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Contains fulltext : 232751.pdf (Publisher’s version ) (Closed access) Clinical research in venous thromboembolism (VTE) is hindered by variability in the collection and reporting of data and outcomes. A consistent data language facilitates efficiencies, leads to higher quality data, and permits between-study comparisons and evidence synthesis. The International Society on Thrombosis and Haemostasis (ISTH) launched an international task force of more than 50 researchers to develop common data elements for clinical research in venous thromboembolism. The project was organized in seven working groups, each focusing on a topic area: General Core Data Elements; Anticoagulation and Other Therapies; Chronic VTE and Functional Outcomes; Diagnosis of VTE; Malignancy; Perioperative; and Predictors of VTE. The groups met via teleconference to collaboratively identify key data elements and develop definitions and data standards that were structured in a project-specific taxonomy. A Steering Committee met by teleconference and in-person to determine the overall scope of the project and resolve questions arising from the working groups. ISTH held an open public comment period to enable broader stakeholder involvement and feedback. The common data elements were then refined by the working groups to create a set of 512 unique data elements that are publicly available at http://isth.breakthrough.healthcare. The ISTH VTE Common Data Elements are intended to be a living project with ongoing curation, future expansion, and adaptation to meet the needs of the thrombosis and hemostasis research community.

The interdependence among eating behaviour (EB), physical activity (PA) and sedentary time (ST) suggests simultaneously identifying homogeneous profiles and describing their changes. This study aimed to (1) identify cross-sectional lifestyle behaviour profiles and their 2-year changes among French school-age adolescents and (2) identify factors associated with these profiles and changes. Longitudinal data from adolescents who participated in the PRomotion de l’ALIMentation et de l’Activité Physique trial were used. PA and ST were assessed with the International Physical Activity Questionnaire and EB with a FFQ. Profiles at baseline and their changes were identified by latent transition analysis. Multinomial logistic regression models were used to identify factors associated with profiles and their changes. Among 2390 adolescents included (14–18 years), five baseline profiles that differed mainly in EB were identified: ‘healthy diet and high PA (7·9 %)’, ‘big eater and moderate to high PA (23·8 %)’, ‘healthy diet and low PA (31·2 %)’, ‘restrictive diet and moderate PA (20·6 %)’ and ‘sugar products, nibbling and moderate PA (16·5 %)’. Young adolescents, those who were overweight or obese and socially advantaged, were more in the ‘healthy diet and low PA’ than others. Boys, older and socially less advantaged adolescents exhibited more ‘unfavourable’ than ‘mixed’ changes, while adolescents with overweight or obesity had less ‘unfavourable’ than ‘mixed’ changes. In conclusion, adolescents were twice the number in the least than the most favourable profile. Findings highlighted the importance of EB among adolescents and suggest taking adolescents’ sociodemographic and weight characteristics into account in interventions aimed at acting on adolescents’ behaviours.

Ocean warming is driving species poleward, causing a ‘tropicalization’ of temperate ecosystems around the world. Increasing abundances of tropical herbivores on temperate reefs could accelerate declines in habitat-forming seaweeds with devastating consequences for these important marine ecosystems. Here we document an expansion of rabbitfish (Siganus fuscescens), a tropical herbivore, on temperate reefs in Western Australia following a marine heatwave and demonstrate their impact on local kelp forests (Ecklonia radiata). Before the heatwave there were no rabbitfish and low rates of kelp herbivory but after the heatwave rabbitfish were common at most reefs and consumption of kelp was high. Herbivory increased 30-fold and kelp abundance decreased by 70% at reefs where rabbitfish had established. In contrast, where rabbitfish were absent, kelp abundance and herbivory did not change. Video-analysis confirmed that rabbitfish were the main consumers of kelp, followed by silver drummers (Kyphosus sydneyanus), a temperate herbivore. These results represent a likely indirect effect of the heatwave beyond its acute impacts, and they provide evidence that range-shifting tropical herbivores can contribute to declines in habitat-forming seaweeds within a few years of their establishment.

Made available in DSpace on 2019-10-06T16:13:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 Palosuojelurahasto Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) No-take marine reserves (NTRs), i.e. areas with total fishing restrictions, have been established worldwide aiming to promote biodiversity and ecosystem conservation. Brazil has 3.3% of its exclusive economic zone protected by 73 different NTRs, however, most of them currently lack scientific knowledge and understanding of their ecological role, particularly regarding rocky reefs in subtropical regions. In this context, this study aimed to contrast a network of NTRs with comparable fished sites across a coastal biogeographic gradient to investigate the effect of fishing and habitat variability on the abundance and body size of rocky reef fish. We used Baited Remote Underwater stereo-Video (stereo-BRUVs) and Diver Operated stereo-Video (stereo-DOVs) systems to simultaneously sample reef fish and habitat. Model selection and results identified habitat and biogeographic variables, such as distance from shore, as important predictor variables, explaining several aspects of the fish assemblage. The effect of protection was important in determining the abundance and body size of targeted species, in particular for epinephelids and carangids. Conversely, species richness was correlated with habitat complexity but not with protection status. This is the first study using these survey methods in the Southwestern Atlantic, demonstrating how a network of NTRs can provide benchmarks for biodiversity conservation and fisheries management. Instituto de Biociências Universidade Estadual Paulista (UNESP) Campus de Rio Claro Laboratório de Pesquisa em Elasmobrânquios Instituto de Biociências Universidade Estadual Paulista (UNESP) Campus do Litoral Paulista UWA Oceans Institute and School of Biological Sciences University of Western Australia (UWA) Laboratório de Ecologia e Conservação Marinha Instituto do Mar Universidade Federal de São Paulo (UNIFESP) Departamento de Ciências do Meio Ambiente Universidade Federal Rural do Rio de Janeiro (UFRRJ) Campus Três Rios Instituto de Biociências Universidade Estadual Paulista (UNESP) Campus de Rio Claro Laboratório de Pesquisa em Elasmobrânquios Instituto de Biociências Universidade Estadual Paulista (UNESP) Campus do Litoral Paulista Palosuojelurahasto: #1043_20151 CNPq: 308430/2015-8

