Production and characterization of monoclonal antibodies raised against recombinant human granzymes A and B and showing cross reactions with the natural proteins

The human serine proteases granzymes A and B are expressed in cytotoplasmic granules of activated cytotoxic T lymphocytes and natural killer cells. Recombinant granzyme A and granzyme B proteins were produced in bacteria, purified and then used to raise specific mouse monoclonal antibodies. Seven monoclonal antibodies (mAb) were raised against granzyme A, which all recognized the same or overlapping epitopes. They reacted specifically in an immunoblot of interleukin-2 (IL-2) stimulated PBMNC with a disulfide-linked homodimer of 43 kDa consisting of 28 kDa subunits. Seven mAb against granzyme B were obtained, which could be divided into two groups, each recognizing a different epitope. On an immunoblot, all mAb reacted with a monomer of 33 kDa protein. By immunohistochemistry, these mAb could be used to detect granzymes A and B expression in activated CTL and NK cells. The availability of these mAb may facilitate studies on the role of human cytotoxic cells in various immune reactions and may contribute to a better understanding of the role of granzmes A and B in the cytotoxic response in vivo

Motor Cortex Representation of the Upper-Limb in Individuals Born without a Hand

The body schema is an action-related representation of the body that arises from activity in a network of multiple brain areas. While it was initially thought that the body schema developed with experience, the existence of phantom limbs in individuals born without a limb (amelics) led to the suggestion that it was innate. The problem with this idea, however, is that the vast majority of amelics do not report the presence of a phantom limb. Transcranial magnetic stimulation (TMS) applied over the primary motor cortex (M1) of traumatic amputees can evoke movement sensations in the phantom, suggesting that traumatic amputation does not delete movement representations of the missing hand. Given this, we asked whether the absence of a phantom limb in the majority of amelics means that the motor cortex does not contain a cortical representation of the missing limb, or whether it is present but has been deactivated by the lack of sensorimotor experience. In four upper-limb amelic subjects we directly stimulated the arm/hand region of M1 to see 1) whether we could evoke phantom sensations, and 2) whether muscle representations in the two cortices were organised asymmetrically. TMS applied over the motor cortex contralateral to the missing limb evoked contractions in stump muscles but did not evoke phantom movement sensations. The location and extent of muscle maps varied between hemispheres but did not reveal any systematic asymmetries. In contrast, forearm muscle thresholds were always higher for the missing limb side. We suggest that phantom movement sensations reported by some upper limb amelics are mostly driven by vision and not by the persistence of motor commands to the missing limb within the sensorimotor cortex. We propose that prewired movement representations of a limb need the experience of movement to be expressed within the primary motor cortex

Plasma-derived mannose-binding lectin shows a direct interaction with C1-inhibitor

MBL-deficiency has been associated with an increased frequency and severity of infection, in particular in children and under immunocompromized conditions. In an open uncontrolled safety and pharmacokinetic MBL-substitution study using plasma-derived MBL (pdMBL) in MBL-deficient pediatric oncology patients, we found that despite MBL trough levels above 1.0μg/ml MBL functionality was not efficiently restored upon ex vivo testing. PdMBL showed C4-converting activity by itself, indicating the presence of MASPs. Upon incubation of pdMBL with MBL-deficient sera this C4-converting activity was significantly reduced. Depletion of the MASPs from pdMBL, paradoxically, restored the C4-converting activity. Subsequent depletion or inhibition of C1-inh, the major inhibitor of the lectin pathway, in the recipient serum restored the C4-converting activity as well. Complexes between MBL/MASPs and C1-inh (MMC-complexes) were detected after ex vivo substitution of MBL-deficient serum with pdMBL. These MMC-complexes could also be detected in the sera of the patients included in the MBL-substitution study shortly after pdMBL infusion. Altogether, we concluded that active MBL-MASP complexes in pdMBL directly interact with C1-inh in the recipient, leading to the formation of a multimolecular complex between C1-inh and MBL/MASPs, in contrast to the classical pathway where C1r and C1s are dissociated from C1q by C1-inh. Because of the presence of activated MASPs in the current pdMBL products efficient MBL-mediated host protection cannot be expected because of the neutralizing capacity by C1-in

Light-Induced Coalescence of Plasmonic Dimers and Clusters.

