Accidents in practice.
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Accidents, Occupational↗
Biomedical subjects
A Buckley
Publications and source records attributed to A Buckley.
Nucleotide sequence of the envelope glycoprotein of Negishi virus shows very close homology to louping ill virus.
Negishi virus, a member of the family Flaviviridae, was originally isolated in Japan, during an outbreak of Japanese encephalitis. Antigenically, however, Negishi virus resembles the tick-borne rather than the mosquito-borne flaviviruses. Monoclonal antibodies that bind louping ill virus showed a close antigenic relationship between louping ill and Negishi virus. The genes encoding the envelope glycoprotein of Negishi virus (strain 3248/49/P10) and louping ill virus (strain SB526) were cloned and sequenced. They showed a very close homology at both the nucleotide and deduced amino acid levels. Comparison with the known sequence of another strain of louping ill virus (strain 369/T2) and with other tick-borne flaviviruses showed that Negishi virus was more closely related to louping ill virus than to the other tick-borne viruses. The significance of this observation for virus evolution, virus distribution in the environment, and the potential use of nucleotide sequencing for rapid and precise identification of flaviviruses are discussed.
Amino Acid Sequence↗
Monoclonal antibodies identify the NS5 yellow fever virus non-structural protein in the nuclei of infected cells.
Eight monoclonal antibodies (MAbs) derived using yellow fever (YF) virus (French viscerotropic virus strain) labelled the nuclei (wild-type strains) and/or the nucleoli (vaccine strains) of cells infected with different strains of YF virus. The specificity of these antibodies for YF virus-infected cells was confirmed using MAbs that bind only the YF virus envelope glycoprotein. The characteristics of fluorescent labelling in the nuclei and nucleoli of both normal cells and of nuclei separate from cell cytoplasm confirmed that the antigen was inside the nucleus rather than on the outer surface of the nuclear membrane. Virus-specific antigen was also observed in the cytoplasm of infected cells. One additional virus envelope-specific antibody, derived at the same time, identified cytoplasmic antigen exclusively. The eight antibodies that identified nuclear antigen showed no activity in neutralization, haemagglutination inhibition or mouse protection tests. Analysis of their molecular specificities by radioimmunoprecipitation of virus-infected cell lysates showed that they identified the non-structural NS5 antigen of YF virus. These results support the possibility of nuclear involvement in the replicative stages of YF virus infection.
Animals↗
17D yellow fever vaccine virus envelope protein expressed by recombinant baculovirus is antigenically indistinguishable from authentic viral protein.
We have constructed a recombinant baculovirus containing cloned DNA encoding the membrane and envelope (E) proteins of 17D yellow fever vaccine virus. Spodoptera frugiperda cells infected with this recombinant baculovirus produced a 66K protein which corresponded to the estimated size of the protein encoded by the cloned inserted DNA, and a 54K protein with the same molecular size as that of the authentic 17D yellow fever virus E protein. This recombinant 54K protein was labile, producing E protein-specific breakdown products (45K to 36K). Indirect immunofluorescence, using a panel of E protein-specific monoclonal antibodies, showed that the recombinant protein was presented both inside as well as on the surface of cells and was antigenically indistinguishable from the E protein of 17D yellow fever vaccine virus.
Animals↗
The role of ultrasound in evaluation of Takayasu's arteritis.
Duplex ultrasound was used to assess the vascular status and predict the angiographic findings in 3 patients with Takayasu's arteritis. The most striking sonographic feature was the presence of concentric arterial wall thickening. Using pulsed Doppler, stenotic lesions were quantified, occlusive lesions were identified and collateral circulation was demonstrated. A high resistive flow pattern was demonstrated in diseased vessels compared with carotid wave-forms of control subjects. Subtle mural irregularity, minor stenotic lesions and areas of stenosis in branch vessels were missed by duplex evaluation. The thoracic aorta and occasionally major arterial branches in the abdomen were impossible to evaluate with ultrasound. Vascular magnetic resonance imaging was successful in delineating major aortic branches but was inferior to real-time ultrasound in resolving mural thickening. While angiography plays a major role as a baseline assessment of the entire vascular tree, duplex ultrasound can monitor disease progression and the effects of therapy. Serial duplex studies should greatly reduce the need for interval angiographic followup.