Introduction Some studies have suggested that regular cannabis users appear to exhibit a general reckless driving style that may contribute to an inflated estimate of collisions related to driving under the influence of cannabis (DUIC) among this group. Objective This study investigated whether patterns of cannabis use would predict levels of self-reported unsafe driving behaviours and levels of performance observed in a driving simulator. Method The study investigated 48 young cannabis users with respect to frequency of cannabis consumption, alcohol use, driving exposure, self-reported dangerous driving habits, impulsivity, sensation seeking and performance in a driving simulator. Results Cannabis use was positively associated with DUIC, with sensation seeking and with self-reports of risky driving. An ANOVA confirmed a significant effect of levels of consumption of cannabis among participants on self-report risk-taking in driving. Moreover, the observed behaviours during the simulation tasks correlated with risky driving habits, and with the self-reported frequency of driving in the hour following smoking cannabis or immediately after drinking alcohol. Conclusion Since cannabis usage and DUIC appear to be related to an overall reckless style of driving, it is proposed that public safety policies should be more holistic, simultaneously targeting multiple on-road dangerous behaviours for intervention.

Background Case control studies suggest that genetic thrombophilias increase the risk of placenta-mediated pregnancy complications (pregnancy loss, small for gestational age (SGA), preeclampsia and/or placental abruption). Cohort studies have not supported this association but were underpowered to detect small effects. Objective To determine if factor V Leiden (FVL) or the prothrombin gene mutation (PGM) were associated with placenta-mediated pregnancy complications. Patients/Methods A prospective cohort of unselected, consenting pregnant women at three Canadian tertiary care hospitals had blood drawn in the early second trimester and were genotyped for FVL and PGM after delivery. The main outcome measure was a composite of pregnancy loss, SGA

Previously unaccounted cryptic speciation requires revaluating species identification, particularly for fisheries assessment purposes. We describe a rapid and reliable method for distinguishing between phenotypically similar species that utilizes simple otolith morphometry (length, width, thickness and weight) with or without fish length, within a traditional canonical discriminant analysis (CDA). Data were subject to CDA in order to differentiate between the cryptic Etelis carbunculus (ruby snapper) and E. marshi (pygmy ruby snapper). A very high allocation success rate was achieved using otolith morphometry and fork length (99.6% for E. carbunculus and 100% for E. marshi) or otolith morphometry only (98.8% for E. carbunculus and 100% for E. marshi), which indicated the high discriminatory power of this method. The CDA successfully grouped samples of the same species collected from different locations in the eastern central Indian and South Pacific Oceans, indicating the robustness of this technique to discriminate between species, irrespective of their geographic range. This technique can be applied to archived otolith collections to confirm teleost species identification, and likely has broader applications for species identification involving extractive, diet or video-based studies.

The tropicalization of temperate marine ecosystems can lead to increased herbivory rates, reducing the standing stock of seaweeds and potentially causing increases in detritus production. However, long-term studies analysing these processes associated with the persistence of tropical herbivores in temperate reefs are lacking. We assessed the seasonal variation in abundances, macrophyte consumption, feeding modes and defecation rates of the range-extending tropical rabbitfishSiganus fuscescensand the temperate silver drummerKyphosus sydneyanusand herring caleOlisthops cyanomelason tropicalized reefs of Western Australia. Rabbitfish overwintered in temperate reefs, consumed more kelp and other macrophytes in all feeding modes, and defecated more during both summer and winter than the temperate herbivores. Herbivory and defecation increased with rabbitfish abundance, but this was dependent on temperature, with higher rates attained by big schools during summer and lower rates in winter. Still, rabbitfish surpassed temperate herbivores, leading to a fivefold acceleration in the transformation of macrophyte standing stock to detritus, a function usually attributed to sea urchins in kelp forests. Our results suggest that further warming and tropicalization will not only increase primary consumption and affect the habitat structure of temperate reefs but also increase detritus production, with the potential to modify energy pathways.

