Macrophage phagocytosis of SARS-CoV-2-infected cells mediates potent plasmacytoid dendritic cell activation.

Early and strong interferon type I (IFN-I) responses are usually associated with mild COVID-19 disease, whereas persistent or unregulated proinflammatory cytokine responses are associated with severe disease outcomes. Previous work suggested that monocyte-derived macrophages (MDMs) are resistant and unresponsive to SARS-CoV-2 infection. Here, we demonstrate that upon phagocytosis of SARS-CoV-2-infected cells, MDMs are activated and secrete IL-6 and TNF. Importantly, activated MDMs in turn mediate strong activation of plasmacytoid dendritic cells (pDCs), leading to the secretion of high levels of IFN-α and TNF. Furthermore, pDC activation promoted IL-6 production by MDMs. This kind of pDC activation was dependent on direct integrin-mediated cell‒cell contacts and involved stimulation of the TLR7 and STING signaling pathways. Overall, the present study describes a novel and potent pathway of pDC activation that is linked to the macrophage-mediated clearance of infected cells. These findings suggest that a high infection rate by SARS-CoV-2 may lead to exaggerated cytokine responses, which may contribute to tissue damage and severe disease.

Accidental mechanical asphyxia of children in Germany between 2000 and 2008.

Accidents constitute one of the greatest risks to children, yet there are few medical reports that discuss the subject of accidental asphyxia. However, a systematic analysis of all documented cases in Germany over the years 2000-2008 has now been conducted, aiming at identifying patterns of accidental asphyxia, deducing findings, defining avoidance measures and recommending ways of increasing product safety and taking possible precautions. The analysis is based on a detailed retrospective analysis of all 91 relevant autopsy reports from 24 different German forensic institutes. A variety of demographic and morphological data was systematically collected and analysed. In 84 of the 91 cases, the sex of the victim was reported, resulting in a total of 57 boys (68 %) and 27 girls (32 %). The age spread ranged between 1 day and 14 years, with an average of 5.9 years. Most accidents occurred in the first year of life (20 %) or between the ages of 1 and 2 years (13 %). In 46 % of cases, the cause of death was strangulation, with the majority occurring in the home environment. In 31 % of all cases, the cause of death was positional asphyxia, the majority resulting from chest compression. In 23 % of cases, the cause of death was aspiration, mainly of foreign bodies. Today, accidental asphyxiation is a rare cause of death in children in Germany. Nevertheless, the majority of cases could have been avoided. Future incidence can be reduced by implementing two major precautions: increasing product safety and educating parents of potentially fatal risks. Specific recommendations relate to children's beds, toys and food.

Petechial bleedings in sudden infant death.

The autopsy reports of 484 cases of deceased infants (201 females, 283 males) were analysed retrospectively for the existence of external and internal petechial bleedings (PET). The cases were divided into five groups on the basis of the cause of death (sudden infant death syndrome, sepsis, airway infections, asphyxia and trauma). Internal PET (pleural, pericardial, epicardial, thymic and peritoneal) were observed in each group with a lower prevalence in cases of trauma. The highest prevalence of external (cutaneous and conjunctival) PET was detected in cases of asphyxia (38% and 31%, respectively). However, even if with low prevalence, such bleedings were detected in every group. Factors like sex, age, cardiopulmonary resuscitation and its duration did not influence the presence of PET. The detection of external PET at autopsy is a suspicious finding that suggests asphyxia. Because of the possible natural origin of these bleedings, the medicolegal investigation has to be as complete as possible and has to include histology as mandatory.

Initial operation and data processing on a system for real-time evaluation of Thomson scattering signals on the Large Helical Device.

A scalable system for real-time analysis of electron temperature and density based on signals from the Thomson scattering diagnostic, initially developed for and installed on the NSTX-U experiment, was recently adapted for the Large Helical Device and operated for the first time during plasma discharges. During its initial operation run, it routinely recorded and processed signals for four spatial points at the laser repetition rate of 30 Hz, well within the system's rated capability for 60 Hz. We present examples of data collected from this initial run and describe subsequent adaptations to the analysis code to improve the fidelity of the temperature calculations.

