Production of antibodies to canine IL-1beta and canine TNF to assess the role of proinflammatory cytokines

IL-1 and TNF are important proinflammatory cytokines implicated in both antimicrobial host defense and pathogenesis of diseases with an immune-mediated and/or inflammatory component. Respective studies in the dog have been hampered by the unavailability of reagents allowing the specific measurement of canine cytokine proteins and the effect of canine cytokine neutralization by Ab. Starting with recombinant canine (rcan) IL-1beta and rcanTNF, four polyclonal antisera and 22 mAb specific for rcanIL-1beta and rcanTNF were generated. Their usefulness in neutralization assays was determined. Using cytokine-containing supernatants of canine cells in bioassays, polyclonal antisera neutralized either canine IL-1beta or TNF. TNF was also neutralized by three antibodies developed in this study and one commercial mAb. The usefulness of monoclonal and polyclonal Ab in canine cytokine-specific Ab capture ELISA's was assessed. This resulted in the identification of a commercial mAb combination and one pair developed in this study allowing low levels of TNF to be detected by antibody capture ELISA. The detection limit was 141 pg/ml rcanTNF for both combinations. Using rcanIL-1beta as an antigen allowed the detection of lower concentrations of rcanIL-1beta (20 pg/ml, on the average) by a pair of polyclonal antisera than when monoclonals were used. By using such IL-1beta-specific and TNF-specific ELISA's, the respective cytokines were detected in supernatants of canine PBMC stimulated with LPS or heat-killed Listeria monocytogenes and interferon-gamma combined. Thus, monoclonal and polyclonal reagents were identified allowing the quantitation of canine IL-1beta and TNF production in vitro, and the neutralization of these cytokines.

Intravital and whole-organ imaging reveals capture of melanoma-derived antigen by lymph node subcapsular macrophages leading to widespread deposition on follicular dendritic cells.

Aberrant antigens expressed by tumor cells, such as in melanoma, are often associated with humoral immune responses, which may in turn influence tumor progression. Despite recent data showing the central role of adaptive immune responses on cancer spread or control, it remains poorly understood where and how tumor-derived antigen (TDA) induces a humoral immune response in tumor-bearing hosts. Based on our observation of TDA accumulation in B cell areas of lymph nodes (LNs) from melanoma patients, we developed a pre-metastatic B16.F10 melanoma model expressing a fluorescent fusion protein, tandem dimer tomato, as a surrogate TDA. Using intravital two-photon microscopy (2PM) and whole-mount 3D LN imaging of tumor-draining LNs in immunocompetent mice, we report an unexpectedly widespread accumulation of TDA on follicular dendritic cells (FDCs), which were dynamically scanned by circulating B cells. Furthermore, 2PM imaging identified macrophages located in the subcapsular sinus of tumor-draining LNs to capture subcellular TDA-containing particles arriving in afferent lymph. As a consequence, depletion of macrophages or genetic ablation of B cells and FDCs resulted in dramatically reduced TDA capture in tumor-draining LNs. In sum, we identified a major pathway for the induction of humoral responses in a melanoma model, which may be exploitable to manipulate anti-TDA antibody production during cancer immunotherapy.

Prevalence estimation of tick-borne encephalitis virus (TBEV) antibodies in dogs from Finland using novel dog anti-TBEV IgG MAb-capture and IgG immunofluorescence assay based on recombinant TBEV subviral particles

Tick-borne encephalitis (TBE) is one of the most dangerous human neurological infections occurring in Europe and Northern parts of Asia with thousands of cases and millions vaccinated against it. The risk of TBE might be assessed through analyses of the samples taken from wildlife or from animals which are in close contact with humans. Dogs have been shown to be a good sentinel species for these studies. Serological assays for diagnosis of TBE in dogs are mainly based on purified and inactivated TBEV antigens. Here we describe novel dog anti-TBEV IgG monoclonal antibody (MAb)-capture assay which is based on TBEV prME subviral particles expressed in mammalian cells from Semliki Forest virus (SFV) replicon as well as IgG immunofluorescence assay (IFA) which is based on Vero E6 cells transfected with the same SFV replicon. We further demonstrate their use in a small-scale TBEV seroprevalence study of dogs representing different regions of Finland. Altogether, 148 dog serum samples were tested by novel assays and results were compared to those obtained with a commercial IgG enzyme immunoassay (EIA), hemagglutination inhibition test and IgG IFA with TBEV infected cells. Compared to reference tests, the sensitivities of the developed assays were 90-100% and the specificities of the two assays were 100%. Analysis of the dog serum samples showed a seroprevalence of 40% on Åland Islands and 6% on Southwestern archipelago of Finland. In conclusion, a specific and sensitive EIA and IFA for the detection of IgG antibodies in canine sera were developed. Based on these assays the seroprevalence of IgG antibodies in dogs from different regions of Finland was assessed and was shown to parallel the known human disease burden as the Southwestern archipelago and Åland Islands in particular had considerable dog TBEV antibody prevalence and represent areas with high risk of TBE for humans.

