Local proteolytic activation after pulmonary trauma is not prevented by high dose steroids

AbstractBeginning in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as a novel pathogen that causes coronavirus disease 2019 (COVID-19). SARS-CoV-2 has infected more than 111 million people worldwide and caused over 2.47 million deaths. Individuals infected with SARS-CoV-2 show symptoms of fever, cough, dyspnea, and fatigue with severe cases that can develop into pneumonia, myocarditis, acute respiratory distress syndrome, hypercoagulability, and even multi-organ failure. Current clinical management consists largely of supportive care as commonly administered treatments, including convalescent plasma, remdesivir, and high-dose glucocorticoids. These have demonstrated modest benefits in a small subset of hospitalized patients, with only dexamethasone showing demonstrable efficacy in reducing mortality and length of hospitalization. At this time, no SARS-CoV-2-specific antiviral drugs are available, although several vaccines have been approved for use in recent months. In this review, we will evaluate the efficacy of preclinical and clinical drugs that precisely target three different, essential steps of the SARS-CoV-2 replication cycle: the spike protein during entry, main protease (MPro) during proteolytic activation, and RNA-dependent RNA polymerase (RdRp) during transcription. We will assess the advantages and limitations of drugs that precisely target evolutionarily well-conserved domains, which are less likely to mutate, and therefore less likely to escape the effects of these drugs. We propose that a multi-drug cocktail targeting precise proteins, critical to the viral replication cycle, such as spike protein, MPro, and RdRp, will be the most effective strategy of inhibiting SARS-CoV-2 replication and limiting its spread in the general population.

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A Subset of High Titer Acquired Hemophilia Patients May Benefit from Treatment with High Dose Factor VIII.

Blood ◽

10.1182/blood.v114.22.3476.3476 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3476-3476

Author(s):

Shannon Meeks ◽

John F Healey ◽

Ernest T Parker ◽

Pete Lollar

Keyword(s):

Factor Viii ◽

Hemophilia A ◽

High Titer ◽

High Dose ◽

Type I ◽

C2 Domain ◽

Type Ii ◽

Acquired Hemophilia ◽

Proteolytic Activation

Abstract Abstract 3476 Poster Board III-413 Approximately 30% of patients with severe hemophilia A will develop inhibitory antibodies (Abs) to factor VIII (fVIII inhibitors). The immune response to fVIII currently is the most significant complication in the management of patients with hemophilia A. In addition, autoimmune Abs to fVIII can develop in non-hemophiliacs, producing acquired hemophilia A, which frequently produces life- or limb-threatening bleeding. Patients with autoimmune hemophilia often have Abs with type II kinetics in which there is incomplete inactivation of fVIII at saturating concentrations of inhibitor. We have characterized the antibody response to the C2 domain of human fVIII in a murine hemophilia model and described 5 structural groups of Abs. Groups A, AB, and B are classical anti-C2 Abs that block fVIII and fVIIIa binding to phospholipid. Groups BC and C consist of non-classical anti-C2 Abs that inhibit the proteolytic activation of fVIII but do not block the binding of fVIII to phospholipid. Subsequently, we identified classical and non-classical anti-C2 Abs in human fVIII inhibitor plasmas. Most murine non-classical Abs have inhibitor titers greater than 10,000 Bethesda units/mg IgG. In a murine in vivo bleeding model, both type I classical C2 Abs, type II non-classical C2 Abs, and a type I anti-A2 Ab produced similar amounts of blood loss that were significantly greater than control mice injected with 180 U/kg of fVIII alone. Increasing the dose of fVIII to 360 U/kg overcame the bleeding diathesis produced by the type II MAbs, but not the type I Abs. These results were consistent with the in vitro Bethesda assay in which a type I anti-A2 Ab, 4A4, completely inhibited both 1 U/mL and 3 U/mL fVIII, while there was 40% residual activity at saturating concentrations of a type II anti-C2 Ab, 2-77, at either concentration of fVIII. To determine if similar in vitro characteristics exist in patients with acquired hemophilia, plasmas from 3 patients with high titer type II inhibitors were studied. All 3 plasmas primarily had C2 domain epitope specificity that included non-classical Abs. Plasma A7 additionally had detectable anti-A2 activity. Recovery of fVIII activity after a 2 h incubation at 37 °C at nominal added concentrations of 1 mL and 3 U/mL fVIII was compared (Table 1). At 3 U/mL added fVIII, recovery of activity in plasmas A4 and A5 was 1.1 U/mL and 0.51 U/mL, respectively, despite the presence of inhibitor titers of 18 and 11 Bethesda units (BU) per mL. The presence of anti-A2 Abs, which typically have type I kinetics, may have contributed to the overall lower recovery of activity in plasma A7. These results suggest that treatment with high-dose fVIII rather than bypassing agents may be warranted in patients with an inhibitor response dominated by non-classical anti-C2 Abs. Table 1 Patient Plasma Inhibitor Titer (BU/mL) Recovered Activity at 1U/mL FVIII (U/mL) Recovered Activity at 3 U/mL FVIII (U/mL) A4 18 0.31 1.1 A5 11 0.18 0.51 A7 62 0.07 0.12 Disclosures: No relevant conflicts of interest to declare.