The severe combined immunodeficiency (scid) mouse. A laboratory model for the analysis of Lyme arthritis and carditis.

We report that the spirochete B. burgdorferi induces progressive polyarthritis and carditis in mice with severe combined immunodeficiency syndrome (scid) but not in normal C.B-17 mice. The onset and severity of the disease were dependent on (a) the viability; (b) the infectivity; and (c) the dose of inoculated B. burgdorferi organisms. Infective spirochetes were isolated from both blood and joints of inoculated scid mice. These findings suggest that B. burgdorferi-induced chronic arthritis and carditis in mice develops independently of lymphocyte function and makes the scid mouse an attractive laboratory model to study the role of the immune system in experimental Lyme Borreliosis.

T cell responses in calcineurin A alpha-deficient mice.

We have created embryonic stem (ES) cells and mice lacking the predominant isoform (alpha) of the calcineurin A subunit (CNA alpha) to study the role of this serine/threonine phosphatase in the immune system. T and B cell maturation appeared to be normal in CNA alpha -/- mice. CNA alpha -/- T cells responded normally to mitogenic stimulation (i.e., PMA plus ionomycin, concanavalin A, and anti-CD3 epsilon antibody). However, CNA alpha -/- mice generated defective antigen-specific T cell responses in vivo. Mice produced from CNA alpha -/- ES cells injected into RAG-2-deficient blastocysts had a similar defective T cell response, indicating that CNA alpha is required for T cell function per se, rather than for an activity of other cell types involved in the immune response. CNA alpha -/- T cells remained sensitive to both cyclosporin A and FK506, suggesting that CNA beta or another CNA-like molecule can mediate the action of these immunosuppressive drugs. CNA alpha -/- mice provide an animal model for dissecting the physiologic functions of calcineurin as well as the effects of FK506 and CsA.

Validation of a commercially available SARS-CoV-2 serological immunoassay.

OBJECTIVES: To validate the diagnostic accuracy of a Euroimmun SARS-CoV-2 IgG and IgA immunoassay for COVID-19. METHODS: In this unmatched (1:2) case-control validation study, we used sera of 181 laboratory-confirmed SARS-CoV-2 cases and 326 controls collected before SARS-CoV-2 emergence. Diagnostic accuracy of the immunoassay was assessed against a whole spike protein-based recombinant immunofluorescence assay (rIFA) by receiver operating characteristic (ROC) analyses. Discrepant cases between ELISA and rIFA were further tested by pseudo-neutralization assay. RESULTS: COVID-19 patients were more likely to be male and older than controls, and 50.3% were hospitalized. ROC curve analyses indicated that IgG and IgA had high diagnostic accuracies with AUCs of 0.990 (95% Confidence Interval [95%CI]: 0.983-0.996) and 0.978 (95%CI: 0.967-0.989), respectively. IgG assays outperformed IgA assays (p=0.01). Taking an assessed 15% inter-assay imprecision into account, an optimized IgG ratio cut-off > 2.5 displayed a 100% specificity (95%CI: 99-100) and a 100% positive predictive value (95%CI: 96-100). A 0.8 cut-off displayed a 94% sensitivity (95%CI: 88-97) and a 97% negative predictive value (95%CI: 95-99). Substituting the upper threshold for the manufacturer's, improved assay performance, leaving 8.9% of IgG ratios indeterminate between 0.8-2.5. CONCLUSIONS: The Euroimmun assay displays a nearly optimal diagnostic accuracy using IgG against SARS-CoV-2 in patient samples, with no obvious gains from IgA serology. The optimized cut-offs are fit for rule-in and rule-out purposes, allowing determination of whether individuals in our study population have been exposed to SARS-CoV-2 or not. IgG serology should however not be considered as a surrogate of protection at this stage.

