Comparison of serological assays for detecting antibodies in ducks exposed to H5 subtype avian influenza virus

ABSTRACT: BACKGROUND: Chicken red blood cells (RBCs) are commonly used in hemagglutination inhibition (HI) tests to measure hemagglutinating antibodies against influenza viruses. The use of horse RBCs in the HI test can reportedly increase its sensitivity when testing human sera for avian influenza antibodies. This study aims to compare the proportion of positives detected and the agreement between two HI tests using either chicken or horse red blood cells for antibody detection in sera of ducks experimentally infected or naturally exposed to Indonesian H5 subtype avian influenza virus. In addition, comparison with a virus neutralisation (VN) test was conducted with the experimental sera. RESULTS: In the experimental study, the proportion of HI antibody-positive ducks increased slightly, from 0.57 when using chicken RBCs to 0.60 when using horse RBCs. The HI tests indicated almost perfect agreement (kappa = 0.86) when results were dichotomised (titre [greater than or equal to] 4 log2), and substantial agreement (weighted kappa = 0.80) for log titres. Overall agreements between the two HI tests were greater than between either of the HI tests and the VN test. The use of horse RBCs also identified a higher proportion of antibody positives in field duck sera (0.08, compared to chicken RBCs 0.02), with also almost perfect agreements for dichotomized results (Prevalence and bias adjusted Kappa (PABAK) = 0.88) and for log titres (weighted PABAK = 0.93), respectively. Factors that might explain observed differences in the proportion of antibody-positive ducks and in the agreements between HI tests are discussed. CONCLUSION: In conclusion, we identified a good agreement between HI tests. However, when horse RBCs were used, a higher proportion of sera was positive (titre [greater than or equal to] 4 log2) than using chicken RBCs, especially during the early response against H5N1 virus. The HRBC-HI might be more responsive in identifying early H5N1 HPAI serological response and could be a recommended assay for avian influenza sero-surveillance in both wild and domestic birds.     

Cutting edge: CD8+CD122+ regulatory T cells produce IL-10 to suppress IFN-gamma production and proliferation of CD8+ T cells

We recently identified CD8+CD122+ regulatory T cells that directly control CD8+ and CD4+ cells without intervention of APCs. In this study, we investigated the effector mechanism of CD8+CD122+ regulatory T cells by using an in vitro regulation system. The profile of cytokine expression revealed that IL-10 was predominantly produced by CD8+CD122+ cells, whereas other cytokines were similarly expressed in CD8+CD122+ cells and CD8+CD122- cells. Suppression of both proliferation and IFN-gamma production by CD8+CD122- cells by CD8+CD122+ cells was blocked by adding anti-IL-10 Ab to the culture but not by adding anti-TGF-beta Ab. When IL-10 was removed from the conditioned medium from CD8+CD122+ cells, the conditioned medium no longer showed regulatory activity. Finally, CD8+CD122+ cells from IL-10-deficient mice had no regulatory activity in vitro and reduced regulatory activity in vivo. Our results clearly indicate that IL-10 is produced by CD8+CD122+ cells and mediates the regulatory activity of these cells.     

Prostate cancer and the genomic revolution: Advances using microarray analyses

The emerging technology of microarray analysis allows the establishment of molecular portraits of prostate cancer and the discovery of novel genes involved in the carcinogenesis process. Many novel genes have already been identified using this technique, and functional analyses of these genes are currently being tested. The combination of microarray analysis with other recently developed high-throughput techniques, such as proteomics, tissue arrays, and gene promoter-methylation, especially using tissue microdissection methods, will provide us with more comprehensive insights into how prostate cancer develops and responds to gene-targeted therapies. Animal models of prostate cancer are being characterized by high throughput techniques to better define the similarities and differences between those models and the human disease, and to determine whether particular models may be useful for specific targeted therapies in pre-clinical studies. Although profiling of mRNA expression provides important information of gene expression, the development of proteomic technologies will allow for an even more precise global insight into cellular signaling and structural alterations during prostate carcinogenesis. Not only will the "omic" revolution change basic science, but it will lead to a new era of molecular medicine.     

Immunolocalization of Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase in the branchial cavity during the early development of the crayfish <Emphasis Type="Italic">Astacus leptodactylus</Emphasis> (Crustacea,Decapoda)

The ontogeny of osmoregulation was examined in the branchial cavity of embryonic and early post-embryonic stages of the crayfish Astacus leptodactylus maintained in freshwater, at the sub-cellular level through the detection of the sodium–potassium adenosine triphosphatase (Na⁺,K⁺-ATPase). The embryonic rate of development was calculated according to the eye index (EI) which was 430–450 m at hatching. The distribution of the enzyme was identified by immunofluorescence microscopy using a monoclonal antibody IgG5 raised against the avian -subunit of the Na⁺,K⁺-ATPase. Immunoreactivity staining, indicating the presence of Na⁺, K⁺-ATPase appeared in the gills of late embryos (EI400 m), i.e. a few days before hatching time, and steadily increased throughout the late embryonic and early post-embryonic development. The appearance of the enzyme correlates with the ability to osmoregulate which also occurs late in the embryonic development at EI 410–420 m and with tissue differentiation within the gill filaments. These observations indicate that the physiological shift from osmoconforming embryos to hyper-regulating late embryos and post-hatching stages in freshwater must originate partly from the differentiation in the gill epithelia of ionocytes which are the site of ion pumping, as suggested by the location of Na⁺,K⁺-ATPase. Only the gills were immunostained and a lack of specific staining was noted in the lamina and the branchiostegites. Therefore, osmoregulation through Na⁺active uptake is likely achieved in embryos at the gill level; all the newly formed gills in embryos function in ion regulation; other parts of the branchial chamber such as the branchiostegites and lamina do not appear to be involved in osmoregulation.     

