Inhibition of the Nef regulatory protein of HIV-1 by a single-domain antibody

The Nef protein of HIV-1 is important for AIDS pathogenesis, but it is not targeted by current antiviral strategies. Here, we describe a single-domain antibody (sdAb) that binds to HIV-1 Nef with a high affinity (K(d) = 2 × 10(-9)M) and inhibited critical biologic activities of Nef both in vitro and in vivo. First, it interfered with the CD4 down-regulation activity of a broad panel of nef alleles through inhibition of the Nef effects on CD4 internalization from the cell surface. Second, it was able to interfere with the association of Nef with the cellular p21-activated kinase 2 as well as with the resulting inhibitory effect of Nef on actin remodeling. Third, it counteracted the Nef-dependent enhancement of virion infectivity and inhibited the positive effect of Nef on virus replication in peripheral blood mononuclear cells. Fourth, anti-Nef sdAb rescued Nef-mediated thymic CD4(+) T-cell maturation defects and peripheral CD4(+) T-cell activation in the CD4C/HIV-1(Nef) transgenic mouse model. Because all these Nef functions have been implicated in Nef effects on pathogenesis, this anti-Nef sdAb may represent an efficient tool to elucidate the molecular functions of Nef in the virus life cycle and could now help to develop new strategies for the control of AIDS.

Impaired antibody memory to varicella zoster virus in HIV-infected children: low antibody levels and avidity*

HIV-infected children have impaired antibody responses after exposure to certain antigens. Our aim was to determine whether HIV-infected children had lower varicella zoster virus (VZV) antibody levels compared with HIV-infected adults or healthy children and, if so, whether this was attributable to an impaired primary response, accelerated antibody loss, or failure to reactivate the memory VZV response.

Synthesis, binding and cellular uptake properties of oligodeoxynucleotides containing cationic bicyclo-thymidine residues

The synthesis and incorporation into oligodeoxynucleotides of two novel derivatives of bicyclothymidine carrying a cationic diaminopropyl or lysine unit in the C(6′)-β position is described. Compared to unmodified DNA these oligonucleotides show Tm-neutral behavior when paired against complementary DNA and are destabilizing when paired against RNA. Unaided uptake experiments of a decamer containing five lys-bcT units into HeLa and HEK293T cells showed substantial internalization with mostly cytosolic distribution which was not observed in the case of an unmodified control oligonucleotide.

Constraining global methane emissions and uptake by ecosystems

Natural methane (CH4) emissions from wet ecosystems are an important part of today's global CH4 budget. Climate affects the exchange of CH4 between ecosystems and the atmosphere by influencing CH4 production, oxidation, and transport in the soil. The net CH4 exchange depends on ecosystem hydrology, soil and vegetation characteristics. Here, the LPJ-WHyMe global dynamical vegetation model is used to simulate global net CH4 emissions for different ecosystems: northern peatlands (45°–90° N), naturally inundated wetlands (60° S–45° N), rice agriculture and wet mineral soils. Mineral soils are a potential CH4 sink, but can also be a source with the direction of the net exchange depending on soil moisture content. The geographical and seasonal distributions are evaluated against multi-dimensional atmospheric inversions for 2003–2005, using two independent four-dimensional variational assimilation systems. The atmospheric inversions are constrained by the atmospheric CH4 observations of the SCIAMACHY satellite instrument and global surface networks. Compared to LPJ-WHyMe the inversions result in a~significant reduction in the emissions from northern peatlands and suggest that LPJ-WHyMe maximum annual emissions peak about one month late. The inversions do not put strong constraints on the division of sources between inundated wetlands and wet mineral soils in the tropics. Based on the inversion results we diagnose model parameters in LPJ-WHyMe and simulate the surface exchange of CH4 over the period 1990–2008. Over the whole period we infer an increase of global ecosystem CH4 emissions of +1.11 Tg CH4 yr−1, not considering potential additional changes in wetland extent. The increase in simulated CH4 emissions is attributed to enhanced soil respiration resulting from the observed rise in land temperature and in atmospheric carbon dioxide that were used as input. The long-term decline of the atmospheric CH4 growth rate from 1990 to 2006 cannot be fully explained with the simulated ecosystem emissions. However, these emissions show an increasing trend of +3.62 Tg CH4 yr−1 over 2005–2008 which can partly explain the renewed increase in atmospheric CH4 concentration during recent years.

Diagnostic value of animal-side antibody assays for rapid detection of Mycobacterium bovis or Mycobacterium microti infection in South American camelids

Tuberculosis (TB) in South American camelids (SAC) is caused by Mycobacterium bovis or Mycobacterium microti. Two serological methods, rapid testing (RT) and the dual-path platform (DPP) assay, were evaluated using naturally infected SAC. The study population included 156 alpacas and 175 llamas in Great Britain, Switzerland, and the United States. TB due to M. bovis (n = 44) or M. microti (n = 8) in 35 alpacas and 17 llamas was diagnosed by gross pathology examination and culture. Control animals were from herds with no TB history. The RT and the DPP assay showed sensitivities of 71% and 74%, respectively, for alpacas, while the sensitivity for llamas was 77% for both assays. The specificity of the DPP assay (98%) was higher than that of RT (94%) for llamas; the specificities of the two assays were identical (98%) for alpacas. When the two antibody tests were combined, the parallel-testing interpretation (applied when either assay produced a positive result) enhanced the sensitivities of antibody detection to 89% for alpacas and 88% for llamas but at the cost of lower specificities (97% and 93%, respectively), whereas the serial-testing interpretation (applied when both assays produced a positive result) maximized the specificity to 100% for both SAC species, although the sensitivities were 57% for alpacas and 65% for llamas. Over 95% of the animals with evidence of TB failed to produce skin test reactions, thus confirming concerns about the validity of this method for testing SAC. The findings suggest that serological assays may offer a more accurate and practical alternative for antemortem detection of camelid TB.