Background A structured screening strategy led to a significant decrease in overweight and obesity prevalence. Inverse relationship between income and the prevalence of obesity is known. Furthermore, public health interventions may increase health inequalities. The PRALIMAP-INES trial was implemented between 2012 and 2015 and aimed to investigate whether a strengthened care management strategy to prevent and reduce overweight could do …

Background Reducing overweight in adolescent requires taking into account social inequalities. The study aims to investigate the participation social gradient in prevention programme. Methods The PRALIMAP-INES trial included 1417 13-18 year adolescents categorised in 2 groups according to their Family Affluence Scale (FAS) score: advantaged (FAS > 5) (n = 808) and less advantaged (LA n = 609). A standard care management …

Did Russia initiate conflict with Georgia in an attempt to restrain Georgia from formally joining the Western coalition, or was the decision more complex? What did Russia hope to gain from initiating conflict? What value did Russia assign to maintaining an interest in regional oil trade routes, in which Georgia straddles an important route to the Caspian Sea region? How was the potential incorporation of Georgia into NATO factored into Russia’s decision? To answer these questions, I used the expected-utility model developed by Bruce Bueno de Mesquita. In this analysis, policy preferences and power measurements were considered to determine that Russia’s decision was rationally selected based on a positive expectedutility calculation. I attempted to explain whether this model’s explanatory and predictive capabilities could accurately elucidate Russia’s conflict decision. Furthermore, within the context of the expected-utility model, what can we expect about Russian-Georgian relations in the future?

Previous standards for assessing the reliability of a measurement tool have lacked consistency. We reviewed the most current American Society for Testing and Materials and International Organisation for Standardisation (ISO) recommendations, and propose an algorithm for orthopaedic surgeons. When assessing a measurement tool, conditions of the experimental set-up and clear formulae used to compile the results should be strictly reported. According to these recent guidelines, accuracy is a confusing word with an overly broad meaning and should therefore be abandoned. Depending on the experimental conditions, one should be referring to bias (when the study protocol involves accepted reference values), and repeatability (sr, r) or reproducibility (SR, R). In the absence of accepted reference values, only repeatability (sr, r) or reproducibility (SR, R) should be provided. Take home message: Assessing the reliability of a measurement tool involves reporting bias, repeatability and/or reproducibility depending on the defined conditions, instead of precision or accuracy. Cite this article: Bone Joint J 2016;98-B2:166–72.

Resume Objectif. – En 2003, 250 000 patients splenectomises ont ete recenses en France. Les patients aspleniques presentent un haut risque d'infections, en particulier vis-a-vis des bacteries encapsulees : pneumocoque, Haemophilus influenzae et meningocoque. Ces infections, caracterisees par leur rapidite d'installation et leur gravite importante, comme en temoigne une morbimortalite elevee, necessitent une prise en charge globale specifique qui a ete revue au cours d'une reunion d'experts en juin 2003 a Paris. Voici les principales conclusions concernant la periode perioperatoire. Type d'etude. – Mise au point. Synthese des donnees. – Deux vaccins antipneumococciques sont a notre disposition : le vaccin polysaccharidique (Pneumo 23®) et le vaccin conjugue (Prenevar®), propose plus recemment. Le vaccin polysaccharidique, d'efficacite variable, et qui couvre un grand nombre de serotypes, est conseille chez l'adulte. Le vaccin conjugue est preconise chez l'enfant de moins de deux ans car il est actif sur les souches resistantes aux antibiotiques et potentialise l'effet de l'antibioprophylaxie. En cas de splenectomie programmee, les vaccinations couvrant le pneumocoque (Pneumo 23®), l'Haemophilus de type b et le meningocoque de type C doivent etre realisees au moins 15 jours avant l'intervention assurant ainsi une meilleure immunogenicite du vaccin. En cas de chirurgie urgente, les vaccins doivent etre administres dans les 30 jours suivant l'intervention. Une injection preoperatoire de cefazoline est preconisee. Un relais par amoxicilline injectable peut etre entrepris jusqu'a la reprise du transit ; un traitement par penicilline V (Oracilline®) est ensuite institue pour une duree minimale de deux ans chez l'adulte et de cinq ans chez l'enfant. Cette antibioprophylaxie, complementaire de la vaccination, assure une diminution du nombre d'infection a germes encapsules.

We previously showed that envelope (gp160)-based vaccines, used in a live recombinant virus priming and subunit protein boosting regimen, protected macaques against intravenous and intrarectal challenges with the homologous simian immunodeficiency virus SIVmne clone E11S. However, the breadth of protection appears to be limited, since the vaccines were only partially effective against intravenous challenge by the uncloned SIVmne. To examine factors that could affect the breadth and the efficacy of this immunization approach, we studied (i) the effect of priming by recombinant vaccinia virus; (ii) the role of surface antigen gp130; and (iii) the role of core antigens (Gag and Pol) in eliciting protective immunity. Results indicate that (i) priming with recombinant vaccinia virus was more effective than subunit antigen in eliciting protective responses; (ii) while both gp130 and gp160 elicited similar levels of SIV-specific antibodies, gp130 was not as effective as gp160 in protection, indicating a possible role for the transmembrane protein in presenting functionally important epitopes; and (iii) although animals immunized with core antigens failed to generate any neutralizing antibody and were infected upon challenge, their virus load was 50- to 100-fold lower than that of the controls, suggesting the importance of cellular immunity or other core-specific immune responses in controlling acute infection. Complete protection against intravenous infection by the pathogenic uncloned SIVmne was achieved by immunization with both the envelope and the core antigens. These results indicate that immune responses to both antigens may contribute to protection and thus argue for the inclusion of multiple antigens in recombinant vaccine designs.

