Measuring vaccine effectiveness against persistent HPV infections: a comparison of different statistical approaches.

BACKGROUND: Persistent high-risk human papillomavirus (HPV) infection is endorsed by the World Health Organization as an intermediate endpoint for evaluating HPV vaccine effectiveness/efficacy. There are different approaches to estimate the vaccine effectiveness/efficacy against persistent HPV infections. METHODS: We performed a systematic literature search in Pubmed to identify statistical approaches that have been used to estimate the vaccine effectiveness/efficacy against persistent HPV infections. We applied these methods to data of a longitudinal observational study to assess their performance and compare the obtained vaccine effectiveness (VE) estimates. RESULTS: Our literature search identified four approaches: the conditional exact test for comparing two independent Poisson rates using a binomial distribution, Generalized Estimating Equations for Poisson regression, Prentice Williams and Peterson total time (PWP-TT) and Cox proportional hazards regression. These approaches differ regarding underlying assumptions and provide different effect measures. However, they provided similar effectiveness estimates against HPV16/18 and HPV31/33/45 persistent infections in a cohort of young women eligible for routine HPV vaccination (range VE 93.7-95.1% and 60.4-67.7%, respectively) and seemed robust to violations of underlying assumptions. CONCLUSIONS: As the rate of subsequent infections increased in our observational cohort, we recommend PWP-TT as the optimal approach to estimate the vaccine effectiveness against persistent HPV infections in young women. Confirmation of our findings should be undertaken by applying these methods after longer follow-up in our study, as well as in different populations.

Intravenous dexamethasone fails to prolong psoas compartment block when assessed by objective pinprick sensory testing: a prospective, randomised, dose-dependent, placebo-controlled equivalency trial.

BACKGROUND: Recent studies have concluded that i.v. dexamethasone can prolong the duration of peripheral nerve blockade. We hypothesized that a 4 mg dose would equally prolong the duration of psoas compartment blocks (PCBs) when compared with 8 mg, and that both doses would prolong the duration when compared with placebo. METHODS: This was a prospective, randomized, placebo-controlled, dose-dependent, equivalency trial with 115 patients undergoing total hip arthroplasty. The patients received a PCB. Subsequently, 15 patients received i.v. normal saline (placebo), 50 patients received i.v. dexamethasone 4 mg, and 50 patients received i.v. dexamethasone 8 mg. The primary outcome was the duration in hours of PCB, determined by serial pinprick assessments. Secondary outcomes included pain scores, time to first analgesic, and opioid consumption. An intention-to-treat-analysis (ITA) and per-protocol analysis (PPA) were performed. RESULTS: The ITA showed that block duration in the 4 and 8 mg groups was equivalent [mean (standard deviation), 18.5 h (8.0) vs 18.1 h (7.1)]. However, neither group differed from placebo [19.6 h (6.7), (4 mg vs placebo), P=0.97; (8 mg vs placebo), P=0.77)]. Postoperative pain scores and opioid consumption were not different between groups. Time to first analgesic was not different between the 4 and 8 mg groups, or the 4 mg and placebo groups. The 8 mg group, however, had a longer time to first analgesic (median of 533 vs 432 min, P=0.047) when compared with placebo, although the significance was not observed in the PPA (P=0.058). CONCLUSIONS: I.V. dexamethasone did not prolong PCB when duration was objectively assessed, or decrease total opioid consumption. However, dexamethasone 8 mg prolonged the time to first analgesic. CLINICAL TRIAL REGISTRATION: NCT 02464176.

Accuracy of first-trimester ultrasound in diagnosis of tubal ectopic pregnancy in the absence of an obvious extrauterine embryo: systematic review and meta-analysis.

