A Comparative Study of the RAPINA and the Virus-Neutralizing Test (RFFIT) for the Estimation of Antirabies-Neutralizing Antibody Levels in Dog Samples.

The mass vaccination of dogs against rabies is a highly rational strategy for interrupting the natural transmission of urban rabies. According to the World Organization for Animal Health (OIE) and the World Health Organization (WHO), the immunization of at least 70% of the total dog population minimizes the risk of endemic rabies. Knowledge of the virus-neutralizing antibody (VNA) level against the rabies virus (RABV) is required to evaluate protective immunity and vaccine coverage of dogs in the field. The rapid focus fluorescent inhibition test (RFFIT) and the fluorescent antibody virus neutralization (FAVN) test are recommended by OIE and WHO to determine the VNA levels in serum. However, these tests are cell culture based and require the use of live viruses and specialized equipment. The rapid neutralizing antibody test (RAPINA) is a novel, immunochromatographic test that uses inactivated virus to estimate the VNA level qualitatively. It is a simple, rapid and inexpensive, although indirect, assay for the detection of VNA levels. The RAPINA has shown good positive and negative predictive values and a high concordance with the RFFIT results. In this study, we compared the performance of the two tests for evaluating the vaccination status of dogs in the Philippines, Thailand and Japan. A total of 1135 dog sera were analysed by the RAPINA and compared to the VNA levels determined by the RFFIT. The overall positive and negative predictive values of the RAPINA were 96.2-99.3% and 84.5-94.8%, respectively, with a concordance (kappa) of 0.946-0.97 among the three countries. The RAPINA results were highly homologous and reproducible among different laboratories. These results suggest that this test is appropriate to survey vaccination coverage in countries with limited resources.

Immunoglobulin G enzyme-linked immunosorbent assay using truncated nucleoproteins of Reston Ebola virus.

We developed an immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA), using partial recombinant nucleoproteins (rNP) of Reston Ebola virus (EBO-R) and Zaire Ebola virus (EBO-Z). We examined the reaction of 10 sera from cynomolgus macaques naturally infected with EBO-R to each of the partial rNP in the IgG ELISA. All the sera reacted to the C-terminal halves of the rNP of both EBO-R and EBO-Z. Most of the sera reacted to the RdeltaC (amino acid (aa) 360-739), and Rdelta6 (aa 451-551) and/or Rdelta8 (aa 631-739) at a higher dilution than to the corresponding truncated rNPs of EBO-Z. The results indicate that this IgG ELISA is useful for detecting EBO-R specific antibody, and may have a potential to discriminate EBO-R infection from other subtypes.

Partial HIF-1alpha deficiency impairs pulmonary arterial myocyte electrophysiological responses to hypoxia.

Chronic hypoxia depolarizes and reduces K+ current in pulmonary arterial smooth muscle cells (PASMCs). Our laboratory previously demonstrated that hypoxia-inducible factor-1 (HIF-1) contributed to the development of hypoxic pulmonary hypertension. In this study, electrophysiological parameters were measured in PASMCs isolated from intrapulmonary arteries of mice with one null allele at the Hif1a locus encoding HIF-1alpha [Hif1a(+/-)] and from their wild-type [Hif1a(+/+)] littermates after 3 wk in air or 10% O2. Hematocrit and right ventricular wall and left ventricle plus septum weights were measured. Capacitance, K+ current, and membrane potential were measured with whole cell patch clamp. Similar to our laboratory's previous results, hypoxia-induced right ventricular hypertrophy and polycythemia were blunted in Hif1a(+/-) mice. Hypoxia increased PASMC capacitance in Hif1a(+/+) mice but not in Hif1a(+/-) mice. Chronic hypoxia depolarized and reduced K+ current density in PASMCs from Hif1a(+/+) mice. In PASMCs from hypoxic Hif1a(+/-) mice, no reduction in K+ current density was observed, and depolarization was significantly blunted. Thus partial deficiency of HIF-1alpha is sufficient to impair hypoxia-induced depolarization, reduction of K+ current density, and PASMC hypertrophy.

Resolution, detection, and characterization of redox conformers of human HSF1.