We previously reported that immunization with recombinant simian immunodeficiency virus SIVmne envelope (gp160) vaccines protected macaques against an intravenous challenge by the cloned homologous virus, E11S. In this study, we confirmed this observation and found that the vaccines were effective not only against virus grown on human T-cell lines but also against virus grown on macaque peripheral blood mononuclear cells (PBMC). The breadth of protection, however, was limited. In three experiments, 3 of 10 animals challenged with the parental uncloned SIVmne were completely protected. Of the remaining animals, three were transiently virus positive and four were persistently positive after challenge, as were 10 nonimmunized control animals. Protection was not correlated with levels of serum-neutralizing antibodies against the homologous SIVmne or a related virus, SIVmac251. To gain further insight into the protective mechanism, we analyzed nucleotide sequences in the envelope region of the uncloned challenge virus and compared them with those present in the PBMC of infected animals. The majority (85%) of the uncloned challenge virus was homologous to the molecular clone from which the vaccines were made (E11S type). The remaining 15% contained conserved changes in the V1 region (variant types). Control animals infected with this uncloned virus had different proportions of the two genotypes, whereas three of four immunized but persistently infected animals had >99% of the variant types early after infection. These results indicate that the protective immunity elicited by recombinant gp160 vaccines is restricted primarily to the homologous virus and suggest the possibility that immune responses directed to the V1 region of the envelope protein play a role in protection.

Antisera to 21 synthetic peptides containing hydrophilic sequences of simian immunodeficiency virus strain mac251 (SIVmac251) gp120 and gp32 were tested for the ability to neutralize SIVmac251. Goat antisera raised to peptides SP-1 and SP-1V containing the carboxy-terminal portion of the V3 domain of SIVmac251 gp120 between amino acids 327 and 339 inhibited syncytium formation (90% inhibition at a 1/1024 dilution) and cell killing of CEMx174 cells by SIVmac251 (50% inhibition of cell killing at a dilution of 1/5832), SIVDeltaB670 (1/568), and SIVsmH4 (1/740). Neutralizing antibodies to SIVmac251, SIVDeltaB670, and SIVsmH4 could be adsorbed by peptides containing a neutralizing V3 sequence of SIVmac251 gp120 (GLVFHSQPIND, amino acids 329–339) but not by peptides lacking this sequence. This V3 neutralizing region corresponds to a homologous V3 neutralizing site within HIV-2 gp120 reported by Björlinget al.1991,Proc. Natl. Acad. Sci. USA88, 6082–6086, 1994,J. Immunol.152, 1952–1959). Antibodies in 20 of 31 sera obtained from rhesus macaques infected with SIVmac251 reacted with a peptide containing the entire V3 sequence of SIVmac251 gp120, whereas no sera contained antibodies reacting with the V3 neutralizing site between amino acids 329 and 339. Low levels of antibody-mediated recognition and subsequent lack of selective pressure against this linear V3 neutralizing site might in part explain why this region is not a dominant neutralizing site and also why sequences within V3 do not vary during the course of SIV infection.

Using pathogenic simian immunodeficiency virus (SIV) infection of macaques as a model, we explored the limits of the protective immunity elicited by recombinant subunit vaccines and examined factors that affect their efficacy. Envelope gp 160 vaccines, when used in a live recombinant virus-priming and subunit-protein-boosting regimen, protected macaques against a low-dose, intravenous infection by a cloned homologous virus SIV mne E11S. The same regimen was also effective against intrarectal challenge by the same virus and against intravenous challenge by E11S grown on primary macaque peripheral blood mononuclear cells (PBMC). However, only limited protection was observed against uncloned SIV mne . Priming with live recombinant virus was more effective than immunization with subunit gp 160 alone, indicating a potential advantage of native antigen presentation and the possible role of cell-mediated immunity in protection. Whole gp 160 was more effective than the surface antigen (gp130), even though both antigens elicited similar levels of neutralizing antibodies. Animals immunized with the core ( gag-pol ) antigens failed to generate any neutralizing antibody and were all infected following challenge. However, their proviral load was 10–100-fold lower than that of the control animals, indicating that immune mechanisms such as cytotoxic T lymphocytes (CTL) may play a role. Finally, animals immunized with both the core and the envelope antigens generated significant protective immunity, even with relatively low neutralizing antibodies. Taken together, these results indicate that multiple mechanisms may contribute to protection. It may therefore be advantageous to incorpotate multiple antigens in the design of recombinant subunit vaccines against acquired immunodeficiency syndrome (AIDS).