OBJECTIVES: To determine the accuracy of ultrasound in the diagnosis of a tubal ectopic pregnancy in the absence of an obvious extrauterine embryo. METHODS: This was a systematic review conducted in accordance with the PRISMA statement and registered with PROSPERO. We searched MEDLINE, EMBASE and The Cochrane Library for relevant citations from database inception to July 2014. Studies were selected in a two-stage process and their data extracted by two reviewers. Accuracy measures were calculated for each ultrasound sign, i.e. empty uterus, pseudosac, adnexal mass and free fluid in the pouch of Douglas, alone and in various combinations. Individual study estimates were plotted in summary receiver-operating characteristics curves and forest plots for examination of heterogeneity. The quality of included studies was assessed. RESULTS: Thirty-one studies including 5858 women were selected from 19,959 citations. Following meta-analysis, an empty uterus on ultrasound was found to predict an ectopic pregnancy with a sensitivity of 81.1% (95% CI, 42.1-96.2%) and specificity of 79.5% (95% CI, 68.9-87.1%). The corresponding performance of the pseudosac, adnexal mass and free fluid were: 5.5% (95% CI, 3.3-9.0%) and 94.2% (95% CI, 75.9-98.8%); 63.5% (95% CI, 48.5-76.3%) and 91.4% (95% CI, 83.6-95.7%); and 47.2% (95% CI, 33.2-61.7%) and 92.3% (95% CI, 85.6-96.0%), respectively. CONCLUSION: Visualization of an empty uterus, adnexal mass, free fluid or a pseudosac has poor sensitivity for the diagnosis of a tubal pregnancy when an obvious extrauterine embryo is absent, but it has good specificity. We can therefore infer that ultrasound is more useful for 'ruling in' a tubal pregnancy than 'ruling out' one. However, the findings were limited by the poor quality of some included studies and heterogeneity in the index test and reference standard.

The impact of insecticide-treated cloth targets on the survival of Stegomyia polynesiensis (= Aedes polynesiensis) under laboratory and semi-field conditions in French Polynesia.

The impact of deltamethrin-impregnated cloth targets on Stegomyia polynesiensis (= Aedes polynesiensis) (Marks) (Diptera: Culicidae) was assessed under laboratory and semi-field settings in French Polynesia. Stegomyia polynesiensis females were released into small laboratory cages and large field cages containing either a deltamethrin-treated or an untreated navy blue cloth, and mosquito knock-down and mortality were assessed. The 24-h mortality rate in mosquitoes exposed to the insecticide-treated target in small cages was 98.0%. These mosquitoes also demonstrated significantly higher levels of knock-down than those exposed to the untreated target. Mortality in field cages was assessed at 24 and 48 h. The 24-h mortality rate in mosquitoes exposed to the control target was 31.2%, whereas that in those exposed to the deltamethrin-treated target was 54.3%. The 48-h mortality rate was also elevated in mosquitoes exposed to the deltamethrin-treated target, but this result did not differ significantly from that observed in mosquitoes exposed to the control target. The significant suppression of female S. polynesiensis by deltamethrin-treated resting targets in this study indicates that these targets could play a role in the control of an important disease vector in the South Pacific region.

Accuracy of first-trimester ultrasound in diagnosis of intrauterine pregnancy prior to visualization of the yolk sac: a systematic review and meta-analysis.

OBJECTIVES: To evaluate the diagnostic accuracy of ultrasound in predicting the location of an intrauterine pregnancy before visualization of the yolk sac is possible. METHODS: This was a systematic review conducted in accordance with the PRISMA statement and registered with PROSPERO. We searched MEDLINE, EMBASE and The Cochrane Library for relevant citations. Studies were selected in a two-stage process and their data extracted by two reviewers. Accuracy measures were calculated for each ultrasound sign, i.e. gestational sac, double decidual sac sign, intradecidual sign, chorionic rim sign and yolk sac. Individual study estimates were plotted in summary receiver-operating characteristics curves and forest plots for examination of heterogeneity. The quality of included studies was assessed. RESULTS: Seventeen studies including 2564 women were selected from 19 959 potential papers. Following meta-analysis, the presence of a gestational sac on ultrasound examination was found to predict an intrauterine pregnancy with a sensitivity of 52.8% (95% CI, 38.2-66.9%) and specificity of 97.6% (95% CI, 94.3-99.0%). The corresponding performance of the double decidual sac sign, intradecidual sign, chorionic rim sign and yolk sac were: 81.8% (95% CI, 68.1-90.4%) and 97.3% (95% CI, 76.1-99.8%); 66.1% (95% CI, 58.9-72.8%) and 100% (95% CI, 91.0-100%); 79.9% (95% CI, 73.0-85.7%) and 97.1% (95% CI, 89.9-99.6%); and 42.2% (95% CI, 27.7-57.9%) and 100% (95% CI, 54.1-100%), respectively. CONCLUSION: Visualization of a gestational sac, double decidual sac sign, intradecidual sign or chorionic rim sign increases the probability of an intrauterine pregnancy but is not as accurate for diagnosis as the detection of the yolk sac. However, the findings were limited by the small number and poor quality of the studies included and heterogeneity in the index test and reference standard.

