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At least 217 records · Page 12

Factor VIII moiety of recombinant Factor VIII Fc fusion protein impacts Fc effector function and CD16 + NK cell activation

Recombinant Factor VIII-Fc fusion protein (rFVIIIFc) is an enhanced half-life therapeutic protein product used for the management of hemophilia A. Recent studies have demonstrated that rFVIIIFc interacts with Fc gamma receptors (FcγR) resulting in the activation or inhibition of various FcγR-expressing immune cells. We previously demonstrated that rFVIIIFc, unlike recombinant Factor IX-Fc (rFIXFc), activates natural killer (NK) cells via Fc-mediated interactions with FcγRIIIA (CD16). Additionally, we showed that rFVIIIFc activated CD16 + NK cells to lyse a FVIII-specific B cell clone. Here, we used human NK cell lines and primary NK cells enriched from peripheral blood leukocytes to study the role of the FVIII moiety in rFVIIIFc-mediated NK cell activation. Following overnight incubation of NK cells with rFVIIIFc, cellular activation was assessed by measuring secretion of the inflammatory cytokine IFNγ by ELISA or by cellular degranulation. We show that anti-FVIII, anti-Fc, and anti-CD16 all inhibited indicating that these molecules were involved in rFVIIIFc-mediated NK cell activation. To define which domains of FVIII were involved, we used antibodies that are FVIII domain-specific and demonstrated that blocking FVIII C1 or C2 domain-mediated membrane binding potently inhibited rFVIIIFc-mediated CD16 + NK cell activation, while targeting the FVIII heavy chain domains did not. We also show that rFVIIIFc binds CD16 with about five-fold higher affinity than rFIXFc. Based on our results we propose that FVIII light chain-mediated membrane binding results in tethering of the fusion protein to the cell surface, and this, together with increased binding affinity for CD16, allows for Fc-CD16 interactions to proceed, resulting in NK cellular activation. Our working model may explain our previous results where we observed that rFVIIIFc activated NK cells via CD16, whereas rFIXFc did not despite having identical IgG1 Fc domains.

59 BASIC BIOLOGICAL SCIENCES↗

Hydrodynamic Effects on Biofilm Development and Recombinant Protein Expression

Hydrodynamics play an important role in the rate of cell attachment and nutrient and oxygen transfer, which can affect biofilm development and the level of recombinant protein production. In the present study, the effects of different flow conditions on the development of Escherichia coli biofilms and the expression of a model recombinant protein (enhanced green fluorescent protein, eGFP) were examined. Planktonic and biofilm cells were grown at two different flow rates in a recirculating flow cell system for 7 days: 255 and 128 L h−1 (corresponding to a Reynolds number of 4600 and 2300, respectively). The fluorometric analysis showed that the specific eGFP production was higher in biofilms than in planktonic cells under both hydrodynamic conditions (3-fold higher for 255 L h−1 and 2-fold higher for 128 L h−1). In the biofilm cells, the percentage of eGFP-expressing cells was on average 52% higher at a flow rate of 255 L h−1. Furthermore, a higher plasmid copy number (PCN) was obtained for the highest flow rate for both planktonic (244 PCN/cell versus 118 PCN/cell) and biofilm cells (43 PCN/cell versus 29 PCN/cell). The results suggested that higher flow velocities promoted eGFP expression in E. coli biofilms.

Soares, Alexandra↗

Recombinant Vaccine Strain ASFV-G-Δ9GL/ΔUK Produced in the IPKM Cell Line Is Genetically Stable and Efficacious in Inducing Protection in Pigs Challenged with the Virulent African Swine Fever Virus Field Isolate Georgia 2010

We have previously reported that the recombinant African Swine Fever (ASF) vaccine candidate ASFV-G-Δ9GL/ΔUK efficiently induces protection in domestic pigs challenged with the virulent strain Georgia 2010 (ASFV-G). As reported, ASFV-G-Δ9GL/ΔUK induces protection, while intramuscularly (IM), administered at doses of 10 4 HAD 50 or higher, prevents ASF clinical disease in animals infected with the homologous ASFV g strain. Like other recombinant vaccine candidates obtained from ASFV field isolates, ASFV-G-Δ9GL/ΔUK stocks need to be produced in primary cultures of swine macrophages, which constitutes an important limitation in the production of large virus stocks at the industrial level. Here, we describe the development of ASFV-G-Δ9GL/ΔUK stocks using IPKM (Immortalized Porcine Kidney Macrophage) cells, which are derived from swine macrophages. We show that ten successive passages of ASFV-G-Δ9GL/ΔUK in IPKM cells induced small changes in the virus genome. The produced virus, ASFV-G-Δ9GL/ΔUKp10, presented a similar level of replication in swine macrophages cultures to that of the original ASFV-G-Δ9GL/ΔUK (ASFV-G-Δ9GL/ΔUKp0). The protective efficacy of ASFV-G-Δ9GL/ΔUKp10 was evaluated in pigs that were IM-inoculated with either 10 4 or 10 6 HAD 50 of ASFV-G-Δ9GL/ΔUKp10. While animals inoculated with 10 4 HAD 50 present a partial protection against the experimental infection with the virulent parental virus ASFV-G, those inoculated with 10 6 HAD 50 were completely protected. Therefore, as was just recently reported for another ASF vaccine candidate, ASFV-G-ΔI177L, IPKM cells are an effective alternative to produce stocks for vaccine strains which only grow in swine macrophages.