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Electron Beam Damage of Biomolecules Assessed by Energy Loss Spectroscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100069855 ◽

1978 ◽

Vol 36 (3) ◽

pp. 61-69

Author(s):

M. Isaacson ◽

M.L. Collins ◽

M. Listvan

Keyword(s):

Electron Microscopy ◽

Radiation Damage ◽

Structural Integrity ◽

Analytical Electron Microscopy ◽

High Dose ◽

Dose Rates ◽

Special Cases ◽

Analytical Electron ◽

Atom Migration ◽

Beam Damage

Over the past five years it has become evident that radiation damage provides the fundamental limit to the study of blomolecular structure by electron microscopy. In some special cases structural determinations at very low doses can be achieved through superposition techniques to study periodic (Unwin & Henderson, 1975) and nonperiodic (Saxton & Frank, 1977) specimens. In addition, protection methods such as glucose embedding (Unwin & Henderson, 1975) and maintenance of specimen hydration at low temperatures (Taylor & Glaeser, 1976) have also shown promise. Despite these successes, the basic nature of radiation damage in the electron microscope is far from clear. In general we cannot predict exactly how different structures will behave during electron Irradiation at high dose rates. Moreover, with the rapid rise of analytical electron microscopy over the last few years, nvicroscopists are becoming concerned with questions of compositional as well as structural integrity. It is important to measure changes in elemental composition arising from atom migration in or loss from the specimen as a result of electron bombardment.

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Electron-diffraction studies in synthetic polymer materials

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100075579 ◽

1983 ◽

Vol 41 ◽

pp. 362-365

Author(s):

D.T. Grubb

Keyword(s):

Electron Diffraction ◽

Synthetic Polymer ◽

Polymer Materials ◽

Crystallographic Structure ◽

High Dose ◽

Electron Dose ◽

Dose Rates ◽

First Case ◽

Diffraction Patterns ◽

The Many

Diffraction studies in polymeric and other beam sensitive materials may bring to mind the many experiments where diffracted intensity has been used as a measure of the electron dose required to destroy fine structure in the TEM. But this paper is concerned with a range of cases where the diffraction pattern itself contains the important information.In the first case, electron diffraction from paraffins, degraded polyethylene and polyethylene single crystals, all the samples are highly ordered, and their crystallographic structure is well known. The diffraction patterns fade on irradiation and may also change considerably in a-spacing, increasing the unit cell volume on irradiation. The effect is large and continuous far C94H190 paraffin and for PE, while for shorter chains to C 28H58 the change is less, levelling off at high dose, Fig.l. It is also found that the change in a-spacing increases at higher dose rates and at higher irradiation temperatures.

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Microprobe analysis of inclusion bodies related to two benzofuran derivatives

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100127785 ◽

1987 ◽

Vol 45 ◽

pp. 672-673

Author(s):

T. L. Benning ◽

P. Ingram ◽

J. D. Shelburne

Keyword(s):