Determinants of male Aedes aegypti and Aedes polynesiensis (Diptera: Culicidae) response to sound: efficacy and considerations for use of sound traps in the field.

Understanding the mating competitiveness of male mosquitoes in field settings is essential to programs relying on the mass release of modified male mosquitoes, yet studies on male ecology have been hampered by the lack of a convenient trapping method. An existing promising method makes use of the innate attraction of males to female flight tones. Here, we present laboratory, greenhouse, and field experiments on the efficacy of sound traps for the collection of Aedes aegypti (L.) and Aedes polynesiensis Marks, and laboratory experiments with Ae. aegypti on the effects of male age, size, and mating status on responsiveness to a range of frequencies. Age and mating status influenced the overall responsiveness to sound, whereas male size did not. There were no interactions between these factors and sound frequency. A Centers for Disease Control and Prevention miniature light trap modified to produce a tone of 465 Hz collected 76.2% of Ae. aegypti males in laboratory cages, and 49.7% of males in a greenhouse enclosure. In two sets of experiments in laboratory cages, 50.8 and 46.5% of male Ae. polynesiensis were captured with a trap producing a tone of 440 Hz. In the field, CDC miniature light traps or BG-Sentinel traps fitted with a portable speaker producing tones of 440 or 465 Hz captured significantly more male Ae. polynesiensis when placed near a male swarm than did traps that did not produce sound. When the trap was placed at a distance of 16.5 m from the nearest swarm, there was no significant difference in the number of males caught between control and sound-producing traps. The numbers of Ae. aegypti males captured were low under all circumstances in the field.

Swarming behavior of Aedes polynesiensis (Diptera: Culicidae) and characterization of swarm markers in American Samoa.

We characterize the swarming behavior of male Aedes polynesiensis (Marks) in American Samoa. Instead of swarming around a blood host, males used the base of certain trees as a marker. Repeated sampling proved nondestructive and allowed us to investigate the impact of static (e.g., tree species) and dynamic (e.g., barometric pressure) characters on the likelihood of swarm presence and intensity. Tree circumference and oviposition activity (number of Ae. polynesiensis reared from oviposition cups) were significant positive predictors of the number of males in a swarm. Tree circumference and diameter were significantly positively associated, and canopy height was significantly negatively associated, with swarm occurrence. Comparisons between males swarming early and late during the swarming period allowed for insight into swarm composition in terms of male size and the amount of putative fluid (e.g., nectar) in the crop, indicators of energetic reserves. Males collected during the late period had significantly larger wings and less crop contents than did males of the early cohort. Because the ecology of male Ae. polynesiensis remains understudied, we consider how the current results could facilitate further studies related to applied autocidal strategies as well as the evolution of host-based mating behavior.

Landing response of Aedes (Stegomyia) polynesiensis mosquitoes to coloured targets.

