Phagocyte migration and cellular stress induced in liver, lung, and intestine during sleep loss and sleep recovery

Sleep is understood to possess recuperative properties and, conversely, sleep loss is associated with disease and shortened life span. Despite these critical attributes, the mechanisms and functions by which sleep and sleep loss impact health still are speculative. One of the most consistent, if largely overlooked, signs of sleep loss in both humans and laboratory rats is a progressive increase in circulating phagocytic cells, mainly neutrophils. The destination, if any, of the increased circulating populations has been unknown and, therefore, its medical significance has been uncertain. The purpose of the present experiment was to determine the content and location of neutrophils in liver and lung tissue of sleep-deprived rats. These are two principal sites affected by neutrophil migration during systemic inflammatory illness. The content of neutrophils in the intestine also was determined. Sleep deprivation in rats was produced for 5 and 10 days by the Bergmann-Rechtschaffen disk method, which has been validated for its high selectivity under freely moving conditions and which was tolerated and accompanied by a deep negative energy balance. Comparison groups included basal conditions and 48 h of sleep recovery after 10 days of sleep loss. Myeloperoxidase (MPO), an enzyme constituent of neutrophils, was extracted from liver, lung, and intestinal tissues, and its activity was determined by spectrophotometry. Leukocytes were located in vasculature and interstitial spaces in the liver and the lung by immunohistochemistry. Heme oxygenase-1, also known as heat shock protein-32 and a marker of cellular stress, and corticosterone also were measured. The results indicate neutrophil migration into extravascular liver and lung tissue concurrent with cell stress and consistent with tissue injury or infection induced by sleep loss. Plasma corticosterone was unchanged. Recovery sleep was marked by increased lung heme oxygenase-1, increased intestinal MPO activity, and abnormally low corticosterone, suggesting ongoing reactive processes as a result of prior sleep deprivation.     

Cytokine-mediated downregulation of vasopressin V(1A) receptors during acute endotoxemia in rats

The reduced pressure response to vasopressin during acute sepsis has directed our interest to the regulation of vasopressin V(1A) receptors. Rats were injected with lipopolysaccharide for induction of experimental gram-negative sepsis. V(1A) receptor gene expression was downregulated in the liver, lung, kidney, and heart during endotoxemia. Inasmuch as the concentrations of proinflammatory cytokines such as interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma were highly increased during sepsis, the influence of these cytokines on V(1A) receptor expression was investigated in primary cultures of hepatocytes and in the aortic vascular smooth muscle cell line A7r5. V(1A) receptor expression was downregulated by the cytokines in a nitric oxide-independent manner. Blood pressure dose-response studies after injection of endotoxin showed a diminished responsiveness to the selective V(1) receptor agonist Phe(2),Ile(3),Orn(8)-vasopressin. Our data show that sepsis causes a downregulation of V(1A) receptors and suggest that this effect is likely mediated by proinflammatory cytokines. We propose that this downregulation of V(1A) receptors contributes to the attenuated responsiveness of blood pressure in response to vasopressin and, therefore, contributes to the circulatory failure in septic shock.     

Abundance and aggregation egg of Aedes aegypti L. and Aedes albopictus (Skuse) (Diptera: Culicidae) in the north and northwest of the State of Paraná, Brazil

The abundance and aggregation of eggs of Aedes aegypti L. and Aedes albopictus Skuse was evaluated in the municipalities of Cambé, Ibipor?, Jacarezinho, Maringá and Paranavaí, in the State of Paraná, Brazil by means of oviposition traps. Of the 225 installed traps, 100 were registered as positive for eggs; 4140 eggs were collected, thus demonstrating an highly aggregate distribution. Both species were registered in Cambé, Jacarezinho, Maringá and Paranavaí. Ae. albopictus was generally less abundant and was not present in Ibipor? nor in the oviposition traps of a second collection of Maringá. The relation between sexes for Ae. aegypti was approximately 1:1. In the comparison of the number of adults collected between the two species, a negative correlation was obtained in the samples of Maringá and Cambé, what was attributed the seasonality of these populations. The coexistence of these species indicates that both are under pressure by the control programs, therefore specific evaluations are necessary.     

