c-fos and c-myc induction is shared in the two different lineages. Factor-independent lines derived from CT6 cells show no mitogenic response to IL 2, yet binding of IL 2 with its receptor in the cells was capable of inducing the expression of c-fos and c-myc. In factor-independent cell lines, c-myc was uniformly expressed at high constitutive levels, suggesting that c-myc abrogates growth factor requirements of these cells. The levels of c-myc expression in the factor-independent lines was not due to an autocrine production of IL 2 but may be a consequence of constitutively activated IL 2 receptors.     

Identity of common phosphoprotein substrates stimulated by interleukin 2 and diacylglycerol suggests a role of protein kinase C for IL 2 signal transduction

Interleukin 2 (IL 2) is a polypeptide growth factor essential for the proliferation and differentiation of T lymphocytes, large granulocytic lymphocytes, and, potentially, cells of the antibody-producing lineage, B lymphocytes. Many of the biological properties of IL 2 may be mimicked or potentiated by a potent class of tumor promoters, phorbol esters. Phorbol esters have recently been shown to associate with and activate a unique phospholipid/Ca2+-dependent phosphotransferase, protein kinase C (PK-C). Utilizing two-dimensional gel electrophoresis, we have compared the IL 2 and diacylglycerol-induced protein phosphorylation patterns of several IL 2-dependent murine cell lines. Both IL 2 and synthetic diacylglycerol, 1-oleyl-2-acetylglycerol (OAG), stimulated phosphorylation of a number of protein substrates in intact cells compared to unstimulated controls. Three groups of substrates were identified; the first showed increased phosphorylation following stimulation with either IL 2 or OAG, while the second and third groups showed increased phosphorylation following stimulation with IL 2 but not OAG, and with OAG but not IL 2, respectively. Here, we characterize the kinetics of phosphorylation of one cellular substrate, p68, which appears to be phosphorylated in response to direct activators of PK-C or lymphoid or myeloid growth factors in their respective lineage cell lines. The observation that IL 2 also stimulates a unique series of phosphoproteins in addition to those induced by direct PK-C activators suggests that IL 2 may initiate additional protein kinase activities, unrelated to PK-C, which may also be critical for the ligand-receptor signal transduction process regulating growth and gene expression.     

5-Azacytidine treatment of a murine cytotoxic T cell line alters interferon-gamma gene induction by interleukin 2

Genetic susceptibility to multiple sclerosis (MS) in Caucasians was previously shown to be correlated to the presence of given alleles at the HLA-DR and Gm loci. We now demonstrate that the humoral immune response in MS central nervous system (CNS) is modulated by both loci: the levels of IgG1 subclass and IgG1 allotypes in cerebrospinal fluid of MS patients depend on both their Gm genotype and their HLA-DR2 or HLA-DR7 phenotype. That HLA-DR molecules may either participate in a preferential recruitment of IgG1 allotype-producing B cells in MS CNS or act after such a selective homing is discussed. These results demonstrate that both HLA and Gm loci are synergistically involved in the modulation of the humoral immune response.     

Differential effects of positive and negative proliferative stimuli on murine cytolytic and helper T-cell clones

Studies were performed to determine whether substances could be identified which exhibited differential regulatory effects--either positive or negative--on the growth of murine alloreactive cytolytic (Tc) and helper (Th) cloned T-cell lines. The following lines of evidence suggested that Tc and Th proliferate in response to the same growth factor (GF). (1) When GF-containing fluids from cultures of phorbol myristic acetate (PMA)-activated EL4 thymoma were fractionated by a variety of biochemical techniques. Tc and Th eluted together. (2) Absorption of GF-containing supernatants with either cloned Tc or cloned Th depleted GF activity for each to a similar extent, and GF eluted from either Tc or Th to which it had adsorbed supported the proliferation of Tc and Th equally well. (3) Lectin-depleted supernatants from cultures of concanavalin A (Con A)-activated Th stimulated the proliferation of Th as well as Tc. (4) Recombinant human interleukin (IL-2) supported the growth of Tc and Th with equal efficiency. On the other hand, the following observations indicated that Tc and Th differed in their responses to inhibitors of GF-driven proliferation. (1) Con A at greater than or equal to 0.3 micrograms/ml inhibited the GF-driven proliferation of each of three Th lines but not either of two Tc lines. To the contrary, Con A enhanced GF-dependent proliferation of Tc. (2) Like Con A, allogeneic splenocytes selectively depressed GF-driven proliferation of Th but not Tc. (3) A substance generated during the acid elution of GF from cells, possibly a modified fetal calf serum component, greatly reduced the GF-driven proliferation of Tc but not Th. These results suggest that differential control of the proliferation of Tc and Th in cellular immune responses may be achieved via negative regulatory signals and raise the possibility that substances which can selectively depress the proliferation of specific T-cell subsets might be found which would be of therapeutic value.     

HIV-1 and its envelope glycoprotein down-regulate chemotactic ligand receptors and chemotactic function of peripheral blood monocytes

Peripheral blood monocytes from AIDS patients exhibit defective migratory responses to chemotactic stimuli in vitro and to inflammatory sites in vivo. In studies presented here, normal monocytes were infected with the HIV-1/HTLV-IIIBa-L isolate in vitro and evaluated for chemotactic responsiveness. Within 2 days after viral exposure, but before evidence of virus production in the monocytes, chemotactic activity was significantly impaired. Decreased chemotactic activity was associated with modulation of receptors for the chemotactic ligands, C5a and FMLP, on the monocyte cell surface. In addition to HIV-1, monocytes treated with purified HIV-1 envelope glycoprotein gp120 demonstrated a comparable modulation of chemotactic ligand receptors and migratory function. In addition, the HIV-1 or HIV-1 gp120-treated monocytes were induced to undergo differentiation as monitored by HLA-DR expression. Immunoprecipitation of the gp120 with a specific antibody reversed its effects on monocyte chemotaxis and HLA-DR expression. Taken together, these data indicate that the initial interaction of HIV-1 with the monocyte is not passive, but that the binding of HIV-1 and/or HIV-1 gp120 to the CD4R on monocytes transduces a signal leading to transient monocyte activation.     

Control of barley root respiration

Evidence from barley [ Hordeum distichum (L.) Lam. cv. Maris Mink], and from many other species, suggests that respiration is controlled by either supply of carbohydrate or demand for ATP. The relationship between root respiration rate (measured as O₂ consumption or CO₂ production) and ethanol-soluble carbohydrate content altered with time following selective pruning, and the change could not be accounted for by buffering of the cytoplasmic carbohydrate concentration by sugars in the vacuole. Exogenous sucrose supplied to the roots prevented any decline of the respiration rate in shoot-pruned plants, and if supplied for 24 h stimulated the respiration rate after any treatment. Root extension responded to sucrose in a similar manner. We suggest that respiration is under fine control by adenylates, but the capacity of the respiratory system is fixed by the supply of sucrose, possibly via coarse control of the respiratory machinery, or of the processes requiring metabolic energy.