Virus neutralization assays for human respiratory syncytial virus using airway organoids

Neutralizing antibodies are considered a correlate of protection against severe human respiratory syncytial virus (HRSV) disease. Currently, HRSV neutralization assays are performed on immortalized cell lines like Vero or A549 cells. It is known that assays on these cell lines exclusively detect neutralizing antibodies (nAbs) directed to the fusion (F) protein. For the detection of nAbs directed to the glycoprotein (G), ciliated epithelial cells expressing the cellular receptor CX3CR1 are required, but generation of primary cell cultures is expensive and labor-intensive. Here, we developed a high-throughput neutralization assay based on the interaction between clinically relevant HRSV grown on primary cells with ciliated epithelial cells, and validated this assay using a panel of infant sera. To develop the high-throughput neutralization assay, we established a culture of differentiated apical-out airway organoids (Ap-O AO). CX3CR1 expression was confirmed, and both F- and G-specific monoclonal antibodies neutralized HRSV in the Ap-O AO. In a side-by-side neutralization assay on Vero cells and Ap-O AO, neutralizing antibody levels in sera from 125 infants correlated well, although titers on Ap-O AO were consistently lower. We speculate that these lower titers might be an actual reflection of the neutralizing antibody capacity in vivo. The organoid-based neutralization assay described here holds promise for further characterization of correlates of protection against HRSV disease.</p

Transcranial sonography for diagnosis of Parkinson's disease

<p>Abstract</p> <p>Background</p> <p>In idiopathic Parkinson's disease (IPD) transcranial sonography (TCS) represents an alternative diagnostic method to verify clinical diagnosis. Although the phenomenon of an increased echogenicity of the Substantia nigra (SN) is well known this method is still not widly used in the diagnostic workup. Until now reliability of this method is still a matter of debate, partly because data only existed from a few laboratories using the same ultrasound machine. Therefore our study was conducted to test the reliability of this method by using a different ultrasound device and examining a large population of control and IPD subjects by two examiners to calculate interobserver reliability.</p> <p>Method</p> <p>In this study echogenicity of SN was examined in 199 IPD patients and 201 control subjects. All individuals underwent a neurological assessment including Perdue pegboard test and Webster gait test. Using a Sonos 5500 ultrasound device area of SN was measured, echogenicity of raphe, red nuclei, thalamus, caudate and lenticular nuclei, width of third and lateral ventricle were documented.</p> <p>Results</p> <p>We found a highly characteristic enlargement of the SN echogenic signal in IPD. The cut-off value for the SN area was established using a ROC curve with a sensitivity of 95% corresponding to an area of SN of 0.2 cm²and was found to be equivalent to the cut-off values of other studies using different ultrasound devices.</p> <p>Conclusions</p> <p>Our study shows that TCS is a reliable and highly sensitive tool for differentiation of IPD patients from individuals without CNS disorders.</p

Age-Related Immunity to Meningococcal Serogroup C Vaccination: An Increase in the Persistence of IgG2 Correlates with a Decrease in the Avidity of IgG

Contains fulltext : 97618.pdf (publisher's version ) (Open Access)Background All children and adolescents between 1 and 19 years of age in The Netherlands received a single meningococcal serogroup C conjugate (MenCC) vaccine in 2002. During follow-up 4–5 years later, the persistence of MenC polysaccharide-specific IgG was found to be dependent on age of vaccination with higher IgG levels in the oldest immunized age categories. Methods and Findings Two cross-sectional population-based serum banks, collected in 1995/1996 and in 2006/2007, were used for this study. We measured MenC polysaccharide-specific IgM, the IgG1 and IgG2 subclasses and determined the avidity of the IgG antibodies. We report that the age-related persistence of IgG after immunization with the MenCC vaccine seemed to result from an increase of IgG2 levels with age, while IgG1 levels remained stable throughout the different age-cohorts. Furthermore, an age-related increase in IgM levels was observed, correlating with the persistence of IgG antibodies with age. It is noteworthy that the increase in IgG2 correlated with a reduced IgG-avidity with age. Conclusion These date indicate that the classical characteristics of a T-cell-dependent antibody response as elicited by protein based vaccines might not be completely applicable when conjugate vaccines are administered to older children and adolescents up to 18 years of age. The response elicited by the MenCC vaccine seemed to be more a mixture of both T cell dependent and T cell independent responses in terms of humoral immunological characteristics

Immunity against Neisseria meningitidis Serogroup C in the Dutch Population before and after Introduction of the Meningococcal C Conjugate Vaccine