Spontaneous sister chromatid exchange in normal bone marrow and Ph-positive chronic myelocytic leukemia.

The frequency of spontaneous sister chromatid exchange was studied in normal marrow derived from 38 healthy donors and 40 untreated patients with chronic phase CML. The sister chromatid exchange frequency was significantly lower in the leukemic cells (range, 2.32 +/- 1.31 to 4.76 +/- 2.37 per metaphase; mean, 3.18 +/- 0.49) than in normal marrow (range, 2.36 +/- 1.44 to 5.54 +/- 2.24 per metaphase; mean, 3.92 +/- 0.72). The contraction status of chromosomes was comparable in normal and Ph-positive metaphases. The reduction of sister chromatid exchange in leukemic cells was seen in all chromosome groups. The analysis of cell cycle specific proliferation according to the typical staining patterns of metaphases due to the number of cell cycles during which bromodesoxyuridine was substituted, revealed longer cell cycle times for the leukemic cells.

Oligomycin sensitivity of mitochondrial sulfhydryl groups.

Dithionitrobenzoate has been used to titrate sulfhydryl groups of rat liver mitochondria in glutamate buffer, pH 7.4. Reaction with oligomycin and different SH reagents preceded the SH titration. Under these conditions it was found that 2-mercaptopropionylcne and N-ethylmaleimide reacted in an oligomycin-sensitive manner, so that the control values (in the absence of SH reagent) were obtained. Similar concentrations of mersalyl and of N-(N-acetyl-4-sulfamoylphenyl) maleimide, in the presence of oligomycin, enhanced reactivity toward Nbs2. The concentration range of oligomycin-sensitive SH groups was thus defined between approx. 5 and 9 nmol reagent/mg mitochondrial protein. In this way, a differentiation between SH grops, which are implicated in phosphate transport antd those, which react in an oligomycin-sensitive manner, and which are probably connected with the coupling mechanism was achieved.

Lipid-protein interactions at the erythrocyte membrane. Different influence of glucose and sorbose on membrane lipid transition.

When observed over a temperature range, erythrocyte membrane lipids undergo a transition at 18-20 degrees C (Zimmer, G. and Schirmer, H. (1974) biochim. Biophys. Acta 345, 314-320). This observation has prompted an investigation of the effects that substrate binding has on the transition of the red cell membrane. Glucose and sorbose were compared, since transport kinetics of these sugars still pose unresolved questions. In membranes, preloaded with glucose, the break at the transition temperature was intensified, while it was abolished or reversed in membranes preloaded with sorbose. These results were corroborated using different solubilization procedures (sonication, sodium dodecyl sulfate treatment) of the membranes, and also different techniques (viscosimetry, 90 degrees light scattering, 1-anilino-naphthalene-8-sulfonate fluorescence). In extracted membrane lipids, viscosimetry indicated a break at transition temperature after preloading with either glucose or sorbose. Disc electrophoresis revealed a different binding pattern of the two sugars. It is suggested, that the amplification of the discontinuity in red cell membranes by glucose and the abolition or reversal of the break by sorbose are mediated by membrane protein- and/or membrane lipid-protein interaction.

An interspecies approach to the investigation of the red cell membrane glucose transporter.

Glucose transport differs in red cells of various species. The following sequence of transport velocities was found: man greater than newborn pig greater than rat, dog greater than cattle greater than pig. No correlation was found between the amount of protein in band 3 and glucose transport activity. By contrast, a very clear peak in the band 4.5 region was found for newborn pigs, whereas adult pigs did not exhibit a corresponding peak in the electrophoresis. Thus further evidence is provided by our investigations in favour of band 4.5 region for glucose transport activity in red cells.

Prostaglandin E2 directly protects isolated rat gastric surface cell membranes against bile salts.