Aedes polynesiensis Marks (Diptera: Culicidae) is the primary vector of lymphatic filariasis (LF) in the island countries and territories of the South Pacific. In the development of a novel control tool, the response of Ae. polynesiensis to six different colours (three solid fabrics, two patterned fabrics and a plastic tarp) was measured using a digital photographic system. Adult mosquitoes were placed into an environmental chamber and allowed to choose between a white target and one of six experimental targets. Mosquito landing frequency and landing duration were calculated. Adult female Ae. polynesiensis preferred all of the experimental targets to the white control target. Mosquito landing frequency was highest for the solid targets (black, navy blue and red) followed in turn by the two colour pattern targets and the polyethylene target. Mosquito landing duration was greater for experimental targets when compared with white control targets. Mosquito landing frequencies did not change over time during the course of the assay. The response of male Ae. polynesiensis was also measured when exposed to a 100% cotton black target. Male mosquitoes preferred the black target to the white control target, although at levels lower than that observed in female mosquitoes. The results suggest that future investigations evaluating the visual responses of Ae. polynesiensis mosquitoes are warranted, with a special emphasis on semi-field and field-based experiments.

Infection, growth and maintenance of Wolbachia pipientis in clonal and non-clonal Aedes albopictus cell cultures.

Insect cell lines provide useful in vitro models for studying biological systems, including interactions between mosquitoes and obligate intracellular endosymbionts such as Wolbachia pipientis. The Aedes albopictus Aa23 cell line was the first cell line developed to allow examination of Wolbachia infections. However, Wolbachia studies using Aa23 can be complicated by the presence of different cell types in the cell line and the substantial temporal variation in infection level. Two approaches were examined to ameliorate infection variability. In the first approach, multiple Aa23 passaging regimes were tested for an effect on infection variability. Fluorescence in situ hybridization (FISH) staining was used to characterize Wolbachia infection level over time. The results demonstrate an impact of passaging method on Wolbachia infection level, with some methods resulting in loss of infection. None of the passaging methods succeeded in effectively mitigating infection level variation. In a second approach, the clonal C7-10 A. albopictus cell line was infected with Wolbachia from Aa23 cells and Drosophila simulans (Riverside), resulting in cell lines designated C7-10B and C7-10R, respectively. Characterization via FISH staining showed greater stability and uniformity of Wolbachia infection in C7-10R relative to the infection in C7-10B. Characterization of the Aa23, C7-10B and C7-10R lines is discussed as a tool for the study of Wolbachia-host cell interactions.

Methodology and development of a high-protein plant-based cheese alternative.

Animal-based food products, such as meat and dairy, contribute the most to greenhouse gas emissions in the food sector. This, coupled with the demonstrably worsening climate crisis, means that there needs to be a shift to more sustainable alternatives in the form of plant-based foods. In particular, the plant-based cheese alternative industry is relevant, as the products lack critical functionalities and nutrition compared to their dairy-based counterparts. Waxy starch, plant-protein isolate, and coconut oil were combined to create a novel high-protein (18% w/w) plant-based cheese alternative. We determined that when using native waxy starch, we can enhance its existing viscoelastic properties by modulating gelatinization through adding plant protein and fat. Texture profile analysis indicated that the cheese analogues could reach hardness levels of 15-90N, which allowed samples to be tailored to a broader range of dairy products. We determined that plant proteins and fat can behave as particulate fillers, enhance network strength, and create strategic junction points during starch retrogradation. The degree of melt and stretch of the high-protein plant-based analogues were 2-3 times greater than those observed for commercial plant-based cheese alternatives and significantly more similar to dairy cheese. The rheological melting kinetics saw that the high-protein plant-based cheese alternative displayed more viscous properties with increasing temperature. Tan δ (G"/G') at 80 °C was used as an indicator for sample meltability where, values ≥1 indicate better melt and more viscous systems. The high-protein plant-based cheese alternative reached Tan δ values upwards to 0.7, whereas commercial plant-based cheese alternatives only reached tan δ values around 0.1. Ultimately, the novel high-protein plant-based cheese alternative demonstrates the use of simple ingredients to form complex food systems.

Wolbachia effects on host fitness and the influence of male aging on cytoplasmic incompatibility in Aedes polynesiensis (Diptera: Culicidae).

