Steric exclusion chromatography for the purification of recombinant baculovirus

Steric exclusion chromatography (SXC) has already proven to be a valuable tool in the purification of proteins and virus particles. An important benefit of the method is the fast and simple procedure at mild chromatography conditions as no harsh binding and elution buffers are needed. The sample is initially mixed with a polyethylene glycol (PEG) containing buffer of choice. The steric exclusion of a macromolecule from the polyethylene glycol and the stationary phase allows a selective retention of the product, depending, among others, mainly on its size as well as on the molecular weight and concentration of the PEG. Here, SXC was set up in order that smaller process contaminants, i.e. host cell proteins and DNA, did not bind to the stationary phase, in contrast to the targeted larger virus particles. These were subsequently eluted reducing the PEG concentration in the mobile phase. Regenerated cellulose was used as stationary phase to purify VSV-G pseudotyped AcMNPV baculoviruses derived from Spodoptera frugiperda cells (Sf9 cells) by SXC. The purified virus particles are used as gene transfer tools for human mesenchymal stroma cells. For this purpose, the baculovirus was clarified prior to the SXC by sequential centrifugation (4700 gmax). The SXC conditions were optimized in terms of yield and purity by a design of experiment approach considering the PEG molecular weight, its concentration and the ionic strength of the elution buffer as critical process parameters. Within the design space virus recovery was ≥70%. Without further nuclease treatment the depletion of double-stranded DNA was \u3e90% and the amount of host cell proteins were reduced \u3e90% in the virus fraction. In conclusion, SXC can drastically reduce the process development in terms of time and equipment requirements for the purification of recombinant baculoviruses, as well as for the achieved purity which is superior over classical methods

Role of complement in in vitro and in vivo lung inflammatory reactions

Complement is one of the integral buttresses of the inflammatory response. In addition to host defense activities, proinflammatory properties of several complement components are described. This overview elucidates the role of complement in inflammatory reactions in vitro and in vivo, focusing on the complement activation products, C5a, and the membrane attack complex, C5b‐9. Using several approaches, the impact of these complement components in mechanisms relevant to neutrophil recruitment is emphasized. In addition, the participation of complement in endothelial superoxide generation and its essential requirement for full expression of lung injury is demonstrated, as are the involved intracellular signal transduction pathways. Understanding the mechanisms of complement‐induced proinflammatory effects may provide a basis for future therapeutic blockade of complement and/or its activation products. J. Leukoc. Biol. 64: 40–48; 1998.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142061/1/jlb0040.pd

Regulatory effects of interleukin‐11 during acute lung inflammatory injury

The role of interleukin‐11 (IL‐11) was evaluated in the IgG immune complex model of acute lung injury in rats. IL‐11 mRNA and protein were both up‐regulated during the course of this inflammatory response. Exogenously administered IL‐11 substantially reduced, in a dose‐dependent manner, the intrapulmonary accumulation of neutrophils and the lung vascular leak of albumin. These in vivo anti‐inflammatory effects of IL‐11 were associated with reduced NF‐κB activation in lung, reduced levels of tumor necrosis factor α (TNF‐α) in bronchoalveolar lavage (BAL) fluids, and diminished up‐regulation of lung vascular ICAM‐1. It is interesting that IL‐11 did not affect BAL fluid content of the CXC chemokines, macrophage inflammatory protein‐2 (MIP‐2) and cytokine‐inducible neutrophil chemoattractant (CINC); the presence of IL‐11 did not affect these chemokines. However, BAL content of C5a was reduced by IL‐11. These data indicate that IL‐11 is a regulatory cytokine in the lung and that, like other members of this family, its anti‐inflammatory properties appear to be linked to its suppression of NF‐κB activation, diminished production of TNF‐α, and reduced up‐regulation of lung vascular ICAM‐1. J. Leukoc. Biol. 66: 151–157; 1999.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141937/1/jlb0151.pd