We describe here an experimental protocol for the resolution, detection, and quantitation of the reduced and oxidized conformers of human heat shock factor 1 (hHSF1) and report on the effects in vitro and in vivo of redox-active agents on the redox status, structure, and function of hHSF1. We showed that diamide, a reagent that promotes disulfide bond formation, caused a loss of immunorecognition of the monomeric hHSF1 protein in a standard Western blot detection procedure. Modification of the Western blot procedure to include dithiothreitol in the equilibration and transfer buffers after gel electrophoresis allowed for the detection of a compact, intramolecularly disulfide cross-linked oxidized hHSF1 (ox-hHSF1) in the diamide-treated sample. The effect of diamide was blocked by pretreatment with N-ethylmaleimide and was reversed by dithiothreitol added to the sample prior to gel electrophoresis. Incubation with nitrosoglutathione at 42 degrees C also promoted the conversion of HSF1 to ox-HSF1; at 25 degrees C, however, nitrosoglutathione was by itself without effect but blocked the formation of ox-hHSF1 in the presence of diamide. The disulfide cross-linked ox-hHSF1 was monomeric and resistant to the in vitro heat-induced trimerization and activation. The possibility that ox-HSF1 may occur in oxidatively stressed cells was evaluated. Treatment of HeLa cells with 2 mm l-buthionine sulfoximine promoted the formation of ox-HSF1 and blocked the heat-induced activation of HSF DNA binding activity. Our result suggests that hHSF1 may have integrated redox chemistry of cysteine sulfhydryl into its functional responses.

Epidemiology of Ebola (subtype Reston) virus in the Philippines, 1996.

Ebola (subtype Reston [EBO-R]) virus infection was detected in macaques imported into the United States from the Philippines in March 1996. Studies were initiated in the Philippines to identify the source of the virus among monkey-breeding and export facilities, to establish surveillance and testing, and to assess the risk and significance of EBO-R infections in humans who work in these facilities. Over a 5-month period, acutely infected animals were found at only one facility, as determined using Ebola antigen detection. Three of 1732 monkeys and 1 of 246 animal handlers tested had detectable antibodies; all were from the same facility, which was the source of infected monkeys imported to the United States. Virus transmission, which was facilitated by poor infection-control practices, continued for several months in one facility and was stopped only when the facility was depopulated. None of the 246 employees of the facilities or 4 contacts of previously antibody-positive individuals reported an Ebola-like illness. This investigation suggests that human EBO-R infection is rare.

Heat shock response, heat shock transcription factor and cell aging.

A characteristic feature of aging is a progressive impairment in the ability to adapt to environmental challenges. The purpose of this article is to review the evidence of an attenuated response to heat and physiological stresses in a number of mammalian aging model systems, including the human diploid fibroblasts in culture, whole animals and animal-derived cells and cell cultures, as well as peripheral blood mononuclear cells obtained from human donors. Analyses of the regulation and function of heat shock factor 1 (HSF1), a transcription factor that mediates the response to heat shock, showed that while the relative abundance of both the hsf1 transcript and the HSF1 protein did not change as a function of age, the responsiveness of HSF1 to heat-induced activation, as measured by its trimerization and ability to bind to the heat shock element consensus sequence, was inversely related to the age of the cells used. Given the fundamentally important role of heat shock proteins (HSPs) in many aspects of protein homeostasis and signal transduction it seems likely that the inability, or compromised ability, of aging cells and organisms to activate HSF1 and produce HSPs in response to stress would contribute to the well-known increase in morbidity and mortality of the aged when challenged.

Attenuated heat shock transcriptional response in aging: molecular mechanism and implication in the biology of aging.

A characteristic feature of aging is a progressive impairment in the ability to adapt to environmental challenges. The purpose of this review is to present the experimental evidence of an attenuated heat shock transcriptional response to heat and physiological stresses in a number of aging mammalian model systems. These include the human diploid fibroblasts in culture, whole animals and animal derived cells and cell cultures, as well as peripheral blood mononuclear cells obtained from human donors. The possibility that age-dependent changes in cellular redox status, as exemplified by the increased production of reactive oxygen inter-mediates and accumulation of oxidatively-modified proteins, affects the regulation and function of the heat shock factor 1 (HSF1) and contributes to the attenuated heat shock transcriptional response in aging cells and organisms is discussed. Given the fundamentally important role of HSPs in many aspects of protein homeostasis and signal transduction, it seems likely that the inability, or compromised ability, of aging cells and organisms to produce HSPs in response to stress would contribute to the well known increase in morbidity and mortality of the aged when challenged.

The axonal glycoprotein TAG-1 is an immunoglobulin superfamily member with neurite outgrowth-promoting activity.

Pathfinding of axons in the developing nervous system is thought to be mediated by glycoproteins expressed on the surface of embryonic axons and growth cones. One molecule suggested to play a role in axonal growth is TAG-1, a 135 kd glycoprotein expressed transiently on the surface of subsets of neurons in the developing mammalian nervous system. We isolated a full-length cDNA clone encoding rat TAG-1. TAG-1 has six immunoglobulin-like domains and four fibronectin type III-like repeats and is structurally similar to other immunoglobulin-like proteins expressed on developing axons. Neurons maintained in vitro on a substrate of TAG-1 extend long neurites, suggesting that this protein plays a role in the initial growth and guidance of axons in vivo. TAG-1 is anchored to the neuronal membrane via a glycosyl phosphatidylinositol linkage and is also released from neurons, suggesting that TAG-1 also functions as a substrate adhesion molecule when released into the extracellular environment.