Rat gastric surface cell membranes were prepared and the effect of taurocholic acid assessed by ESR spectroscopy using the 16-doxylstearic acid spin label. Taurocholic acid increased the polar part of the spectra, indicating an augmented amount of spin label molecules with a polar environment. Concomitantly, mobility of the spin label molecule was augmented. The effect of taurocholic acid was completely prevented by the previous addition of prostaglandin E2. This suggests a direct protective efficiency of prostaglandin E2 on rat gastric surface cell membranes without the metabolic participation of intact cells.

Ursodeoxycholate stabilizes phospholipid-rich membranes and mimics the effect of cholesterol: investigations on large unilamellar vesicles.

Ursodeoxycholate is used to treat primary biliary cirrhosis and is incorporated into hepatocyte plasma membranes. Its steroid nucleus binds to the apolar domain of the membrane, in a similar position to cholesterol. Therefore the question arises whether ursodeoxycholate has a similar effect on membrane structure and stability as cholesterol. Using differential scanning calorimetry the thermotropic behavior of egg phosphatidylcholine and dimyristoylphosphatidylcholine were studied after incubation with cholesterol or ursodeoxycholate. Large unilamellar vesicles were prepared with cholesterol contents of 0-50%. Following incubation of these vesicles with different amounts of ursodeoxycholate, vesicle stability in a gravitational field was investigated by measuring the phospholipid and cholesterol release. Vesicle size was studied by laser light scattering after incubation with cheno- and ursodeoxycholate, and the release of entrapped carboxyfluorescein was measured by means of fluorescence spectroscopy. Increasing cholesterol diminished the enthalpy of the phase transition in the membrane. Ursodeoxycholate decreased the enthalpy of the phase transition at even lower concentrations. Lipid release from vesicles in a high gravitational field diminished with increasing cholesterol content of the vesicles. Ursodeoxycholate had a comparable effect, which increased as the cholesterol content of the vesicles was decreased. Chenodeoxycholate damaged vesicles, whereas ursodeoxycholate did not. Cholesterol and ursodeoxycholate (below its critical micellar concentration) decreased the carboxyfluorescein release from vesicles induced by chenodeoxycholate. Thus like cholesterol, ursodeoxycholate is incorporated into phospholipid model membranes and reduces the change in enthalpy of the gel to liquid-crystalline phase transition. Like cholesterol ursodeoxycholate also maintains membrane stability and prevents membrane damage induced by mechanical and chemical stress.

The effect of bile salts and calcium on isolated rat liver mitochondria.

Intact mitochondria were incubated with and without calcium in solutions of chenodeoxycholate, ursodeoxycholate, or their conjugates. Glutamate dehydrogenase, protein and phospholipid release were measured. Alterations in membrane and organelle structure were investigated by electron paramagnetic resonance spectroscopy. Chenodeoxycholate enhanced enzyme liberation, solubilized protein and phospholipid, and increased protein spin label mobility and the polarity of the hydrophobic membrane interior, whereas ursodeoxycholate and its conjugates did not damage mitochondria. Preincubation with ursodeoxycholate or its conjugate tauroursodeoxycholate for 20 min partially prevented damage by chenodeoxycholate. Extended preincubation even with 1 mM ursodeoxycholate could no longer prevent structural damage. Calcium (from 0.01 mM upward) augmented the damaging effect of chenodeoxycholate (0.15-0.5 mM). The combined action of 0.01 mM calcium and 0.15 mM chenodeoxycholate was reversed by ursodeoxycholate only, not by its conjugates tauroursodeoxycholate and glycoursodeoxycholate. In conclusion, ursodeoxycholate partially prevents chenodeoxycholate-induced glutamate dehydrogenase release from liver cell mitochondria by membrane stabilization. This holds for shorter times and at concentrations below 0.5 mM only, indicating that the different constitution of protein-rich mitochondrial membranes does not allow optimal stabilization such as has been seen in phospholipid- and cholesterol-rich hepatocyte cell membranes, investigated previously.