To assess the characteristics of hybrid receptors composed of one kinase-inactive alpha beta-insulin half-receptor and one endogenous alpha beta-insulin-like growth factor 1 (IGF-1) or insulin half-receptor, a cell line expressing an insulin receptor truncated by 365 amino acids (HIR delta 978) was studied, which lacks most of the cytoplasmic beta-subunit. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis under nonreducing conditions revealed four distinct receptor species: endogenous receptors, the more rapidly migrating HIR delta 978 homodimer, and two intermediate species representing HIR delta 978/IGF-1 hybrid receptors and HIR delta 978/IR hybrid receptors. In vivo ligand-binding affinity of the hybrid receptors was studied by receptor-ligand cross-linking, and the delta 978/IGF-1R hybrid receptor was found to have a high affinity for IGF-1, whereas its affinity for insulin was low. Autophosphorylation studies of lectin-purified receptors revealed that neither the HIR delta 978 holoreceptor nor the hybrid receptors underwent autophosphorylation in response to either ligand, despite the presence of intact IGF-1 or insulin half-receptors in the hybrids. Neither hybrid receptor underwent ligand-induced endocytosis, as assessed with the bioactive photoaffinity probes B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin and N-epsilon B28-monoazidobenzoyl-IGF-1. In conclusion, the HIR delta 978/IGF-1R hybrid receptor has a high in vivo affinity for IGF-1 but not for insulin. Neither the delta 978/IGF-1R nor the delta 978/IR hybrids undergo autophosphorylation or ligand-induced endocytosis in response to either ligand, indicating that intramolecular trans-, rather than cis-, signal transduction is important in mediating autophosphorylation and endocytosis.

Insulin stimulates tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and She in Rat1 fibroblasts overexpressing wild type insulin receptors. We investigated the relative role of IRS-1 and She in insulin activation of guanine nucleotide releasing factor (GNRF) and p21ras-GTP formation. The time course of insulin-stimulated tyrosine phosphorylation of IRS-1 was rapid, whereas Shc phosphorylation was relatively slow. Growth factor receptor bound protein-2 (Grb2) associated with IRS-1 rapidly and gradually dissociated after 5 min, whereas Grb2 association with Shc was slower and reached a maximum at 10 min after insulin stimulation. Thus, the kinetics of Grb2 association with IRS-1 and She corresponded closely to the time course of tyrosine phosphorylation of IRS-1 and Shc, respectively. Importantly, 3-13-fold more Grb2 was associated with Shc than with IRS-1. In addition, the kinetics of insulin-stimulated GNRF activity and p21ras-GTP formation corresponded more closely to the time course of Shc phosphorylation than to the kinetics of IRS-1 phosphorylation. Furthermore, immunoprecipitation of Shc proteins from cell lysates of insulin-stimulated cells removed 67% of the GNRF activity, whereas precipitation of IRS-1 had a negligible effect on GNRF activity. Thus, although both IRS-1 and Shc associate with Grb2, the current results indicate that Shc plays a more important role than IRS-1 in insulin stimulation of GNRF activity and subsequent p21ras-GTP formation.

To assess the function of the cytoplasmic domain of the insulin receptor (IR) beta subunit, we have studied a mutant IR truncated by 365 aa (HIR delta 978), thereby deleting > 90% of the cytoplasmic domain. HIR delta 978 receptors were processed normally to homodimers that were expressed at the cell surface where they bind insulin with normal affinity. Although these truncated IRs were inactive with respect to ligand-induced internalization and autophosphorylation, insulin stimulated endogenous substrate (pp185) phosphorylation significantly more in HIR delta 978 cells than in untransfected Rat1 cells. Importantly, despite absence of the beta-subunit cytoplasmic domain, fibroblasts expressing HIR delta 978 receptors displayed enhanced sensitivity to insulin for stimulation of glucose incorporation into glycogen, alpha-aminoisobutyric acid uptake, thymidine incorporation, and S6 kinase activity compared with parental fibroblasts. Insulin also induced the expression of the protooncogene c-fos and the early growth response gene Egr-1 in HIR delta 978 cells far greater than in parental Rat1 fibroblasts. Furthermore, an agonistic monoclonal antibody specific for the human IR stimulated insulin action in fibroblasts expressing wild-type human IR but had no effect on HIR delta 978 cells. In conclusion, the HIR delta 978 truncated IRs appear to confer enhanced insulin sensitivity by augmenting the signaling properties of the endogenous rodent IRs.

Two Macaca fascicularis with preexisting immunity to vaccinia virus were immunized twice with recombinant vaccinia virus expressing SIVmne gp160. Their SIV-specific antibody responses were lower than that of vaccinia-naive animals immunized similarly. Upon repeated boosting with gp160, the SIV-specific antibody titers in vaccinia-primed animals reached similar levels as vaccinia-naive animals and with comparable neutralizing titers. Both animals were protected against repeated intravenous challenge with low-dose SIVmne E11S. These results are significant because SIVmne E11S infection in M. fascicularis is pathogenic and leads to AIDS-like diseases.

Simultaneous exposure to chemicals which can oxidize the haemoglobin of the red blood cell to methaemoglobin is common. Although the effects of some of these agents have been documented individually, little research considers the interactive effects. In-vitro experiments on the treated blood of female Dorset sheep assessed the interactive capacity of chlorite, copper and nitrite to affect methaemoglobin formation. All combinations of doses which produced 2.5, 5, 10% methaemoglobin were tested in all possible combinations (a total of 80), as were the controls. This included data on each chemical alone, each two-way combination and the three-way combination. The response is largely additive (the sum of the individual effects) except for one of the two-way interactions, chlorite/nitrite (P < . 01), which showed antagonism. Chlorite may oxidize nitrite which could explain the less-than-additive response. Overall, the result of combining these agents on methaemoglobin was additive.