Preclinical in vitro and in vivo characterization of the fully human monoclonal IgM antibody KBPA101 specific for Pseudomonas aeruginosa serotype IATS-O11

Pseudomonas aeruginosa infection in ventilator-associated pneumonia is a serious and often life-threatening complication in intensive care unit patients, and new treatment options are needed. We used B-cell-enriched peripheral blood lymphocytes from a volunteer immunized with a P. aeruginosa O-polysaccharide-toxin A conjugate vaccine to generate human hybridoma cell lines producing monoclonal antibodies specific for individual P. aeruginosa lipopolysaccharide serotypes. The fully human monoclonal antibody secreted by one of these lines, KBPA101, is an IgM/kappa antibody that binds P. aeruginosa of International Antigenic Typing System (IATS) serotype O11 with high avidity (5.81 x 10(7) M(-1) +/- 2.8 x 10(7) M(-1)) without cross-reacting with other serotypes. KBPA101 specifically opsonized the P. aeruginosa of IATS O11 serotype and mediated complement-dependent phagocytosis in vitro by the human monocyte-like cell line HL-60 at a very low concentration (half-maximal phagocytosis at 0.16 ng/ml). In vivo evaluation of KBPA101 demonstrated a dose-response relationship for protection against systemic infections in a murine burn wound sepsis model, where 70 to 100% of animals were protected against lethal challenges with P. aeruginosa at doses as low as 5 microg/animal. Furthermore, a high efficacy of KBPA101 in protection from local respiratory infections in an acute lung infection model in mice was demonstrated. Preclinical toxicology evaluation on human tissue, in rabbits, and in mice did not indicate any toxicity of KBPA101. Based on these preclinical findings, the first human clinical trials have been initiated.

Improvement of non-paraneoplastic voltage-gated potassium channel antibody-associated limbic encephalitis without immunosuppressive therapy

We describe a 61-year-old patient with clinical evidence of limbic encephalitis who improved with anticonvulsant treatment only, that is, without the use of immunosuppressive agents. Three years following occurrence of anosmia, increasing memory deficits, and emotional disturbances, he presented with new-onset temporal lobe epilepsy, with antibodies binding to neuronal voltage-gated potassium channels and bitemporal hypometabolism on FDG-PET scan; the MRI scan was normal. This is most likely a case of spontaneous remission, illustrating that immunosuppressive therapy might be suspended in milder courses of limbic encephalitis. It remains open whether treatment with anticonvulsant drugs played an additional beneficiary role through the direct suppression of seizures or, additionally, through indirect immunomodulatory side effects.

Anti-interleukin-5 antibody treatment (mepolizumab) in active eosinophilic oesophagitis: a randomised, placebo-controlled, double-blind trial

Eosinophilic oesophagitis (EoO) is a clinicopathological condition defined by proton pump inhibitor-refractory oesophageal symptoms combined with oesophageal eosinophilia. The pharmacodynamic effect of mepolizumab (a humanised anti-interleukin-5 monoclonal antibody) in EoO was evaluated.

Multiplex suspension array for human anti-carbohydrate antibody profiling

Glycan-binding antibodies form a significant subpopulation of both natural and acquired antibodies and play an important role in various immune processes. They are for example involved in innate immune responses, cancer, autoimmune diseases, and neurological disorders. In the present study, a microsphere-based flow-cytometric immunoassay (suspension array) was applied for multiplexed detection of glycan-binding antibodies in human serum. Several approaches for immobilization of glycoconjugates onto commercially available fluorescent microspheres were compared, and as the result, the design based on coupling of end-biotinylated glycopolymers has been selected. This method requires only minute amounts of glycans, similar to a printed glycan microarray. The resulting glyco-microspheres were used for detection of IgM and IgG antibodies directed against ABO blood group antigens. The possibility of multiplexing this assay was demonstrated with mixtures of microspheres modified with six different ABO related glycans. Multiplexed detection of anti-glycan IgM and IgG correlated well with singleplex assays (Pearson's correlation coefficient r = 0.95-0.99 for sera of different blood groups). The suspension array in singleplex format for A/B trisaccharide, H(di) and Le(x) microspheres corresponded well to the standard ELISA (r > 0.94). Therefore, the described method is promising for rapid, sensitive, and reproducible detection of anti-glycan antibodies in a multiplexed format.

Mannan-binding lectin deficiency results in unusual antibody production and excessive experimental colitis in response to mannose-expressing mild gut pathogens

In Crohn's disease (CD) the deficiency of mannan-binding lectin (MBL) is associated with an increased prevalence of anti-Saccharomyces cerevisiae antibodies (ASCA) and with complicated phenotypes of the disease. However, the role of MBL in intestinal inflammation is currently unclear. A study was undertaken to analyse local MBL expression in human intestine and the consequences of MBL deficiency in experimental colitis and yeast infection.

Loss of pace capture on the ablation line: a new marker for complete radiofrequency lesions to achieve pulmonary vein isolation

Catheter ablation procedures for atrial fibrillation (AF) often involve circumferential antral isolation of pulmonary veins (PV). Inability to reliably identify conduction gaps on the ablation line necessitates placing additional lesions within the intended lesion set.

Sequence capture and next-generation resequencing of multiple tagged nucleic acid samples for mutation screening of urea cycle disorders

Molecular genetic testing is commonly used to confirm clinical diagnoses of inherited urea cycle disorders (UCDs); however, conventional mutation screenings encompassing only the coding regions of genes may not detect disease-causing mutations occurring in regulatory elements and introns. Microarray-based target enrichment and next-generation sequencing now allow more-comprehensive genetic screening. We applied this approach to UCDs and combined it with the use of DNA bar codes for more cost-effective, parallel analyses of multiple samples.

Report from a consensus conference on antibody-mediated rejection in heart transplantation

The problem of AMR remains unsolved because standardized schemes for diagnosis and treatment remains contentious. Therefore, a consensus conference was organized to discuss the current status of antibody-mediated rejection (AMR) in heart transplantation.

Systemic antibody responses to gut commensal bacteria during chronic HIV-1 infection

Human systemic antibody responses to commensal microbiota are not well characterised during health and disease. Of particular interest is the analysis of their potential modulation caused by chronic HIV-1 infection which is associated with sustained enteropathy and systemic B cell disturbances reflected by impaired B cell responses and chronic B cell hyperactivity. The mechanisms underlying B cell hyperactivation and the specificities of the resulting hypergammaglobulinaemia are only poorly understood.

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.