Columba: an integrated database of proteins,structures, and annotations

Background  

Structural and functional research often requires the computation of sets of protein structures based on certain properties of the proteins, such as sequence features, fold classification, or functional annotation. Compiling such sets using current web resources is tedious because the necessary data are spread over many different databases. To facilitate this task, we have created COLUMBA, an integrated database of annotations of protein structures.     

Survival, osmoregulation and oxygen consumption of YOY coastal largemouth bass,Micropterus salmoides (Lacepede) exposed to saline media

The effect of salinity on survival, osmoregulation and oxygen consumption was determined on coastal young-of-the-year (YOY) largemouth bassMicropterus salmoides by exposing them to saline media of 0, 4, 8, 12, and 16‰. The data indicated a decrease in survival with longer exposure time and increased salinity. There were no significant differences in plasma osmolality with increased salinity from 0 to 8‰ but osmolality was significantly greater at 12‰ than 0‰ and osmolality from 16‰ was greater than all other salinity treatments. No significant differences in hematocrits were detected between 0 and 12‰, but hematocrits in the 16‰ treatment were significantly reduced compared to all other treatments. YOYM. salmoides are good osmoregulators up to 8‰ but increased salinity caused measurable osmoregulatory dysfunction. Oxygen consumption rate increased significantly as salinity increased, suggesting that adaptation of this species to hypersaline media is in part accompanied by increased energy expenditure.     

Intramural conduction system gradients and electrogram regularity during ventricular fibrillation

Introduction

The His-Purkinje system has been shown to harbor triggers for ventricular fibrillation (VF) initiation. However, the substrate responsible for VF maintenance remains elusive. We hypothesized that standard, electrode-based, point-to-point mapping would yield meaningful insight into site-specific patterns and organization which may shed light on the critical substrate for maintenance of VF.

Charge heterogeneity: Basic antibody charge variants with increased binding to Fc receptors

We identified active isoforms of the chimeric anti-GD2 antibody, ch14.18, a recombinant antibody produced in Chinese hamster ovary cells, which is already used in clinical trials.^1,2,3 Ladenstein R, Weixler S, Baykan B, Bleeke M, Kunert R, Katinger D, Pribill I, Glander P, Bauer S, Pistoia V, et al. Ch14.18 antibody produced in CHO cells in relapsed or refractory Stage 4 neuroblastoma patients: a SIOPEN Phase 1 study. MAbs 2013; 5:801-9; PMID:23924804; http://dx.doi.org/10.4161/mabs.25215 Desai AV, Fox E, Smith LM, Lim AP, Maris JM, Balis FM. Pharmacokinetics of the chimeric anti-GD2 antibody, ch14.18, in children with high-risk neuroblastoma. Cancer Chemother Pharmacol 2014; 74:1047-55; PMID:25212536; http://dx.doi.org/10.1007/s00280-014-2575-9 Siebert N, Eger C, Seidel D, Jüttner M, Zumpe M, Wegner D, Kietz S, Ehlert K, Veal GJ, Siegmund W, et al. Pharmacokinetics and pharmacodynamics of ch14.18/CHO in relapsed/refractory high-risk neuroblastoma patients treated by long-term infusion in combination with IL-2. MAbs 2016; 8:604-16; PMID:26785755; http://dx.doi.org/10.1080/19420862.2015.1130196  We separated the antibody by high resolution ion-exchange chromatography with linear pH gradient elution into acidic, main and basic charge variants on a preparative scale yielding enough material for an in-depth study of the sources and the effects of microheterogeneity. The binding affinity of the charge variants toward the antigen and various cell surface receptors was studied by Biacore. Effector functions were evaluated using cellular assays for antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. Basic charge variants showed increased binding to cell surface receptor FcγRIIIa, which plays a major role in regulating effector functions. Furthermore, increased binding of the basic fractions to the neonatal receptor was observed. As this receptor mediates the prolonged half-life of IgG in human serum, this data may well hint at an increased serum half-life of these basic variants compared to their more acidic counterparts. Different glycoform patterns, C-terminal lysine clipping and N-terminal pyroglutamate formation were identified as the main structural sources for the observed isoform pattern. Potential differences in structural stability between individual charge variant fractions by nano differential scanning calorimetry could not been detected. Our in-vitro data suggests that the connection between microheterogeneity and the biological activity of recombinant antibody therapeutics deserves more attention than commonly accepted.     

Functionalization of fullerene via the Bingel reaction with α-chlorocarbanions: an ONIOM approach

Two-layer ONIOM method at the ONIOM(B3LYP/6-31G(d):PM6) level of theory was applied to study the cycloaddition reaction of α-chlorocarbanions (CR₂Cl^─, where R is H, Cl, CH₃ CN, and NO₂) and fullerene. The results show that the reaction pathways depend on the electron withdrawing functional groups or the electron donating functional groups contained in the α-chlorocarbanions. The energy profile analysis reveals that functionalization of fullerene by CCl₃ ^─, C(CH₃)₂Cl^─, and CH₂Cl^─ is more favorable than by C(CN)₂Cl^─ and C(NO₂)₂Cl^─ in terms of the thermodynamic point of view.