Contains fulltext : 88187.pdf (publisher's version ) (Open Access)BACKGROUND: In 2002 a Meningococcal serogroup C (MenC) conjugate vaccine, with tetanus toxoid as carrier protein, was introduced in the Netherlands as a single-dose at 14 months of age. A catch-up campaign was performed targeting all individuals aged 14 months to 18 years. We determined the MenC-specific immunity before and after introduction of the MenC conjugate (MenCC) vaccine. METHODS AND FINDINGS: Two cross-sectional population-based serum banks, collected in 1995/1996 (n = 8539) and in 2006/2007 (n = 6386), were used for this study. The main outcome measurements were the levels of MenC polysaccharide(PS)-specific IgG and serum bactericidal antibodies (SBA) after routine immunization, 4-5 years after catch-up immunization or by natural immunity. There was an increasing persistence of PS-specific IgG and SBA with age in the catch-up immunized cohorts 4-5 years after their MenCC immunization (MenC PS-specific IgG, 0.25 microg/ml (95%CI: 0.19-0.31 microg/ml) at age 6 years, gradually increasing to 2.34 microg/ml,(95%CI: 1.70-3.32 microg/ml) at age 21-22 years). A comparable pattern was found for antibodies against the carrier protein in children immunized above 9 years of age. In case of vaccination before the age of 5 years, PS-specific IgG was rapidly lost. For all age-cohorts together, SBA seroprevalence (> or =8) increased from 19.7% to 43.0% in the pre- and post-MenC introduction eras, respectively. In non-immunized adults the SBA seroprevalence was not significantly different between the pre- and post-MenC introduction periods, whereas PS-specific IgG was significantly lower in the post-MenC vaccination (GMT, age > or =25 years, 0.10 microg/ml) era compared to the pre-vaccination (GMT, age > or =25 years, 0.43 microg/ml) era. CONCLUSION: MenCC vaccination administered above 5 years of age induced high IgG levels compared to natural exposure, increasing with age. In children below 14 months of age and non-immunized cohorts lower IgG levels were observed compared to the pre-vaccination era, whereas functional levels remained similar in adults. Whether the lower IgG poses individuals at increased risk for MenC disease should be carefully monitored. Large-scale introduction of a MenCC vaccine has led to improved protection in adolescents, but in infants a single-dose schedule may not provide sufficient protection on the long-term and therefore a booster-dose early in adolescence should be considered

Taking Two-Photon Excitation to Exceptional Path-Lengths in Photonic Crystal Fiber

The well-known, defining feature of two-photon excitation (TPE) is the tight, three-dimensional confinement of excitation at the intense focus of a laser beam. The extremely small excitation volume, on the order of 1 μm3 (1 femtoliter), is the basis of far-reaching applications of TPE in fluorescence imaging, photodynamic therapy, nanofabrication, and three-dimensional optical memory. Paradoxically, the difficulty of detecting photochemical events in such a small volume is a barrier to the development of the two-photon-activated molecular systems that are essential to the realization of such applications. We show, using two-photon-excited fluorescence to directly visualize the excitation path, that confinement of both laser beam and sample solution within the 20 μm hollow core of a photonic crystal fiber permits TPE to be sustained over an extraordinary path-length of more than 10 cm, presenting a new experimental paradigm for ultrasensitive studies of two-photon-induced processes in solution. (Figure Presented).We are grateful to the Koerber Foundation (Germany) and the EPSRC (UK) for financial support. G.O.S.W. is a recipient of an EPSRC Prize Postdoctoral Fellowshi

Trade in live reptiles, its impact on wild populations, and the role of the European market

Of the 10,272 currently recognized reptile species, the trade of fewer than 8% are regulated by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) and the European Wildlife Trade Regulations (EWTR). However, the International Union for Conservation of Nature (IUCN) Red List has assessed 45% of the world's reptile species and determined that at least 1390 species are threatened by “biological resource use”. Of these, 355 species are intentionally targeted by collectors, including 194 non-CITES-listed species. Herein we review the global reptile pet trade, its impacts, and its contribution to the over-harvesting of species and populations, in light of current international law. Findings are based on an examination of relevant professional observations, online sources, and literature (e.g., applicable policies, taxonomy [reptile database], trade statistics [EUROSTAT], and conservation status [IUCN Red List]). Case studies are presented from the following countries and regions: Australia, Central America, China, Galapagos Islands (Ecuador), Germany, Europe, India, Indonesia (Kalimantan), Islamic Republic of Iran, Japan, Madagascar, Mexico, New Zealand, the Philippines, South Africa, Sri Lanka, Vietnam, Western Africa, and Western Asia. The European Union (EU) plays a major role in reptile trade. Between 2004 and 2014 (the period under study), the EU member states officially reported the import of 20,788,747 live reptiles. This review suggests that illegal trade activities involve species regulated under CITES, as well as species that are not CITES-regulated but nationally protected in their country of origin and often openly offered for sale in the EU. Further, these case studies demonstrate that regulations and enforcement in several countries are inadequate to prevent the overexploitation of species and to halt illegal trade activities