The endosymbiotic bacteria Wolbachia manipulate host reproduction by inducing a form of sterility known as cytoplasmic incompatibility (CI), promoting the invasion of infection into natural host populations. CI has received attention for use in applied strategies to control insect vectors of disease. Thus, to understand both naturally occurring Wolbachia invasions and evaluate potential applied strategies, it is important to understand Wolbachia interactions with its host, including impacts on fitness and the CI level. In this study, we examined for an effect of Wolbachia on survivorship, developmental time, sex ratio, longevity, fecundity, and egg hatch of Aedes polynesiensis Marks, which is the primary vector of Wuchereria bancrofti in the South Pacific. In this study, we have compared strains of A. polynesiensis that are naturally and artificially infected with Wolbachia and additional strains that are aposymbiotic (Wolbachia removed to generate an uninfected strain). Artificially infected strains were observed to have increased larval mortality and decreased adult longevity when compared with aposymbiotic strains. Naturally infected strains were observed to have decreased larval mortality, pupal mortality, increased adult longevity, and a larger adult size when compared with aposymbiotic strains. Artificially infected males that were 4 wk old were able to induce high rates of CI, similar to young males. We discuss the results in relation to the natural spread of Wolbachia and Wolbachia-based applied strategies to modify A. polynesiensis populations.

SYTO11 staining vs FISH staining: a comparison of two methods to stain Wolbachia pipientis in cell cultures.

AIMS: The Aedes albopictus C7-10 cell line was infected with Wolbachia strains wRi and wAlbB to create C7-10R and C7-10B cell lines, respectively. We compared two different methods, fluorescence in situ hybridization staining and SYTO11 staining, to describe these new Wolbachia infections in C7-10. METHODS AND RESULTS: Both staining methods were as efficient to stain Wolbachia. A formula was developed to quantify Wolbachia infection. The infection levels in C7-10B and C7-10R differed. The live stain SYTO11 was found to be useful to visualize Wolbachia in replicating host cells. Its potential cytotoxic effect at high concentration was investigated. CONCLUSIONS: C7-10 supported two Wolbachia infections, constituting new tools to study Wolbachia-host interactions. The different infection levels suggest that wRi and wAlbB have different requirements for their survival in C7-10 host cell line. Observation of SYTO11-stained live cells gave new insights on Wolbachia segregation pattern during host cell mitosis. SIGNIFICANCE AND IMPACT OF THE STUDY: Wolbachia-induced phenotypes in their arthropod and worm hosts could potentially be used to control pest populations. However, the mechanisms underlying these phenotypes are difficult to study because of Wolbachia's intracellular lifestyle. The Wolbachia infections in C7-10 described here could be used as in vitro models to investigate Wolbachia biology.

The effect of a new topical local anaesthetic delivery system on forearm skin blood flow reactivity.

Different topical local anaesthetics have varying effects on skin blood flow and vascular reactivity. We compared the vasoactive properties of Rapydan, a new topical local anaesthetic, with those of Ametop and EMLA creams in 20 healthy volunteers. Blood flow and vascular reactivity in the forearm skin were assessed by laser Doppler flowmetry and the transient hyperaemic response ratio respectively, before and after the application of EMLA (for 60 min), Ametop (for 30 and 60 min) and Rapydan (for 30 min). Application of EMLA had no effect on skin blood flow (median (IQR [range]) change from baseline -0.9% (-63 to 414 [-38.5 to 51.3] %, p = 1.0)) or mean (SD) transient hyperaemic response ratio (from 2.86 (0.86) to 3.17 (1.3), p = 0.38). The application of Ametop for 60 min produced a greater median (IQR [range]) increase in blood flow from baseline (508 (-55 to 998 [148-649]) %) than Rapydan applied for 30 min 160 (-77 to 997 [45-301]) %, p = 0.001), and a similar decrease in mean (SD) transient hyperaemic response ratio (from 2.69 (1.16) to 1.08 (0.26) and from 2.83 (0.84) to 1.49 (0.93) respectively, p = 0.57).