The properties of nanoplasmonic structures depend strongly on their geometry, creating the need for high-precision control and characterization. Here, by exploiting the low activation energy of gold atoms on nanoparticle surfaces, we show how laser irradiation reshapes nanoparticle dimers. Time-course dark-field microspectroscopy allows this process to be studied in detail for individual nanostructures. Three regimes are identified: facet growth, formation of a conductive bridge between particles, and bridge growth. Electromagnetic simulations confirm the growth dynamics and allow measurement of bridge diameter, found to be highly reproducible and also self-limiting. Correlations in spectral resonances for the initial and final states give insight into the energy barriers for bridge growth. Dark-field microscopy shows that coalescence of multiple gaps in nanoparticle clusters can be digitally triggered, with each gap closing after discrete increases in irradiation power. Such control is important for light-induced nanowire formation or trimming of electronic and optoelectronic devices

Unraveling the Effect of a Potentiating Anti-Factor H Antibody on Atypical Hemolytic Uremic Syndrome-Associated Factor H Variants

The complement system plays an important role in our innate immune system. Complement activation results in clearance of pathogens, immune complex, and apoptotic cells. The host is protected from complement-mediated damage by several complement regulators. Factor H (FH) is the most important fluid-phase regulator of the alternative pathway of the complement system. Heterozygous mutations in FH are associated with complement-related diseases such as atypical hemolytic uremic syndrome (aHUS) and age-related macular degeneration. We recently described an agonistic anti-FH mAb that can potentiate the regulatory function of FH. This Ab could serve as a potential new drug for aHUS patients and alternative to C5 blockade by eculizumab. However, it is unclear whether this Ab can potentiate FH mutant variants in addition to wild-type (WT) FH. In this study, the functionality and potential of the agonistic Ab in the context of pathogenic aHUS-related FH mutant proteins was investigated. The binding affinity of recombinant WT FH and the FH variants, W1183L, V1197A, R1210C, and G1194D to C3b was increased upon addition of the potentiating Ab and similarly, the decay-accelerating activity of all mutants is increased. The potentiating anti-FH Ab is able to restore the surface regulatory function of most of the tested FH mutants to WT FH levels on a human HAP-1 cell line and on sheep erythrocytes. In conclusion, our potentiating anti-FH is broadly active and able to enhance both WT FH function as well as most aHUS-associated FH variants tested in this study

Full Control of Plasmonic Nanocavities Using Gold Decahedra-on-Mirror Constructs with Monodisperse Facets.

Bottom-up assembly of nanoparticle-on-mirror (NPoM) nanocavities enables precise inter-metal gap control down to ≈ 0.4 nm for confining light to sub-nanometer scales, thereby opening opportunities for developing innovative nanophotonic devices. However limited understanding, prediction, and optimization of light coupling and the difficulty of controlling nanoparticle facet shapes restricts the use of such building blocks. Here, an ultraprecise symmetry-breaking plasmonic nanocavity based on gold nanodecahedra is presented, to form the nanodecahedron-on-mirror (NDoM) which shows highly consistent cavity modes and fields. By characterizing > 20 000 individual NDoMs, the variability of light in/output coupling is thoroughly explored and a set of robust higher-order plasmonic whispering gallery modes uniquely localized at the edges of the triangular facet in contact with the metallic substrate is found. Assisted by quasinormal mode simulations, systematic elaboration of NDoMs is proposed to give nanocavities with near hundred-fold enhanced radiative efficiencies. Such systematically designed and precisely-assembled metallic nanocavities will find broad application in nanophotonic devices, optomechanics, and surface science

The contribution of the alternative pathway in complement activation on cell surfaces depends on the strength of classical pathway initiation.

We evaluated the contribution of the AP to complement responses initiated through the CP on human RBCs by serum of AIHA patients and recombinant antibodies. Moreover, we studied complement activation on Neisseria meningitidis and Escherichia coli. The effect of the AP was examined using either AP-depleted sera or antibodies against factor B and factor D

The contribution of the alternative pathway in complement activation on cell surfaces depends on the strength of classical pathway initiation

Abstract Objectives The complement system is an important component of innate immunity. The alternative pathway (AP) amplification loop is considered an essential feed forward mechanism for complement activation. However, the role of the AP in classical pathway (CP) activation has only been studied in ELISA settings. Here, we investigated its contribution on physiologically relevant surfaces of human cells and bacterial pathogens and in antibody‐mediated complement activation, including in autoimmune haemolytic anaemia (AIHA) setting with autoantibodies against red blood cells (RBCs). Methods We evaluated the contribution of the AP to complement responses initiated through the CP on human RBCs by serum of AIHA patients and recombinant antibodies. Moreover, we studied complement activation on Neisseria meningitidis and Escherichia coli. The effect of the AP was examined using either AP‐depleted sera or antibodies against factor B and factor D. Results We show that the amplification loop is redundant when efficient CP activation takes place. This is independent of the presence of membrane‐bound complement regulators. The role of the AP may become significant when insufficient CP complement activation occurs, but this depends on antibody levels and (sub)class. Our data indicate that therapeutic intervention in the amplification loop will most likely not be effective to treat antibody‐mediated diseases. Conclusion The AP can be bypassed through efficient CP activation. The AP amplification loop has a role in complement activation during conditions of modest activation via the CP, when it can allow for efficient complement‐mediated killing