To identify the principal neutralization determinant (PND) of simian immunodeficiency virus (SIV), antisera were generated using recombinant gp110 [the SIV analog of the human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein, gp120], gp140, several large recombinant and proteolytic envelope fragments, and synthetic peptides of the SIVmac251 isolate. When purified under conditions that retain its native structure, gp110 bound CD4 and elicited antisera that neutralized SIVmac251 with high titer. Native gp110 also completely inhibited neutralizing antibody in sera from SIVmac251-infected macaques. In contrast, denatured gp110 and gp140, large envelope fragments, and synthetic peptides (including peptides analogous to the HIV-1 PND) elicited very low or undetectable neutralizing antibody titers and did not inhibit neutralizing antibody in infected macaque sera. Enzymatically deglycosylated gp110 efficiently absorbed neutralizing antibodies from macaque sera, showing that neutralizing antibodies primarily bind the protein backbone. A 45-kDa protease digest product, mapping to the carboxyl-terminal third of gp110, also completely absorbed neutralizing antibodies from infected macaque sera. These results show that the PND(s) of this SIV isolate depends on the native conformation and that linear peptides corresponding to the V3 loop of SIV envelope, in contrast to that of HIV-1, do not elicit neutralizing antibody. This may affect the usefulness of SIVmac for evaluating HIV-1 envelope vaccine approaches that rely on eliciting neutralizing antibody.

Simian immunodeficiency virus (SIV) was used as a model to study the protective efficacy of an immunization regimen currently being evaluated as candidate vaccines against HIV in human subjects. Four Macaca fascicularis were first immunized with recombinant vaccinia virus expressing the envelope glycoprotein gp160 of SIVmne and then boosted with subunit gp160. Both cell-mediated and humoral immune responses against SIV, including neutralizing antibodies, were elicited. The macaques were shown to be protected from a homologous virus infection as determined by serology, lymphocyte cocultivation, polymerase chain reactions and in vivo transmission analyses. Four unimmunized control animals were readily infected. However, viremia in infected control animals could decrease substantially following the initial phase of infection so that persistent infection might not be readily detectable.

Simian immunodeficiency virus (SIV) is a primate lentivirus related to human immunodeficiency viruses and is an etiologic agent for acquired immunodeficiency syndrome (AIDS)-like diseases in macaques. To date, only inactivated whole virus vaccines have been shown to protect macaques against SIV infection. Protective immunity was elicited by recombinant subunit vaccines. Four Macaca fascicularis were immunized with recombinant vaccinia virus expressing SIVmne gp160 and were boosted with gp160 produced in baculovirus-infected cells. All four animals were protected against an intravenous challenge of the homologous virus at one to nine animal-infectious doses. These results indicate that immunization with viral envelope antigens alone is sufficient to elicit protective immunity against a primate immunodeficiency virus. The combination immunization regimen, similar to one now being evaluated in humans as candidate human immunodeficiency virus (HIV)-1 vaccines, appears to be an effective way to elicit such immune responses.

The influence of human anti-human immunodeficiency virus type 1 (HIV-1) antibody on HIV-1 infection of freshly isolated normal human peritoneal macrophages and blood monocytes was examined. Each of 14 HIV antibody-positive human serum samples was found to block the infection of four virus isolates (human T-cell lymphotropic virus type IIIBa-L [HTLV-IIIBa-L], HTLV-IIIB, D.U. 6587-7, and D.U. 7887-8) at serum dilutions ranging from 10(-1) to 10(-2). Three of these isolates (HTLV-IIIBa-L, D.U. 6587-7, and D.U. 7887-8) infected cultures of monocytes and macrophages rapidly and produced high levels of virus reverse transcriptase and p24 antigen. A fourth virus isolate (HTLV-IIIB) infected the monocytes and macrophages more slowly and produced low levels of viral protein. More dilute HIV antibody-positive sera had no significant effect on the overall level or rate of virus infection or expression. Complement did not appear to influence the course of infection by any combination of antisera or virus examined. Successful HIV-1 infection of the peritoneal macrophages and blood monocytes under the conditions tested showed strict dependence on CD4 since a recombinant CD4 polypeptide and an anti-CD4 monoclonal antibody effectively blocked the process.

Sensitive and reproducible assays for SIV infection and syncytium formation have been developed in which high titers of neutralizing and fusion-inhibiting antibodies can be recorded. These assays will contribute toward our understanding of the role of humoral responses in SIV vaccine strategies.

Infection with attenuated simian immunodeficiency virus (SIV) in rhesus macaques has been shown to raise antibodies capable of neutralizing an animal challenge stock of primary SIVmac251 in CEMx174 cells that correlate with resistance to infection after experimental challenge with this virulent virus (M. S. Wyand, K. H. Manson, M. Garcia-Moll, D. C. Montefiori, and R. C. Desrosiers, J. Virol. 70:3724–3733, 1996). Here we show that these neutralizing antibodies are not detected in human and rhesus peripheral blood mononuclear cells (PBMC). In addition, neutralization of primary SIVmac251 in human and rhesus PBMC was rarely detected with plasma samples from a similar group of animals that had been infected either with SIVmac239Δnef for 1.5 years or with SIVmac239Δ3 for 3.2 years, although low-level neutralization was detected in CEMx174 cells. Potent neutralization was detected in CEMx174 cells when the latter plasma samples were assessed with laboratory-adapted SIVmac251. In contrast to primary SIVmac251, laboratory-adapted SIVmac251 did not replicate in human and rhesus PBMC despite its ability to utilize CCR5, Bonzo/STRL33, and BOB/gpr15 as coreceptors for virus entry. These results illustrate the importance of virus passage history and the choice of indicator cells for making assessments of neutralizing antibodies to lentiviruses such as SIV. They also demonstrate that primary SIVmac251 is less sensitive to neutralization in human and rhesus PBMC than it is in established cell lines. Results obtained in PBMC did not support a role for neutralizing antibodies as a mechanism of protection in animals immunized with attenuated SIV and challenged with primary SIVmac251.