IgE, IgGa, IgGb and IgG(T) serum antibody levels in offspring of two sires affected with equine recurrent airway obstruction

Equine recurrent airway obstruction (RAO) is a chronic lower airway disease of the horse caused by hypersensitivity reactions to inhaled stable dust, including mould spores such as Aspergillus fumigatus. The goals of this study were to investigate whether total serum IgE levels and allergen-specific IgE and IgG subclasses are influenced by genetic factors and/or RAO and whether quantitative trait loci (QTL) could be identified for these parameters. The offspring of two RAO-affected sires (S1: n=56 and S2: n=65) were grouped by stallion and disease status, and total serum IgE levels and specific IgE, IgGa, IgGb and IgG(T) levels against recombinant Aspergillus fumigatus 7 (rAspf7) were measured by ELISA. A panel of 315 microsatellite markers covering the 31 equine autosomes were used to genotype the stallions and their offspring. A whole-genome scan using half-sib regression interval mapping was performed for each of the IgG and IgE subclasses. There was no significant effect of disease status or sire on total IgE levels, but there was a significant effect of gender and age. rAspf7-specific IgGa levels were significantly higher in RAO-affected than in healthy horses. The offspring of S1 had significantly higher rAspf7-specific IgGa and IgE levels than those of S2. Five QTLs were significant chromosome-wide (P<0.01). QTLs for rAspf7-specific IgGa and IgE were identified on ECA 1, for rAspf7-specific IgGa and IgGb on ECA 24 and for rAspf7 IgGa on ECA 26. These results provide evidence for effects of disease status and genetics on allergen-specific IgGa and IgE.

Somatostatin receptor subtype 2A immunohistochemistry using a new monoclonal antibody selects tumors suitable for in vivo somatostatin receptor targeting

High overexpression of somatostatin receptors in neuroendocrine tumors allows imaging and radiotherapy with radiolabeled somatostatin analogues. To ascertain whether a tumor is suitable for in vivo somatostatin receptor targeting, its somatostatin receptor expression has to be determined. There are specific indications for use of immunohistochemistry for the somatostatin receptor subtype 2A, but this has up to now been limited by the lack of an adequate reliable antibody. The aim of this study was to correlate immunohistochemistry using the new monoclonal anti-somatostatin receptor subtype 2A antibody UMB-1 with the gold standard in vitro method quantifying somatostatin receptor levels in tumor tissues. A UMB-1 immunohistochemistry protocol was developed, and tumoral UMB-1 staining levels were compared with somatostatin receptor binding site levels quantified with in vitro I-[Tyr]-octreotide autoradiography in 89 tumors. This allowed defining an immunohistochemical staining threshold permitting to distinguish tumors with somatostatin receptor levels high enough for clinical applications from those with low receptor expression. The presence of >10% positive tumor cells correctly predicted high receptor levels in 95% of cases. In contrast, absence of UMB-1 staining truly reflected low or undetectable somatostatin receptor expression in 96% of tumors. If 1% to 10% of tumor cells were stained, a weak staining intensity was suggestive of low somatostatin receptor levels. This study allows for the first time a reliable recommendation for eligibility of an individual patient for in vivo somatostatin receptor targeting based on somatostatin receptor immunohistochemistry. Under optimal methodological conditions, UMB-1 immunohistochemistry may be equivalent to in vitro receptor autoradiography.

Diagnostic value of biopsies in identifying cytoplasmic antineutrophil cytoplasmic antibody-negative localized Wegener's granulomatosis presenting primarily with sinonasal disease

A substantial proportion of Wegener's disease (WG) patients present with localized disease of the upper airways, i.e., sinonasal and other ear/nose/throat (ENT) symptoms. Because of the oligosymptomatic presentation a timely diagnosis of this potentially fatal disease is challenging. This study evaluates diagnostic peculiarities between WG in its localized and generalized form of the disease.

[Interdisciplinary AWMF guideline for the treatment of primary antibody deficiencies]

Currently, management of antibody deficient patients differs significantly among caregivers. Evidence and consensus based (S3) guidelines for the treatment of primary antibody deficiencies were developed to improve the management of these patients.

myo-Inositol uptake is essential for bulk inositol phospholipid but not glycosylphosphatidylinositol synthesis in Trypanosoma brucei

myo-Inositol is an essential precursor for the production of inositol phosphates and inositol phospholipids in all eukaryotes. Intracellular myo-inositol is generated by de novo synthesis from glucose 6-phosphate or is provided from the environment via myo-inositol symporters. We show that in Trypanosoma brucei, the causative pathogen of human African sleeping sickness and nagana in domestic animals, myo-inositol is taken up via a specific proton-coupled electrogenic symport and that this transport is essential for parasite survival in culture. Down-regulation of the myo-inositol transporter using RNA interference inhibited uptake of myo-inositol and blocked the synthesis of the myo-inositol-containing phospholipids, phosphatidylinositol and inositol phosphorylceramide; in contrast, it had no effect on glycosylphosphatidylinositol production. This together with the unexpected localization of the myo-inositol transporter in both the plasma membrane and the Golgi demonstrate that metabolism of endogenous and exogenous myo-inositol in T. brucei is strictly segregated.