Adolescent↗
Antibody-dependent enhancement of yellow fever and Japanese encephalitis virus neurovirulence.
Antibody-dependent enhancement of yellow fever virus neurovirulence, as measured by a reduction in the average survival time of groups of mice, was demonstrated with wild-type or vaccine strains of yellow fever virus and with Japanese encephalitis virus using intraperitoneally administered monoclonal antibodies specific for the viral E glycoprotein of yellow fever virus. Enhancement of virulence could be induced by neutralizing, non-neutralizing or protective antibodies if the virus was allowed to establish a productive infection in the mouse brain before the antibody was administered. The implications of antibody-dependent enhancement in flaviviruses are discussed.
Animals↗
Use of a monoclonal antibody specific for wild-type yellow fever virus to identify a wild-type antigenic variant in 17D vaccine pools.
Monoclonal antibodies (MAbs) against the Asibi wild-type strain of yellow fever (YF) virus were prepared and characterized. One of the MAbs (designated MAb 117) was shown, by cross-immunofluorescence tests with flaviviruses, to be specific for wildtype YF virus. This MAb was used in indirect immunofluorescence tests to identify wild-type antigenic variants in several different YF vaccine pools. Simultaneously, a vaccine-specific MAb prepared previously (MAb 864) was used to identify YF strain 17D vaccine type variants in the wild-type Asibi virus preparation. One variant, isolated by plaque purification from a 17D vaccine pool, possessed the wild-type epitope and was neurovirulent in infant mice whereas other variants, lacking the wild-type epitope but with vaccine-specific epitopes (identified by MAb 411), were avirulent in infant mice. Avirulent variants were able to infect mice and induce antibody. Virus-specific antigen was still detected in the brains of these mice 4 weeks after inoculation, suggesting that persistent infections were developing. These results to the potential risk of selection of wild-type variants in YF vaccine preparations. They also point to the potential risk of selection of wild-type variants in YF vaccine preparations and re-emphasize the need for modernization of techniques and more effective control measures to be taken during the production of YF vaccine.
Animals↗
Recombinant basic fibroblast growth factor accelerates wound healing.
Basic fibroblast growth factor (bFGF) stimulates extracellular matrix metabolism, growth, and movement of mesodermally derived cells. We have previously shown that collagen content in polyvinyl alcohol sponges increased after bFGF treatment. We hypothesized that bFGF-treated incisional wounds would heal more rapidly. After intraperitoneal pentobarbital anesthesia, male, 200- to 250-g, Sprague-Dawley rats (n = 27) each underwent two sets of paired, transverse, dorsal incisions closed with steel sutures. On Day 3 postwounding, 0.4 ml of bFGF (recombinant, 400 ng. Synergen) or normal saline was injected into one of each paired incisions. Animals were killed with ether on postwounding Days 5, 6, and 7 and their dorsal pelts were excised. Fresh or formalin-fixed wound strips were subjected to tensile strength measurements using a tensiometer. Breaking energy was calculated. Wound collagen content (hydroxyproline) was measured in wound-edge samples following hydrolysis using high-performance liquid chromatography. There was an overall significant increase in fresh wound tensile strength (13.7 +/- 1.06 vs 19.1 +/- 1.99 g/mm, P less than 0.01) and wound breaking energy (476 +/- 47 vs 747 +/- 76 mm2, P less than 0.001) in bFGF-treated incisions. There was an increase in wound collagen content which was not statistically significant and there was no difference in fixed incisional tensile strength. Histologic examination showed better organization and maturation in bFGF wounds. Recombinant bFGF accelerates normal rat wound healing. This may be due to earlier accumulation of collagen and fibroblasts and/or to greater collagen crosslinking in bFGF-treated wounds.
Animals↗
Microinjection and expression of an infectious proviral clone and subgenomic envelope construct of a human immunodeficiency virus.