Mediators of Microvascular Injury in Dermal Burn Wounds

In previous studies we have demonstrated that second-degree thermal injury of skin in rats leads to secondary effects, such as systemic complement activation, C5a-mediated activation of blood neutrophils, their adhesion-molecule-guided accumulation in lung capillaries and the development of acute pulmonary injury, largely caused by neutrophil-derived toxic oxygen metabolites. In the dermal burn wound, however, pathophysiologic events are less well understood. The injury is fully developed at four hours post-burn. To further elucidate the pathogenesis of the “late phase” dermal vascular damage, rats were depleted of neutrophils or complement by pretreatment with rabbit antibody against rat neutrophils or with cobra venom factor, respectively. In other experiments, rats were treated with blocking antibodies to IL-6, IL-1, and TNFα immediately following thermal burning or were pretreated with hydroxyl radical scavengers (dimethyl sulfoxide, dimethyl thiourea). Extravasation of 125 I-labeled bovine serum albumin into the burned skin was studied, as well as, skin myeloperoxidase levels. The studies revealed that, like in secondary lung injury, neutrophils and toxic oxygen metabolites, are required for full development of microvascular injury. In contrast, however, development of dermal vascular damage in thermally injured rats was not affected by complement depletion. Our data suggest that the development of microvascular injury in the dermal burn wound is complement-independent, involves the pro-inflammatory cytokines IL-1, TNFα and IL-6, and may result from reactive oxygen metabolites generated by neutrophils accumulating in the burn wound.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44523/1/10753_2004_Article_415307.pd

Silicon detector for a Compton Camera in Nuclear Medical Imaging

Electronically collimated gamma ca\-me\-ras based on Com\-pton scattering in silicon pad sensors may improve imaging in nuclear medicine and bio-medical research. The work described here concentrates on the silicon pad detector developed for a prototype Compton camera. The silicon pad sensors are read out using low noise VLSI CMOS chips and novel fast triggering chips. Depending on the application a light weight and dense packaging of sensors and its readout electronics on a hybrid is required. We describe the silicon pad sensor and their readout with the newly designed hybrid. %The silicon detector of a Compton camera %may contain up to $10^5$~analogue channels requiring %a fast and low cost data acquisition system. We also describe a modular and low-cost data acquisition system (CCDAQ) based on a digital signal processor which is interfaced to the EPP port of personal computers. Using the CCDAQ and the hybrids energy spectra of gamma-ray photons from technetium ($^{\rm 99m}_{43}$Tc) and americium ($^{241}_{95}$Am) were acquired with an energy resolution of 2.45~keV FWHM for the 140.5~keV photo-absorption line of $^{\rm 99m}_{43}$Tc. For all pads the discrimination threshold in the trigger chip was between (15 and 25)~keV

Measurement of inclusive D*+- and associated dijet cross sections in photoproduction at HERA

Inclusive photoproduction of D*+- mesons has been measured for photon-proton centre-of-mass energies in the range 130 < W < 280 GeV and a photon virtuality Q^2 < 1 GeV^2. The data sample used corresponds to an integrated luminosity of 37 pb^-1. Total and differential cross sections as functions of the D* transverse momentum and pseudorapidity are presented in restricted kinematical regions and the data are compared with next-to-leading order (NLO) perturbative QCD calculations using the "massive charm" and "massless charm" schemes. The measured cross sections are generally above the NLO calculations, in particular in the forward (proton) direction. The large data sample also allows the study of dijet production associated with charm. A significant resolved as well as a direct photon component contribute to the cross section. Leading order QCD Monte Carlo calculations indicate that the resolved contribution arises from a significant charm component in the photon. A massive charm NLO parton level calculation yields lower cross sections compared to the measured results in a kinematic region where the resolved photon contribution is significant.Comment: 32 pages including 6 figure

R & D for collider beauty physics at the LHC

We propose an R&D program for the development of a Beauty trigger and innovative elements of the associated spectrometer. A series of short test runs is proposed at the SPS p-pbar Collider with the minimal spectrometer which will allow a credible B signal to be obtained in an invariant mass spectrum of reconstructed B mesons. The program builds on the success of the recent collider run of the P238 Collaboration, in which clean signals from beam-beam interactions were observed in a large silicon strip microvertex detector running 1.5 mm from the circulating beams. A continuing successful R&D program of the type proposed could ultimately lead to a collider experiment at the LHC to study CP Violation and rare B decays