How mechanistic in silico modelling can improve our understanding of TB disease and treatment.

TB is one of the top 10 causes of death worldwide and the leading cause of death from a single infectious agent. Decreasing the length of time for TB treatment is an important step towards the goal of reducing mortality. Mechanistic in silico modelling can provide us with the tools to explore gaps in our knowledge, with the opportunity to model the complicated within-host dynamics of the infection, and simulate new treatment strategies. Significant insight has been gained using this form of modelling when applied to other diseases - much can be learned in infection research from these advances.

Intention of parents to have male children vaccinated with the human papillomavirus vaccine.

BACKGROUND: Although already approved for use in males in some jurisdictions, there is little information about parental attitudes toward having their sons receive the human papillomavirus (HPV) vaccine. The goal of this study was to ascertain parental intentions to vaccinate their sons with an HPV vaccine and to determine factors that predict this intention. METHODS: Parents of children aged 8-18 years were recruited from across Canada through random digit dialling. Participants were asked to respond to a series of questions in the context of a Grade 6 (age 11/12 years old), publicly funded school-based HPV vaccine programme, including their intention to vaccinate their sons with the HPV vaccine. Parents were also asked about a series of characteristics thought to predict intention to vaccinate as well as demographic characteristics. Backwards logistic regression was conducted to calculate adjusted odds ratios (AOR) to identify the factors that are predictive of parents' intention to vaccinate their son(s) against HPV. RESULTS: Of the 1381 respondents with male children, 67.8% (95% CI 65.3 to 70.3) intend to vaccinate their son(s) against HPV. Parents who had positive attitudes toward vaccines and the HPV vaccine in particular (AOR 41.5, 95% CI 9.5 to 181.7), parents who were influenced by subjective norms (AOR 7.8, 95% CI 5.8 to 10.5), parents who felt that the vaccine had limited influence on sexual behaviour (AOR 2.3, 95% CI 1.6 to 3.3) and parents who were aware of HPV (AOR 1.4, 95% CI 1.1 to 2.0) were significantly more likely to report an intention to vaccinate boys against HPV. In contrast, residence in British Columbia compared to Atlantic Canada (AOR 0.4, 95% CI 0.2 to 0.8) and higher education (AOR 0.7, 95% CI 0.5 to 0.9) were negatively associated with intention to vaccinate. Parents who reported an intention to vaccinate their daughters were also highly likely to report an intention to vaccinate their sons (kappa = 0.9, p<0.001). DISCUSSION: The majority of Canadian parents would intend to have their male children receive the HPV vaccine in the context of a publicly funded school-based immunisation programme. Overall attitudes toward vaccine, recommendations from health professionals and impact of the vaccine on sexual practices are important predictors of intention to have a male child receive the HPV vaccine.

Evidence for a genomic imprinting sex determination mechanism in Nasonia vitripennis (Hymenoptera; Chalcidoidea).

Five different models have been proposed for the sex determination mechanism of Chalcidoidea (Hymenoptera). Except for the most recently proposed model (genomic imprinting sex determination; GISD), each of these models has required complicating additions to explain observed phenomena. This report provides the first experimental test of the GISD model while simultaneously examining the four previously proposed models of sex determination. This test utilizes the parasitic wasp Nasonia vitripennis, crossing polyploid females with males harboring the paternal sex ratio chromosome (PSR). The results of this study support the GISD model as the mechanism of sex determination in Chalcidoidea. Specifically, crosses demonstrate that sex determination is independent of embryonic heterozygosity, ploidy, and gametic syngamy but is directly correlated with the embryonic presence of correctly imprinted chromosomes of paternal origin. These crossing experiments also provide information about the poorly characterized mechanisms of PSR, a supernumerary chromosome that induces paternal autosome loss in early embryos. The results demonstrate that the poor transmission of PSR through females is not a result of the ploidy of the host but of an alternative sex-dependent process. Crossing data reveal that PSR consistently induces the loss of the entire paternal complement that it accompanies, regardless of whether this complement is haploid or diploid.