Shc is phosphorylated on Tyr-317, which serves as a docking site for Grb2. To investigate the specific role of Shc phosphorylation and Shc.Grb2 coupling on insulin signaling, we generated expression vectors for wild-type (WT-Shc) and a mutant Shc with a Tyr-317 --> Phe substitution (317Y/F-Shc) and stably transfected them into Rat1 fibroblasts expressing insulin receptors (HIRc). From different clonal cell lines, cells expressing 10 times greater amounts of WT-Shc or 317Y/F-Shc compared with endogenous Shc were chosen for analysis of insulin signaling. Insulin-induced Shc phosphorylation and subsequent association with Grb2 was enhanced in WT-Shc cells. Because of competition between Shc and IRS-1 for interaction with the insulin receptor, insulin-stimulated tyrosine phosphorylation of IRS-1 was decreased in WT-Shc cells compared with that in HIRc cells. Likewise, reduction of endogenous Shc expression by antisense Shc mRNA resulted in increased insulin stimulation of IRS-1 phosphorylation. Although 317Y/F-Shc was also able to interact with insulin receptor, decreased amounts of Shc phosphorylation and Shc association with Grb2 were observed in 317Y/F-Shc cells, indicating that 317Y/F-Shc functions as a dominant-negative mutant. The kinetics of mitogen-activated protein (MAP) kinase activation closely paralleled the kinetics of Shc phosphorylation. Thus, insulin stimulation of MAP kinase activation occurred more rapidly and was prolonged in WT-Shc cells, while the activation was delayed and transient in 317Y/F-Shc cells compared with that in HIRc cells. Importantly, WT-Shc cells displayed enhanced sensitivity to insulin stimulation of thymidine and bromodeoxyuridine incorporation, whereas the sensitivity was decreased in 317Y/F-Shc cells. These results indicate that Shc Tyr-317 phosphorylation plays an important role, via coupling with Grb2 and competition with IRS-1, in signal transduction to MAP kinase by insulin, ultimately leading to mitogenesis in Rat1 fibroblasts.

To compare transesophageal echocardiography (TEE) and magnetic resonance (MR) imaging in the diagnosis of dissection of the thoracic aorta.Thirty-one consecutive patients with clinically suspected aortic dissection and 10 postoperative patients underwent transesophageal color Doppler echocardiography and MR imaging. Imaging results were compared at independent double-blind readings. Final diagnosis was obtained from consensual review of all corroborative studies.MR imaging depicted the intimal flap in 95% of aortic dissections; TEE, in 86% (P.05). In surgical patients, the sensitivity of MR in detection of residual dissection was 100% versus 86% with TEE (P.05). The inferior extent of the dissected lumen was seen only with MR imaging. False-positive results occurred in two cases with TEE and in one with MR imaging.MR imaging is superior to TEE in the evaluation and follow-up of dissection of the thoracic aorta. Because the availability of MR is limited, however, TEE should remain the standard modality for diagnosis.

Both insulin and epidermal growth factor (EGF) significantly increased the activation of p21Ras in Rat-1 fibroblasts transfected with human insulin receptors. As both growth factors have been shown to stimulate guanine nucleotide exchange on p21Ras in permeabilized cells, we examined their effects on total cellular guanine nucleotide exchange factor (GEF) activity in a direct assay, using the rate of dissociation of [3H]GDP from p21Ras as a measure of GEF activity. Insulin increased GEF activity in a time-dependent manner, whereas EGF had no effect on GEF activity at any time point studied. To assess whether EGF stimulates guanine nucleotide exchange by recruiting Grb-2-Sos complexes to the plasma membrane, we measured GEF activity in both the cytosol and the plasma membrane fractions of the insulin- and EGF-treated cells. Insulin increased GEF activity in both fractions (48 +/- 12% [3H]GDP released vs. 24 +/- 6% in control plasma membranes, and 65 +/- 13% vs. 13 +/- 4% in control cytosolic fractions), whereas EGF enhanced only the plasma membrane-associated activity (43 +/- 12% of [3H]GDP release in the plasma membrane fraction and 10 +/- 2% in the cytosol). Western blotting of the subcellular fractions with Grb-2 and Sos antibodies revealed translocation of these elements to the plasma membrane after stimulation of cells with either insulin or EGF. Thus, whereas insulin stimulates guanine nucleotide exchange on Ras by both translocating Grb-2-Sos complexes to the plasma membrane and increasing the GEF activity of Sos, EGF does so by causing a translocation of Grb-2-Sos without increasing GEF activity.