60 APPLIED LIFE SCIENCES↗

Radiofrequency recombination lines from the interstellar medium

Observations of recombination lines form normal H II regions, extended H II regions, nonthermal sources, and the H I medium are discussed. Detection of recombination lines from elements other than hydrogen may provide a means of identifying fossil Stromgren spheres at high temperature.

Dupree, A. K.↗

Effects of wall coatings and temperature on hydrogen atom surface recombination

The efficiency of various surface coatings and materials toward inhibiting hydrogen atom surface recombination was investigated over a temperature range of 77 to 298 K. A flow discharge, mass spectrometer technique was used to make the experimental measurements. Hydrogen atoms were monitored directly, and these measurements were expressed as ratios of mass spectrometer peak heights for atomic and molecular hydrogen. Several of the surface coatings studied were efficient at reducing hydrogen atom surface recombination at room temperature. However, as the temperature was lowered, this efficiency was drastically reduced. Calibration of the mass spectrometer for atomic and molecular hydrogen indicated that mass spectrometer discrimination against hydrogen atoms was severe. Mass spectrometer sensitivity for hydrogen atoms was only about one-sixth of that for molecular hydrogen.

Wong, E. L.↗

Measurements of recombination of electrons with HCO(plus) ions

Recombination coefficients of electrons with HCO(+) ions were determined with a microwave afterglow/mass spectrometer apparatus. Afterglow measurements of electron density decays in neon-hydrogen-carbon monoxide mixtures are correlated with the decay of mass-identified ion currents to the wall of the microwave cavity. At the appropriate partial pressures of hydrogen and carbon monoxide in the mixture, the ion HCO(+) dominates the ion composition and its wall current approximately tracks the electron density decay curve. From recombination controlled electron density decay curves, the values alpha (HCO(+)) = (3.3 + or - 0.5) and (2.0 + or - 0.3) 0.0000001 cu cm/sec are obtained at 205 and 300 K, respectively. The implications of these results for models of polyatomic molecule formation in dense interstellar clouds are briefly discussed.

Leu, M. T.↗

Measurements of recombination of electrons with H3(plus) and H5(plus) ions

The electron-ion recombination coefficients for H3(+) and H5(+) ions were determined by means of a microwave afterglow/mass spectrometer apparatus. Measurements of electron density decays in helium-hydrogen mixtures are correlated with the decay of mass-identified ion currents to the wall of the microwave cavity. At low partial pressures of hydrogen in the mixture, the ion H3(+) dominates the ion composition and the ion wall current tracks the electron density decay curves. From recombination controlled electron density decay curves, the values alpha (H3(+)) = (2.9 + or - 0.3), (2.3 + or - 0.3), and (2.0 + or - 0.2) x 0.0000001 cu cm per sec, are obtained at 205, 300 and 450 K, respectively. At higher partial pressures of hydrogen and low temperatures, where (H5(+)) is the dominant ion, the value alpha (H5(+)) = (3.6 + or - 1.0) x 0.0000001 cu cm per sec is obtained at 205 K. The implications of these results concerning ionization levels in the atmospheres of the outer planets and in the interstellar medium are discussed.

Leu, M. T.↗

The 6300 A O/1-D/ airglow and dissociative recombination

Measurements of night-time 6300 A airglow intensities at the Arecibo Observatory have been compared with dissociative recombination calculations based on electron densities derived from simultaneous incoherent backscatter measurements. The agreement indicates that the nightglow can be fully accounted for by dissociative recombination. The comparisons are examined to determine the importance of quenching, heavy ions, ionization above the F-layer peak, and the temperature parameter of the model atmosphere. Comparable fits between the observed and calculated intensities are found for several available model atmospheres. The least-squares fitting process, used to make the comparisons, produces comparable fits over a wide range of combinations of neutral densities and of reaction constants. Yet, the fitting places constraints upon the possible combinations; these constraints indicate that the latest laboratory chemical constants and densities extrapolated to a base altitude are mutually consistent.