Dominant-negative Ikaros cooperates with BCR-ABL1 to induce human acute myeloid leukemia in xenografts.

Historically, our understanding of mechanisms underlying human leukemogenesis are inferred from genetically engineered mouse models. Relatively, few models that use primary human cells recapitulate the full leukemic transformation as assayed in xenografts and myeloid transformation is infrequent. We report a humanized experimental leukemia model where xenografts develop aggressive acute myeloid leukemia (AML) with disseminated myeloid sarcomas within 4 weeks following transplantation of cord blood transduced with vectors expressing BCR-ABL1 and a dominant-negative isoform of IKAROS, Ik6. Ik6 induced transcriptional programs in BCR-ABL1-transduced progenitors that contained repressed B-cell progenitor programs, along with strong stemness, proliferation and granulocyte-monocytic progenitor (GMP) signatures-a novel combination not induced in control groups. Thus, wild-type IKAROS restrains stemness properties and has tumor suppressor activity in BCR-ABL1-initiated leukemia. Although IKAROS mutations/deletions are common in lymphoid transformation, they are found also at low frequency in AML that progress from a prior myeloproliferative neoplasm (MPN) state. Our experimental system provides an excellent model to gain insight into these rare cases of AML transformation and the properties conferred by IKAROS loss of function as a secondary mutation. More generally, our data points to the importance of deregulated stemness/lineage commitment programs in human myeloid leukemogenesis.

Dynamics of insulin-stimulated translocation of GLUT4 in single living cells visualised using green fluorescent protein.

Insulin increases glucose uptake by promoting the translocation of the GLUT4 isoform of glucose transporters to the plasma membrane. We have studied this process in living single cells by fusing green fluorescent protein (GFP) to the N-terminus (GFP-GLUT4) or C-terminus (GLUT4-GFP), of GLUT4. Both chimeras were expressed in a perinuclear compartment of CHO cells, and in a vesicular distribution through the cytosol. Insulin promoted an increase in plasma membrane fluorescence as a result of net translocation of the chimeras to the cell surface. GLUT4-GFP, but not GFP-GLUT4, was re-internalised upon the removal of insulin suggesting that a critical internalisation signal sequence exists in the N-terminus of GLUT4. The use of GFP thus allows an analysis of GLUT4 trafficking in single living cells.

Selection and optimisation of monoclonal antibodies for a two-site immunoradiometric assay for ACTH.

Four monoclonal antibodies with predominant specificities towards different sequences within the ACTH molecule were investigated in a 2-site immunoradiometric assay (IRMA) for human ACTH. Antibody 3H9 recognises the extreme N-terminal sequence, antibodies 1A12 and 1D1 are specific for the mid N-terminal sequence but differ in that the former cross-reacts with alpha MSH whereas the latter does not, and antibody 2A3 recognises the C-terminal sequence. Combinations of iodinated antibodies with antibodies covalently linked to Sephacryl S300 were tested for their compatibility and potential for a sensitive assay. Two antibody combinations (1D1 plus 3H9 or 1A12) gave no dose-response curve indicating severe steric inhibition, whereas other combinations yielded assays with widely different detection limits (2-2400 ng ACTH/l). The combination of labelled 1D1 and solid-phase 2A3 gave the most sensitive assay and when optimised for antibody concentrations and incubation times the working range was 10-5 X 10(4) ng/l (CV less than 20%). The optimised sequential 2-step IRMA involves incubation of standard or test sample with labelled 1D1 for 18 h at 4 degrees C followed by incubation with solid-phase 2A3 for 2 h at room temperature, after which the labelled complex is separated by the sucrose layering technique. The detection limit of this IRMA was several 100-fold lower than by RIA using the same antibodies. The IRMA detected large molecular weight precursors containing the full ACTH sequence (22 000, 31 000 and 34 000) but not ACTH fragments (1-18, 1-24, 18-39). It is concluded that selected monoclonal antibodies provide a sensitive and rapid 2-site IRMA for intact ACTH and its precursors.