Inclusion Bodies ◽

Uranyl Acetate ◽

Multiple Organ ◽

High Dose ◽

En Bloc ◽

Tissue Samples ◽

Transmission Electron ◽

Organ Systems ◽

Dense Inclusion ◽

Melanin Complex

Two benzofuran derivatives, chlorpromazine and amiodarone, are known to produce inclusion bodies in human tissues. Prolonged high dose chlorpromazine therapy causes hyperpigmentation of the skin with electron-dense inclusion bodies present in dermal histiocytes and endothelial cells ultrastructurally. The nature of the deposits is not known although a drug-melanin complex has been hypothesized. Amiodarone may also cause cutaneous hyperpigmentation and lamellar lysosomal inclusion bodies have been demonstrated within the cells of multiple organ systems. These lamellar bodies are believed to be the product of an amiodarone-induced phospholipid storage disorder. We performed transmission electron microscopy (TEM) and energy dispersive x-ray microanalysis (EDXA) on tissue samples from patients treated with these drugs, attempting to detect the sulfur atom of chlorpromazine and the iodine atom of amiodarone within their respective inclusion bodies.A skin biopsy from a patient with hyperpigmentation due to prolonged chlorpromazine therapy was fixed in 4% glutaraldehyde and processed without osmium tetroxide or en bloc uranyl acetate for Epon embedding.

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Acetaminophen: Neonatal hepatotoxicity due to late pregnancy exposure

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100127906 ◽

1987 ◽

Vol 45 ◽

pp. 718-719

Author(s):

G.A. Miranda ◽

M.A. Arroyo ◽

C.A. Lucio ◽

M. Mongeotti ◽

S.S. Poolsawat

Keyword(s):

Low Dose ◽

Fetal Liver ◽

Late Pregnancy ◽

Toxic Chemicals ◽

Control Group ◽

High Dose ◽

Over The Counter ◽

Liver And Kidney ◽

Neonatal Liver ◽

Childbearing Women

Exposure to drugs and toxic chemicals, during late pregnancy, is a common occurrence in childbearing women. Some studies have reported that more than 90% of pregnant women use at least 1 prescription; of this, 60% used more than one. Another study indicated that 80% of the consumed drugs were not prescribed, and of this figure, 95% were “over-the-counter” drugs. Acetaminophen, the safest of all over-the-counter drugs, has been reported to induce fetal liver necrosis in man and animals and to have abortifacient and embryocidal action in mice. This study examines the degree to which acetaminophen affects the neonatal liver and kidney, when a fatty diet is simultaneously fed to the mother during late pregnancy.Timed Swiss Webster female mice were gavaged during late pregnancy (days 16-19) with fat suspended acetaminophen at a high dose, HD = 84.50 mg/kg, and a low dose, LD = 42.25 mg/kg; a control group received fat alone.

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Analysis of Silicon-On-Insulator Structures Formed By Oxygen Ion Implantation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100118370 ◽

1985 ◽

Vol 43 ◽

pp. 300-301

Author(s):

N. Lewis ◽

E. L. Hall ◽

A. Mogro-Campero ◽

R. P. Love

Keyword(s):

Ion Implantation ◽

Single Crystal ◽

Single Crystal Silicon ◽

Silicon Layer ◽

Device Fabrication ◽

Silicon On Insulator ◽

High Dose ◽

Crystal Silicon ◽

Oxygen Ion ◽

Oxygen Ion Implantation

The formation of buried oxide structures in single crystal silicon by high-dose oxygen ion implantation has received considerable attention recently for applications in advanced electronic device fabrication. This process is performed in a vacuum, and under the proper implantation conditions results in a silicon-on-insulator (SOI) structure with a top single crystal silicon layer on an amorphous silicon dioxide layer. The top Si layer has the same orientation as the silicon substrate. The quality of the outermost portion of the Si top layer is important in device fabrication since it either can be used directly to build devices, or epitaxial Si may be grown on this layer. Therefore, careful characterization of the results of the ion implantation process is essential.

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Evaluation of hippocampus in rhesus monkeys after chronic (1-year) inhalation exposure to marijuana: I. Electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100159539 ◽

1990 ◽

Vol 48 (3) ◽

pp. 398-399

Author(s):

A.M. Andrews ◽

S.W. Wilson ◽

A.C. Scallet ◽

S.F. Ali ◽

J. Bailey ◽

...