An infectious proviral clone of the human immunodeficiency virus (HIV) was microinjected into the cell nucleus in six cell lines derived from caprine, ovine, bovine, or human solid tissue to study the utility of this method in effecting viral gene expression in nonlymphoid cells. Immunofluorescence assays for HIV demonstrated viral gene expression in only 5% of cells (100-200 cells per line) 24-48 h after microinjection; however, no reverse transcriptase activity was detectable, presumably due to a low level of virus release in this limited number of cells. Therefore, to indirectly assess infectious virus release, microinjected cells were cocultured with human T4 antigen-positive lymphocytes (H9) sensitive to HIV infection. Syncytia formation, electron microscopy, reverse transcriptase activity, and radioimmunoassay for HIV p24 were used to monitor viral gene expression in cocultures. HIV was efficiently recovered by cocultivating H9 with microinjected cells 48 h after microinjection, regardless of the tissue type or species of origin. H9 syncytia were visualized in some cocultures as early as day 5 but were readily apparent in all experiments on days 7-10. Syncytia induction in H9 was the earliest and most reliable indicator of infectious virus release. A recombinant construct containing a subgenomic envelope gene derived from the proviral clone of HIV was microinjected into human glioblastoma cells. Twenty-four to 48 h after manipulation, 5-20% of microinjected cells were found by immunofluorescence assay to express low levels of a putative gp120. These results suggest a possible approach to producing virus-free HIV envelope antigens in mammalian cells and may be relevant to subunit vaccine development.
Acquired Immunodeficiency Syndrome↗
Detection of virus-specific antigen in the nuclei or nucleoli of cells infected with Zika or Langat virus.
Two monoclonal antibodies (MAbs) with molecular specificities for either the viral envelope glycoprotein (MAb 541) or the non-structural NS1 glycoprotein (MAb 109) were derived using West Nile and yellow fever (YF) viruses respectively. Their antigenic reactivity with a large number of flaviviruses was tested by indirect immunofluorescence microscopy. Both produced cytoplasmic fluorescent staining patterns with the homologous virus against which they were raised. Additionally, MAb 541 reacted with two substrains of YF virus whereas MAb 109 reacted with Bussuquara, YF and Ntaya viruses. These reactions were exclusively cytoplasmic. Two unexpected patterns of fluorescent labelling were observed when the antibodies were tested with Zika and Langat viruses. MAb 541 produced fluorescent staining of the nuclei, but not the cytoplasm, of cells infected with Zika virus and MAb 109 labelled only the nucleoli of cells infected with Langat virus. Double-labelling experiments showed that the nuclear fluorescent label was confined to virus-infected cells, and antibody absorption experiments with virus-infected cell packs confirmed the virus specificity of the nuclear antigen. The unexpected presence of virus-specific antigen in the nuclei or nucleoli of Zika or Langat virus-infected cells brings into question the role of the nucleus in flavivirus replication.
Animals↗
Mechanisms of accelerated wound repair using epidermal growth factor and basic fibroblast growth factor.
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Animals↗
Patients' awareness of the risks of smoking.
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Adolescent↗
Retinoic acid alters the keratinization of cultured rat sublingual keratinocytes in vitro.
A multilayered, continuously proliferating keratinocyte cell culture has been produced from rat sublingual epithelium. The rate of growth of the cultures was stable throughout long-term culture. Retinoic acid (3.3 microM) inhibited the keratinization of these cultures. Morphological changes included total loss of tonofilaments within 7 days, decrease in desmosomes, an increase in intercellular spaces, absence of thickened plasma membranes, and elongated and more numerous cytoplasmic projections. Exposure to retinoic acid (3.3 microM) for 33 days did not affect the growth rates of the cultures, as estimated from the protein and DNA content per flask. Retinoic acid (3.3 microM) reduced the polyacrylamide gel electrophoresis protein profile within 3 days of treatment and produced reductions in the incorporation of amino acids into keratins of molecular weights 62,000 and 60,000 within 24 h. All five keratin polypeptides showed a reduced incorporation rate after treatment for 3 days. This inhibition was reversible. Protein synthesis of nonkeratins was not detectably affected by retinoid treatment.
Animals↗
Epidermal growth factor increases granulation tissue formation dose dependently.