Activation of adherent vascular neutrophils in the lung during acute endotoxemia

BACKGROUND: Neutrophils constitute the first line of defense against invading microorganisms. Whereas these cells readily undergo apoptosis under homeostatic conditions, their survival is prolonged during inflammatory reactions and they become biochemically and functionally activated. In the present study, we analyzed the effects of acute endotoxemia on the response of a unique subpopulation of neutrophils tightly adhered to the lung vasculature. METHODS: Rats were treated with 5 mg/kg lipopolysaccharide (i.v.) to induce acute endotoxemia. Adherent neutrophils were isolated from the lung vasculature by collagenase digestion and sequential filtering. Agarose gel electrophoresis, RT-PCR, western blotting and electrophoretic mobility shift assays were used to evaluate neutrophil activity. RESULTS: Adherent vascular neutrophils isolated from endotoxemic animals exhibited decreased apoptosis when compared to cells from control animals. This was associated with a marked increase in expression of the anti-apoptotic protein, Mcl-1. Cells isolated 0.5–2 hours after endotoxin administration were more chemotactic than cells from control animals and expressed increased tumor necrosis factor-alpha and cyclooxygenase-2 mRNA and protein, demonstrating that they are functionally activated. Endotoxin treatment of the animals also induced p38 and p44/42 mitogen activated protein kinases in the adherent lung neutrophils, as well as nuclear binding activity of the transcription factors, NF-κB and cAMP response element binding protein. CONCLUSION: These data demonstrate that adherent vascular lung neutrophils are highly responsive to endotoxin and that pathways regulating apoptosis and cellular activation are upregulated in these cells

Targeted Deletion of HIF-1α Gene in T Cells Prevents their Inhibition in Hypoxic Inflamed Tissues and Improves Septic Mice Survival

Sepsis patients may die either from an overwhelming systemic immune response and/or from an immunoparalysis-associated lack of anti-bacterial immune defence. We hypothesized that bacterial superantigen-activated T cells may be prevented from contribution into anti-bacterial response due to the inhibition of their effector functions by the hypoxia inducible transcription factor (HIF-1alpha) in inflamed and hypoxic areas.Using the Cre-lox-P-system we generated mice with a T-cell targeted deletion of the HIF-1alpha gene and analysed them in an in vivo model of bacterial sepsis. We show that deletion of the HIF-1alpha gene leads to higher levels of pro-inflammatory cytokines, stronger anti-bacterial effects and much better survival of mice. These effects can be at least partially explained by significantly increased NF-kappaB activation in TCR activated HIF-1 alpha deficient T cells.T cells can be recruited to powerfully contribute to anti-bacterial response if they are relieved from inhibition by HIF-1alpha in inflamed and hypoxic areas. Our experiments uncovered the before unappreciated reserve of anti-bacterial capacity of T cells and suggest novel therapeutic anti-pathogen strategies based on targeted deletion or inhibition of HIF-1 alpha in T cells

Quadrupole collectivity in Ca 42 from low-energy Coulomb excitation with AGATA

A Coulomb-excitation experiment to study electromagnetic properties of Ca42 was performed using a 170-MeV calcium beam from the TANDEM XPU facility at INFN Laboratori Nazionali di Legnaro. γ rays from excited states in Ca42 were measured with the AGATA spectrometer. The magnitudes and relative signs of ten E2 matrix elements coupling six low-lying states in Ca42, including the diagonal E2 matrix elements of 21+ and 22+ states, were determined using the least-squares code gosia. The obtained set of reduced E2 matrix elements was analyzed using the quadrupole sum rule method and yielded overall quadrupole deformation for 01,2+ and 21,2+ states, as well as triaxiality for 01,2+ states, establishing the coexistence of a weakly deformed ground-state band and highly deformed slightly triaxial sideband in Ca42. The experimental results were compared with the state-of-the-art large-scale shell-model and beyond-mean-field calculations, which reproduce well the general picture of shape coexistence in Ca42