Decreased concentration of myofibrils and myofiber hypertrophy are structural determinants of impaired left ventricular function in patients with chronic heart diseases: a multiple logistic regression analysis.

OBJECTIVES: The aim of this study was to perform a multiple logistic regression analysis to identify independent structural determinants of impaired left ventricular function. BACKGROUND: The association between contractile failure and structural alterations of the myocardium has been demonstrated in several studies, and multiple interactions between myocardial structure and cardiac performance are likely. METHODS: Morphometric data assessed from 130 left ventricular biopsy specimens were analyzed. The endomyocardial specimens were obtained from 57 patients with normal coronary arteries (17 with normal left ventricular ejection fraction and 40 with impaired left ventricular function [dilated cardiomyopathy]), 15 patients with hypertrophic cardiomyopathy and 32 patients with aortic valve disease. Transmural biopsy specimens were assessed in 6 donor hearts before heart transplantation and in 20 patients with left anterior descending coronary artery disease whose specimens were obtained from the left ventricular anterior wall during aortocoronary bypass surgery. Global or regional left ventricular function was evaluated from left cineventriculograms. The volume fraction of cardiac fibrous tissue, intracellular volume fraction of myofibrils, volume fraction of myofibrils related to myocardial tissue (including fibrosis) and myofiber diameters were determined from semithin sections of the biopsy specimens with the use of light microscopic morphometry. RESULTS: Multiple logistic regression analysis revealed decreased volume fraction of myofibrils (p < 0.005) and increased fiber diameter (p < 0.002) as independent determinants of impaired left ventricular function. CONCLUSIONS: These data indicate that, independent of the underlying heart disease, both decreased concentration of contractile proteins and myocyte hypertrophy are independently associated with impaired left ventricular function.

Electron paramagnetic resonance (EPR) imaging in skin: biophysical and biochemical microscopy.

Electron paramagnetic resonance (EPR) is a spectroscopic technique that allows detection of paramagnetic compounds, e.g., free radicals, in skin, and is used for measuring skin membrane fluidity and polarity. EPR imaging is concerned with spatially resolved EPR spectroscopy. We studied EPR images at X-band frequency (9 GHz) in the skin biopsies of hairless mice. Our particular imaging technique utilized a modulated field gradient to obtain cross-sectional images perpendicular to the skin surface. Employing nitroxide free radicals, this approach allows analysis of skin biophysical and biochemical features at the micrometer resolution level. By spin labeling drugs, pharmacokinetic properties of the labeled compound can be monitored in skin. We suggest that EPR imaging has a broad application potential in dermatologic research. In comparison to other spatially visualizing techniques, such as nuclear magnetic resonance (NMR) imaging and ultrasound, EPR imaging has significant advantages, such as high spatial resolution and providing specific biochemical and biophysical information that cannot be obtained by other methods in skin.

Electron paramagnetic resonance studies on nitroxide radical 2,2,5,5-tetramethyl-4-piperidin-1-oxyl (TEMPO) redox reactions in human skin.

Electron paramagnetic resonance (EPR) is currently being explored for the study of living biological systems. Among biophysical and biochemical applications, the study of nitroxide radical interactions with tissue antioxidants and oxidants is of growing interest. Skin is a target organ of the EPR methodology and is frequently exposed to oxidative stress. We investigated the piperidine-type nitroxide 2,2,5,5-tetramethyl-4-piperidin-1-oxyl (TEMPO) because it is skin permeable and readily accepts electrons in biological systems. TEMPO is readily scavenged on the surface of cultured human skin. Pretreatment of skin cultures with butylhydroperoxide, which decreases intracellular ascorbate and glutathione, causes inhibition of nitroxide scavenging. Exposure of skin cultures to dehydroascorbate, which is internalized and converted to ascorbate, leads to stimulation of nitroxide scavenging. In human keratinocytes and fibroblasts, the TEMPO radical is reversibly reduced to the hydroxylamine depending on the oxygen concentration and the availability of intracellular glutathione and ascorbate. Cell exposure to the glutathione synthetase inhibitor buthionine-sulfoximine depleted intracellular glutathione and inhibited nitroxide reduction; exposure to dehydroascorbate or glutathione-monoethylester increased intracellular ascorbate or glutathione concentration and stimulated nitroxide reduction. Quantitative considerations indicate that the major reduction site of TEMPO in skin and skin cells is the cytosol ascorbate/glutathione redox cycle. We suggest that analysis of TEMPO radical scavenging by the EPR technique is a convenient method for measuring skin ascorbate and thiol-dependent antioxidant activity in vitro and in vivo.