Recent studies have shown that epidermal growth factor (EGF) stimulated the rate of formation of granulation tissue in a model of wound repair (A. Buckley, et al., Proc. Nat. Acad. Sci. USA 82: 7340, 1985). Because pharmacologic doses of EGF were used previously, the relationship of EGF concentration to physiologic effects was determined in this study. Rats were implanted with subcutaneous polyvinyl alcohol sponges containing slow-release pellets formulated to release 0, 0.1, 1.0, or 10 micrograms of EGF/day. Tissue response was judged by the degree of histologic organization and vascularity, as well as several quantitative parameters: wet weight, hydroxyproline content, protein content, and DNA concentration. Each of these parameters showed consistent increases by Day 5 after implantation, when inflammation and edema had subsided. Compared with placebo controls, hydroxyproline (collagen) content was significantly increased by as little as 1 microgram/day of EGF, and DNA content was significantly increased by all dose levels of EGF. Endogenous EGF concentration in experimental granulation tissue was found to be fairly constant (30-40 ng/g wet wt); however, the increasing cellularity of the sponges may have reduced the local concentration of free EGF to low levels. Pellets releasing as little as 4 ng/hr of EGF into the surrounding tissue were able to accelerate wound healing, suggesting that the availability of this growth factor may be a rate-limiting step in wound repair.
Animals↗
Differential effects of cartilage-derived growth factor stimulation of collagen secretion by bovine aortic and microvascular endothelial cells.
Cartilage-derived growth factor (CDGF), a protein closely related to basic fibroblast growth factor, is known to have both mitogenic and chemokinetic properties in microvascular endothelial cells (MVEC). Because of the angiogenic properties of CDGF and its rate in accelerating wound repair, the capacity of this factor to stimulate both proliferation and matrix synthesis was compared in distinct populations of vascular endothelial cells: MVEC from bovine adrenal cortex and macrovascular endothelial cells from the bovine aorta (BAEC). No significant differences in the responses to mitogenic stimulation using CDGF (5-100 units/ml) were observed using MVEC and BAEC. Only rapidly dividing MVEC, however, showed significant increases in collagen secretion in the presence of CDGF. The differential responsiveness of these two cell populations to a defined growth factor underscores the phenotypic diversity of endothelium.
Animals↗
Immune enhancement of yellow fever virus neurovirulence for mice: studies of mechanisms involved.
Enhancement of yellow fever virus neurovirulence for mice by specific antibody was studied with the French neurotropic vaccine strain. Experimental conditions for enhancement required mice between 14 and 40 days old and intraperitoneal administration of a selected monoclonal antibody 24 h before or up to 72 h after intracerebral virus challenge. Virus infectivity titrations were similar in brains of antibody-treated and untreated mice. Virus recovered from brains of mice with enhanced viral infections was neither qualitatively nor quantitatively different from standard virus. Humoral immune responses in enhanced infections were normal, macrophages did not become infected and viraemia was not significant. Both hydrocortisone treatment and complement depletion with cobra venom resulted in prolongation of mouse survival times but virulence enhancement persisted. Antithymocyte serum had no effect on enhancement although it reduced the humoral immune response. It is proposed that virulence enhancement is due to the combined effects of virus-specific antibody on infected cells, complement-mediated cytolysis and resultant host anti-cellular activity. There is no analogy between mechanisms effecting increased arbovirus growth in vitro in the presence of specific antibody and increased yellow fever virus neurovirulence in vivo after parenteral administration of antibody.
Age Factors↗
Developmental initiation of elastin gene expression by human fetal skin fibroblasts.
Elastin synthesis is initiated in many organs during the latter part of fetal development. By birth, accumulation of elastin in elastic fibers accounts in large part for the integrity and resilience of skin, blood vessels, and lungs. Developmental studies in several connective tissues of nonhuman vertebrates indicate that elastin synthesis is rapidly initiated during fetal life and that its expression is largely controlled by the abundance of its mRNA. Previous evidence for elastin synthesis in the developing human fetus has been derived from either histologic inference or studies of net accumulation. We now report that the developmental induction of cutaneous elastin synthesis appears to be stably reflected in cell culture. Production of elastin by human skin fibroblasts increased 7- to 14-fold between 17 and 19 weeks of gestation, reaching the levels found in neonatal skin fibroblasts. Consistent with other developmental studies, elastin synthesis was found to be under pretranslational control with relative mRNA levels increasing 6- to 15-fold by 19 weeks of gestation. Under the same circumstances, collagen expression and total protein synthesis were relatively constant among all strains examined. Human skin fibroblasts may thus be a useful system for examining developmentally regulated elastin gene expression.
Elastin↗