Keyword(s):

Synaptic Vesicles ◽

Rhesus Monkeys ◽

Low Dose ◽

Inhalation Exposure ◽

Synaptic Cleft ◽

High Dose ◽

Withdrawal Time ◽

Treatment Groups ◽

Ultrastructural Evidence ◽

Male Rhesus

Exposure of rhesus monkeys (Macaca mulatta) to marijuana via inhalation or to intravenous delta-9-tetrahydrocannabinol (THC), reportedly caused ultrastructural evidence of increased synaptic width. Chronic marijuana smoke in a single rhesus monkey examined after a six month withdrawal time caused ultrastructure changes in the septal, hippocampal and amygdala regions; the synaptic cleft was widened, electron opaque material was found in the cleft and in the pre- and postsynaptic regions, with some clumping of the synaptic vesicles. The objective of our study was to assess neuropathological alterations produced by chronic inhalation of marijuana smoke.Nineteen male rhesus monkeys, 3-5 years of age and weighing 3-8 kg, were divided into four treatment groups: a) sham control, b) placebo smoke (7 days/ week) c) low dose marijuana (2 times/week with 5 days/week sham) and d) high dose marijuana (7 times/week). A smoke exposure consisted of smoke from one cigarette (2.6% THC) burned down to 10 mm butt length. Smoke was administered via smoke generator (ADL II, Arthur D. Little, Inc. Cambridge, MA) and nose-mouth only masks (local production) equipped with one-way valves.

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Defect reduction in oxygen implanted silicon-on-insulator material during high-temperature annealing

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100154998 ◽

1989 ◽

Vol 47 ◽

pp. 604-605

Author(s):

P. Roitman ◽

B. Cordts ◽

S. Visitserngtrakul ◽

S.J. Krause

Keyword(s):

High Temperature ◽

Annealing Temperature ◽

Silicon On Insulator ◽

High Dose ◽

High Temperature Annealing ◽

High Current ◽

Defect Reduction ◽

Annealing Step ◽

Multiple Cycle ◽

Defect Densities

Synthesis of a thin, buried dielectric layer to form a silicon-on-insulator (SOI) material by high dose oxygen implantation (SIMOX – Separation by IMplanted Oxygen) is becoming an important technology due to the advent of high current (200 mA) oxygen implanters. Recently, reductions in defect densities from 109 cm−2 down to 107 cm−2 or less have been reported. They were achieved with a final high temperature annealing step (1300°C – 1400°C) in conjunction with: a) high temperature implantation or; b) channeling implantation or; c) multiple cycle implantation. However, the processes and conditions for reduction and elimination of precipitates and defects during high temperature annealing are not well understood. In this work we have studied the effect of annealing temperature on defect and precipitate reduction for SIMOX samples which were processed first with high temperature, high current implantation followed by high temperature annealing.

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TEM study of buried cobalt disilicide layers formed by ion implantation: Identification of cobalt monosilicide inclusions

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100176010 ◽

1990 ◽

Vol 48 (4) ◽

pp. 576-577

Author(s):

A. De Veirman ◽

J. Van Landuyt ◽

K.J. Reeson ◽

R. Gwilliam ◽

C. Jeynes ◽

...

Keyword(s):

Ion Implantation ◽

Annealing Treatment ◽

Silicon On Insulator ◽

High Dose ◽

Nitrogen Flow ◽

Cobalt Disilicide ◽

Transmission Electron ◽

Beam Heating ◽

Co Concentration ◽

Silicon On Insulator Technology

In analogy to the formation of SIMOX (Separation by IMplanted OXygen) material which is presently the most promising silicon-on-insulator technology, high-dose ion implantation of cobalt in silicon is used to synthesise buried CoSi2 layers. So far, for high-dose ion implantation of Co in Si, only formation of CoSi2 is reported. In this paper it will be shown that CoSi inclusions occur when the stoichiometric Co concentration is exceeded at the peak of the Co distribution. 350 keV Co+ ions are implanted into (001) Si wafers to doses of 2, 4 and 7×l017 per cm2. During the implantation the wafer is kept at ≈ 550°C, using beam heating. The subsequent annealing treatment was performed in a conventional nitrogen flow furnace at 1000°C for 5 to 30 minutes (FA) or in a dual graphite strip annealer where isochronal 5s anneals at temperatures between 800°C and 1200°C (RTA) were performed. The implanted samples have been studied by means of Rutherford Backscattering Spectroscopy (RBS) and cross-section Transmission Electron Microscopy (XTEM).

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