Bromobimane crosslinking studies in oligomycin-sensitive ATPase from beef heart mitochondria. Mr 31 000 protein crosslinked.

Using a bromobimane fluorescent label the Mr 31 000 protein band oligomycin-sensitive (OS)-ATPase from beef heart mitochondria is shown to become much intensified by 2-mercaptopropionylglycine. In the presence of 3.5 nmol/mg protein of the thiol reagent ATP-Pi exchange activity is increased by 90%. With the fluorescent crosslinking reagent dibromobimane (DB) we show that a new fluorescent peak appears between Mr 50 000 and 60 000. ATP-Pi exchange is very much decreased by DB. The results suggest that for regulation of ATP-synthetase activity sulfhydryl groups in the region of the Mr 31 000 protein(s) play an important role.

Nitroxide radical biostability in skin.

Nitroxide radicals are important chemical tools in dermatologic research (e.g., for studying biophysical properties of skin lipids and epidermal membranes with the method of electron paramagnetic resonance, EPR, spectroscopy). However, nitroxides may loose their paramagnetic properties in biological tissues, which could limit their usefulness in biomedical applications. We analyzed the biostability of various chemical types of nitroxide radicals in keratinocytes, epidermis homogenate, and intact skin. EPR signal loss of imidazoline, pyrrolidine, piperidine, and oxazolidine nitroxides is attributed to their reduction to the corresponding hydroxylamine. The rate of nitroxide reduction in skin varies considerably with nitroxide ring structure and substitution. The order of nitroxide stability in isolated human keratinocytes, mouse epidermis homogenate, and intact mouse and human skin is imidazoline > pyrrolidine > di-t-butylnitroxide (DTBN) > piperidine > oxazolidine. Cationic nitroxides are reduced much faster than neutral or anionic probes, presumably due to transmembrane electron shuttle or internalization. The results indicate that imidazoline- and pyrrolidine-type nitroxides should be used when high biostability of nitroxides is needed. Piperidine-type nitroxides are versatile probes for studying one-electron transfer reactions in skin.

Reversal of multidrug resistance and increase in plasma membrane fluidity in CHO cells with R-verapamil and bile salts.

Studies with multidrug resistance modifiers indicate that perturbations of the cell membrane structure may influence P-glycoprotein (P-gp)-mediated drug transport. We describe studies of plasma membrane order using electron-paramagnetic resonance (EPR) in resistant (CH(R)C5) and sensitive (AUXB1) chinese hamster ovary cells treated with R-verapamil and bile salts. Cell growth rates were determined in presence of doxorubicin mitomycin and cisplatin. The plasma membrane order in untreated resistant cells was higher than in the sensitive cells. Both the bile salt taurochenodeoxycholate (TCDC; 0.2-1.6 mM) and R-verapamil (1-3 microM) lowered the membrane order in the CH(R)C5 cells to that in the sensitive cells and reversed the resistance to doxorubicin and mitomycin. The bile salt tauroursodeoxycholate (TUDC; 0.2-3 mM) did not lower membrane order and did not sensitise CH(R)C5 cells. Neither R-verapamil, TCDC nor TUDC reduced the membrane order of the sensitive cells AUXB1 cells. These results support the view that changes in multidrug resistance in Chinese hamster ovary cells and P-gp function are associated with alterations in the fluidity of the plasma membrane.