Generation and spectroscopic and kinetic characterization of methoxy-substituted phenoxyl radicals in solution and on paper

A number of methoxy-substituted phenoxyl radicals have been generated and characterized by laser flash photolysis techniques in solution and on paper. The radicals have been produced by three routes in solution: hydrogen abstraction from phenols by tert-butoxyl radical, photolysis of ?-aryloxyacetophenones, and direct excitation of phenols. Most of the phenoxyl radicals studied have a characteristic absorption near 400 nm; the ortho-substituted radicals have an additional broad absorption in the visible in non-hydroxylic solvents (e.g., 650 nm for 2-methoxyphenoxyl radical). The relative intensities of the 400 nm and the long-wavelength bands vary with substitution pattern and solvent, with the long wavelength band being much weaker in hydroxylic solvents. Direct excitation of phenols provides an alternate source of phenoxyl radicals in some cases. However, for 4-methoxyphenol there are overlapping absorptions due to triplet phenol and phenol radical cation, both of which absorb in the 400-460 nm region. The 4-methoxyphenol radical cation is formed in a biphotonic process and decays by deprotonation to give the phenoxyl radical. The 4-methoxyphenoxyl radical can also be generated on paper from either the ketone or phenol precursor and decays with complex kinetics that depend on the precursor loading and laser intensity. There is no evidence for the formation of significant amounts of long-lived ketone or phenol triplets on paper. The decay kinetics for the phenoxyl radical are modified by the addition of the radical scavenger dithioerythritol. The results indicate both static and dynamic components to the radical scavenging process. The generation of phenoxyl radicals on paper in the presence and absence of scavengers provides the basis for the development of a new method for screening potential photoyellowing inhibitors and understanding their mode of action.On a g\ue9n\ue9r\ue9 un certain nombre de radicaux ph\ue9noxyles substitu\ue9s par des groupes m\ue9thoxyles et on les a caract\ue9ris\ue9s par les techniques de la photolyse laser \ue9clair en solution et sur papier. On a produit les radicaux par trois m\ue9thodes en solution: utilisation du radical tert-butyloxyle pour l'enl\ue8vement d'hydrog\ue9ne \ue0 partir de ph\ue9nols, la photolyse d' ?-aryloxyac\ue9toph\ue9nones et l'excitation directe de ph\ue9nols. La plupart des radicaux ph\ue9noxyles \ue9tudi\ue9s pr\ue9sentent une bande d'absorption caract\ue9ristique vers 400 nm; les radicaux substitu\ue9s en ortho pr\ue9sentent en plus une large bande d'absorption dans le visible dans les solvants non hydroxyliques (environ 650 nm pour le radical 2-m\ue9thoxyph\ue9noxyle). Les intensit\ue9s relatives des bandes \ue0 400 nm et \ue0 longueur d'onde plus \ue9lev\ue9e varient avec le patron de substitution et avec le solvant; la bande \ue0 longueur d'onde plus \ue9lev\ue9e est la plus faible dans les solvants hydroxyliques. Dans certains cas, l'excitation directe des ph\ue9nols est une voie alternative d'obtention de radicaux ph\ue9noxyles. Toutefois, pour le 4-m\ue9thoxyph\ue9nol, il y a superposition de bandes dues au ph\ue9nol triplet et au cation radical ph\ue9nol qui absorbent tous les deux dans la r\ue9gion de 400 \ue0 460 nm. Le cation radical 4-m\ue9thoxyph\ue9nol se forme par un processus biphotonique et il se d\ue9compose par d\ue9protonation pour donner le radical ph\ue9noxyle. Le radical 4-m\ue9thoxyph\ue9noxyle peut aussi \ueatre obtenu sur papier \ue0 partir soit de la c\ue9tone ou du ph\ue9nol appropri\ue9 et il se d\ue9compose avec une cin\ue9tique complexe qui d\ue9pend de la concentration du pr\ue9curseur et de l'intensit\ue9 du laser. Sur le papier, on n'a obtenu aucune indication pouvant sugg\ue9rer qu'il y a formation de quantit\ue9s appr\ue9ciables d'\ue9tats triplets de longue vie de c\ue9tone ou de ph\ue9nol. La cin\ue9tique de la d\ue9composition du radical ph\ue9noxyle est modifi\ue9e par l'addition du pi\ue9ge \ue0 radical dithio\ue9rythritol. La g\ue9n\ue9ration de radicaux ph\ue9noxyles sur papier, en pr\ue9sence ou en l'absence de pi\ue9ges \ue0 radicaux, fournit une base pour le d\ue9veloppement d'une nouvelle m\ue9thode d'\ue9valuer les inhibiteurs potentiels de jaunissement photochimique et de comprendre leur mode d'action.NRC publication: Ye