Wickwar, V. B.↗

Radio recombination line observations of the C II region NGC 2023

The reflection nebula NGC 2023 has been observed at four frequencies in a study of its carbon recombination line emission. The observations show the recombination line region to be about 2 pc in diameter, with an average electron density of about 0.2 per cu cm and electron temperature of about 20 K. The electron density increases exponentially near the B1.5 star HD 37903, the illuminating star of NGC 2023. The most reasonable interpretation of the observational results is that the carbon emission lines arise in a cold C II region surrounding HD 37903. The coexistence of the C II region with a region of enhanced CO emission suggests that the CO in NGC 2023 may be excited by electron collisions.

Knapp, G. R.↗

Dissociative recombination source for O I /1D/ atoms

A study of the nighttime dissociative recombination production of O(1D) is reported. The data were gathered by a rocket payload carrying an ion mass spectrometer, Langmuir probe, retarding potential analyzer, and 6300-A photometer. The specific recombination rate to produce O(1D) atoms is deduced to be (2.8 + or - 1.0) times 10 to the minus 8th cu cm per sec and is 30% of the total laboratory rate. The quenching rate at 250 km is 0.0044 + or - 0.0015 per sec.

Sharp, W. E.↗

Rotational interpretation of recombination line widths in the galactic nucleus

The widths of the radio recombination lines observed in the direction of the galactic center are interpreted as due to pure rotation. This interpretation leads to a mass estimate for the inner 1.6-pc region of our Galaxy. It is noted that recent observations of M 31 suggest the existence of a distinct dynamic entity at its center - the nucleus. The rotational interpretation of the recombination line widths is shown to be consistent with the existence of a nucleus at the center of our own Galaxy which is similar in nature to the nucleus of M 31.

Kellman, S. A.↗

H2 recombination on interstellar grains

From a consideration of relevant theoretical and experimental data it is concluded that H atoms (but not H2 molecules) will be chemisorbed on interstellar graphite grains, with H2 formation proceeding efficiently for graphite grain temperatures less than 70 K. It is argued that graphite grains will act as the principal sites for H2 formation, with a formation rate of about 4 to the minus 17th cu cm per sec. Heating by H2 molecules formed by surface recombination is analyzed in the context of the available experimental data, and a heating rate is derived and compared with other suggested cloud heating mechanisms. It is concluded that H2 recombination will provide the largest heat source in diffuse clouds if the albedo of interstellar dust in the 912-1200 A region is high (about 0.9), whereas if the albedo in this wavelength region is lower (about 0.5), photoelectron ejection from grains will tend to predominate, and can explain observed cloud temperatures with a carbon depletion factor of approximately 2, a factor attributable to a normal interstellar abundance of graphite grains.

Barlow, M. J.↗

Enhancement of dielectronic recombination by plasma electric microfields

A description is presented of an application of techniques from Stark-broadening theory considered by Griem (1974) to the computation of dielectronic recombination rated for multiply charged Fe ions under the influence of plasma electric microfields. Attention is given to a dielectronic recombination process for which the effects of surrounding charged particles have been found to be important at surprisingly low densities.

Jacobs, V. L.↗

Narrow radio recombination lines from ionization fronts

Model weak D-type ionization fronts are used to show that narrow H I radio recombination lines, such as those observed in the directions of several galactic H II regions, may be formed in the outermost layers of an ionization front bounding an H II region. Four model ionization fronts are computed with different values of magnetic-field strength, ionizing photon energy, and initial conditions. The recombination line strengths given by the models are compared with observed line strengths for the H II region NGC 2024, and the observed line profiles are found to be about twice as broad as the model profiles. Some likely explanations for the discrepancy are considered, particularly a variation in front velocity over the observing beam width (Stark broadening is ruled out).

Hill, J. K.↗

Carrier generation, recombination, trapping, and transport in semiconductors with position-dependent composition

The spatial variation of the chemical composition of a semiconductor modifies the ideal one-electron energy band model as well as the Shockley equations for carrier recombination and transport in two important ways. The random component of the spatial variation introduces localized states in the energy gap by perturbing the band states. The nonrandom component gives rise to the position dependences of the conduction and valence band edges or the electron affinity and the energy gap. This paper gives the modifications of the Shockley equations from these two effects as well as an example of the steady-state recombination rate from distributed gap states in junction solar cells

Sah, C.-T.↗