validation-nanobody-antibody-based-vivo-tumor-xenograft-nirf-imaging

Validation of Nanobody and Antibody Based <em>In Vivo</em> Tumor Xenograft NIRF-imaging Experiments in Mice Using <em>Ex Vivo</em> Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

University Medical Center, Hamburg

The effect of choline iodide, bromide and chloride on the kinetics of the electrogenic sodium transport by the Na,K-ATPase was investigated in a model system of ATPase-containing membrane fragments adsorbed on the lipid bilayer membrane. The kinetic parameters of Na(+) transport were determined from short circuit currents after fast release of ATP from its caged precursor. The falling phase of the current transients could be fitted by a single exponential with the time constant, tau (2). Its temperature dependence allowed an estimation of the activation energy of the rate-limiting reaction step, the conformation transition E(1)/E(2). Choline iodide and bromide caused a decrease of the activation energy as well as the overall rate of the process expressed as the pre-exponential factor A of the Arrhenius equation. If choline iodide or bromide were present on the cytoplasmic and extracellular sides of the protein, the temperature dependent changes were more pronounced than when present on the cytoplasmic side only. These results can be explained by an effect of the anions on water structure on the extracellular surface of the protein, where a deep access channel connects the ion-binding sites with the solution. Chloride ions also caused a deceleration of the electrogenic transport, however, in contrast to iodide or bromide, they did not affect the activation energy, and were more effective when added on the cytoplasmic side. This effect can be explained by asymmetric screening of the negative surface charges which leads to a transmembrane electric potential that modifies the ion transfer.

Effect of chaotropic anions on the sodium transport by the Na,K-ATPase.

In this paper, a model of cerebellar function is implemented and evaluated in the control of a robot eye actuated by pneumatic artificial muscles. The investigated control problem is stabilization of the visual image in response to disturbances. This is analogous to the vestibuloocular reflex (VOR) in humans. The cerebellar model is structurally based on the adaptive filter, and the learning rule is computationally analogous to least-mean squares, where parameter adaptation at the parallel fiber/Purkinje cell synapse is driven by the correlation of the sensory error signal (carried by the climbing fiber) and the motor command signal. Convergence of the algorithm is first analyzed in simulation on a model of the robot and then tested online in both one and two degrees of freedom. The results show that this model of neural function successfully works on a real-world problem, providing empirical evidence for validating: 1) the generic cerebellar learning algorithm; 2) the function of the cerebellum in the VOR; and 3) the signal transmission between functional neural components of the VOR.

Cerebellar-inspired adaptive control of a robot eye actuated by pneumatic artificial muscles.

We perform a comprehensive statistical analysis of the standard model (SM) with three and four generations using the latest Higgs search results from LHC and Tevatron, the electroweak precision observables measured at LEP and SLD, and the latest determinations of M(W), m(t), and α(s). For the three-generation case we analyze the tensions in the electroweak fit by removing individual observables from the fit and comparing their predicted values with the measured ones. In particular, we discuss the impact of the Higgs search results on the deviations of the electroweak precision observables from their best-fit values. Our indirect prediction of the top mass is m(t) =175.7(-2.2)(+3.0) GeV at 68.3% C.L., which is in good agreement with the direct measurement. We also plot the preferred area in the M(W)-m(t) plane. The best-fit Higgs boson mass is 126.0 GeV. For the case of the SM with a perturbative sequential fourth fermion generation (SM4) we discuss the deviations of the Higgs signal strengths from their best-fit values. The H → γγ signal strength now disagrees with its best-fit SM4 value at more than 4σ. We perform a likelihood-ratio test to compare the SM and SM4 and show that the SM4 is excluded at 5.3σ. Without the Tevatron data on H → bb the significance drops to 4.8σ.

Impact of a Higgs boson at a mass of 126 GeV on the standard model with three and four fermion generations.

The large size of conventional antibodies impedes tissue penetration and renal elimination, resulting in suboptimal in vivo targeting. Here we assess the utility of nanobodies and nanobody-Fc-fusion proteins as alternatives to monoclonal antibodies as theranostics, using T cell ADP-ribosyltransferase 2 (ART2) as a model antigen for specific targeting of lymph nodes. ART2-specific monovalent nanobody s + 16a (17 kDa), a bivalent Fc-fusion protein of s + 16a (s + 16-mFc, 82 kDa), and conventional antibody Nika102 (150 kDa) were labeled with AlexaFluor680. In vitro binding and inhibitory properties of the three AF680 conjugates were assessed by flow cytometry. For in vivo imaging experiments, AF680 conjugates were intravenously injected in mice lacking (KO) or overexpressing (TG) ART2. We monitored circulating and excreted AF680 conjugates in plasma and urine and performed in vivo near-infrared fluorescence imaging. Nanobody s + 16a(680) and s + 16mFc(680) labeled and inhibited ART2 on T cells in lymph nodes within 10 min. In contrast, mAb Nika102(680) required 2 h for maximal labeling without inhibition of ART2. In vivo imaging revealed specific labeling of ART2-positive lymph nodes but not of ART2-negative lymph nodes with all AF680 conjugates. Even though bivalent s + 16mFc(680) showed the highest labeling efficiency in vitro, the best lymph node imaging in vivo was achieved with monovalent nanobody s + 16a(680) , since renal elimination of unbound s + 16a(680) significantly reduced background signals. Our results indicate that small single-domain nanobodies are best suited for short-term uses, such as noninvasive imaging, whereas larger nanobody-Fc-fusion proteins are better suited for long-term uses, such as therapy of inflammation and tumors.

Alexander Lenz

Department of Diagnostic and Interventional Radiology,
Institute of Immunology

University Medical Center, Hamburg

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

Alexander Lenz

Department of Diagnostic and Interventional Radiology,Institute of Immunology

University Medical Center, Hamburg

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

Department of Diagnostic and Interventional Radiology,
Institute of Immunology