It has been shown previously that the major neutralizing epitopes in simian immunodeficiency virus (SIV) are discontinuous and conformation dependent and that the V3 loop, in contrast to that of human immunodeficiency virus (HIV) type 1, does not by itself elicit neutralizing antibodies (K. Javaherian et al., Proc. Natl. Acad. Sci. USA 89:1418-1422, 1992). We now present data showing that on the basis of fractionation of infected macaque sera, protease digestion of the envelope, and binding properties of two neutralizing monoclonal antibodies to SIV and SIV-HIV chimeric envelope proteins, changes in V3 can disrupt the conformation-dependent neutralization region. The chimeric protein did not produce significant neutralizing antibodies against either SIV or HIV. We also report that neutralizing antibodies elicited by recombinant SIV envelope proteins of mac251 and B670 isolates cross-neutralize. Finally, we show that deglycosylation of the SIV envelope results in a molecule which binds neither soluble CD4 nor the neutralizing monoclonal antibodies being investigated here and does not elicit sera with a significant neutralizing titer.

More than 200 sera from macaques immunized with several different vaccine preparations were tested in various assays with cells of human and macaque origin. Only in instances where whole inactivated SIV preparations were used for immunization, were reactivities found with normal human cells, and this was the case in every instance. Such sera produced a marked clumping of several normal human cell lines and exhibited strong staining of the cell surface in FACS analysis. In the presence of SIVDeltaB670, these sera also enhanced infectivity and fusion formation. When similar tests were performed with macaque cells as targets, such phenomena were not easily discernible. Likewise, there was no trace of such activities in sera from normal animals, animals chronically infected with SIV, or in those from animals which received recombinant viral subunits as vaccines. Finally, we show that in several instances where whole inactivated virus was used as a vaccine, there is a strong correlation between the titer of anticellular activity with protection.

Since the discovery of human immunodeficiency virus (HIV) as the causative agent of AIDS in 1983–84, there has been considerable effort to design an efficacious vaccine. Principally for safety reasons, defined subunit polypeptides and proteins devoid of genomic material have been considered as alternatives to attenuated and/or killed virus. Much of the effort to develope such a component vaccine against HIV has been directed at the envelope gene products. These have included recombinant gp l60 (1,2,3), recombinant gp 120 (4,5,6), native viral gp 120 (7,8,9), bacterially expressed envelope fragments (10), and a large number of synthetic peptides (11–19). In these early studies each of the various reagents were tested for their ability to raise neutralizing antibodies in experimental animals. When successful, resultant sera were usually found to be active only against homologous virus, i.e. were isolate restricted. The results suggested that variable sites on the envelope represented the major target of neutralizing antibody raised by the various experimental immunogens.

A highly immunogenic epitope from a conserved COOH-terminal region of the human immunodeficiency virus (HIV) gp120 envelope protein has been identified with antisera from HIV-seropositive subjects and a synthetic peptide (SP-22) containing 15 amino acids from this region (Ala-Pro-Thr-Lys-Ala-Lys-Arg-Arg-Val-Val-Gln-Arg-Glu-Lys-Arg). Peptide SP-22 absorbed up to 100% of anti-gp120 antibody reactivity from select HIV+ patient sera in immunoblot assays and up to 79% of serum anti-gp120 antibody reactivity in competition RIA. In RIA, 45% of HIV-seropositive subjects had antibodies that bound to peptide SP-22. Human anti-SP-22 antibodies that bound to and were eluted from an SP-22 affinity column reacted with gp120 in RIA and immunoblot assays but did not neutralize HIV or inhibit HIV-induced syncytium formation in vitro, even though these antibodies comprised 70% of all anti-gp120 antibodies in the test serum. In contrast, the remaining 30% of SP-22 nonreactive anti-gp120 antibodies did not react with gp120 in immunoblot assays but did not react in RIA and neutralized HIV in vitro. Thus, approximately 50% of HIV-seropositive patients make high titers of nonneutralizing antibodies to an immunodominant antigen on gp120 defined by SP-22. Moreover, the COOH terminus of gp120 contains the major antigen or antigens identified by human anti-gp120 antibodies in immunoblot assays.

A rapid, simple, reproducible and semi-quantitative assay to measure neutralizing antibodies has been developed. It employs a unique cell line which is exquisitively sensitive to infection with all HIV isolates tested. The assay is amenable to microtiter formulation as well as analysis by automation.

1. The adenosine analog toyocamycin selectively inhibits synthesis of rRNA in mammalian cells, blocking the conversion of the 45-S precursor molecule. In contrast, toyocamycin inhibition in chick embryo cells is less specific, and some rRNA synthesis continues even at relatively high concentrations of toyocamycin. 2. In untreated chick cells rRNA formation was like that in L cells. A broad range of heterogeneous chick cell nuclear RNA's were synthesized most rapidly, followed by the rRNA precursor of about 45 S. This was cleaved to forms of about 38 S, 32 S and, finally 28 S and 18 S. 3. The similarity of normal pathways suggests that the differences in species response to toyocamycin probably reflect different enzyme sensitivities. The inhibitory effect in chick cells appears to be on both synthesis and conversion of the 45-S precursor RNA. 4. The toyocamycin-resistant nucleolar RNA formed in chick cells infected with MC29 virus above that in noninfected cells was principally rRNA precursor with no unequivocal evidence of virus-specific RNA. 5. The differential thermal phenol procedure for separate extraction of nuclear and cytoplasmic RNA's was modified for application to chick cells.