Data‐Independent Acquisition of HLA Class I Peptidomes on the Q Exactive Mass Spectrometer Platform

The characterization of peptides presented by human leukocyte antigen (HLA) class I molecules is crucial for understanding immune processes, biomarker discovery, and the development of novel immunotherapies or vaccines. Mass spectrometry allows the direct identification of thousands of HLA‐bound peptides from cell lines, blood, or tissue. In recent years, data‐independent acquisition (DIA) mass spectrometry methods have evolved, promising to increase reproducibility and sensitivity over classical data‐dependent acquisition (DDA) workflows. Here, we describe a DIA setup on the Q Exactive mass spectrometer, optimized regarding the unique properties of HLA class I peptides. The methodology enables sensitive and highly reproducible characterization of HLA peptidomes from individual cell lines. From up to 16 DDA analyses of 100 million human cells, more than 10 000 peptides could be confidently identified, serving as basis for the generation of spectral libraries. This knowledge enabled the subsequent interrogation of DIA data, leading to the identification of peptide sets with >90% overlap between replicate samples, a prerequisite for the comparative study of closely related specimens. Furthermore, >3000 peptides could be identified from just one million cells after DIA analysis using a library generated from 300 million cells. The reduction in sample quantity and the high reproducibility of DIA‐based HLA peptidome analysis should facilitate personalized medicine applications.     

Varicella‐Zoster virus ORF9 is an antagonist of the DNA sensor cGAS

Varicella‐Zoster virus (VZV) causes chickenpox and shingles. Although the infection is associated with severe morbidity in some individuals, molecular mechanisms that determine innate immune responses remain poorly defined. We found that the cGAS/STING DNA sensing pathway was required for type I interferon (IFN) induction during VZV infection and that recognition of VZV by cGAS restricted its replication. Screening of a VZV ORF expression library identified the essential VZV tegument protein ORF9 as a cGAS antagonist. Ectopically or virally expressed ORF9 bound to endogenous cGAS leading to reduced type I IFN responses to transfected DNA. Confocal microscopy revealed co‐localisation of cGAS and ORF9. ORF9 and cGAS also interacted directly in a cell‐free system and phase‐separated together with DNA. Furthermore, ORF9 inhibited cGAMP production by cGAS. Taken together, these results reveal the importance of the cGAS/STING DNA sensing pathway for VZV recognition and identify a VZV immune antagonist that partially but directly interferes with DNA sensing via cGAS.     

Purification and properties of the germination-inducing factor in the ectomycorrhizal fungus Leccinum aurantiacum (Boletaceae)

A diffusion gradient bioassay on agar strips was developed for determination of the germination-inducing activity of exudates from mycelium of Leccinum aurantiacum (Bull.) S. F. Gray on basidiospores of the same species. The taxon-specific germination-inducing factor, GIF, present in the active exudates, was non-volatile, diffusible, soluble in water, methanol and ethanol, resistant to boiling of short duration, and stable after lyophilization if preserved at −20°C. Gel filtration and adsorption experiments contributed to the chemical and physical characterization of GIF. Passage of an exudate through a Sep-Pak C₁₈ minicolumn removed 99.5% of concomitant substances including germination inhibitors. GIF could then be eluted from the column with methanol without loss of activity.     

Effects of 5-bromo-2-deoxyuridine concentration and alpha-naphthoflavone on the association between smoking and the frequency of sister-chromatid exchanges in lymphocytes from maternal and cord blood

The frequency of sister-chromatid exchanges was analyzed in maternal and cord blood lymphocytes obtained at delivery from 23 nonsmokers and 21 smokers. Lymphocytes were cultured under 3 conditions: in the presence of 100 microM 5-bromo-2-deoxyuridine (BUdR), 20 microM BUdR and 20 microM BUdR with 40 microM alpha-naphthoflavone (ANF). Under all assay conditions, frequencies of SCEs were consistently higher for maternal lymphocytes than for cord lymphocytes. There was no association between SCE values for cultures of the same blood specimen with 100 microM BUdR and 20 microM BUdR. When cultured with 100 microM BUdR, maternal lymphocytes from smokers had a mean SCE frequency of 13.5, which was significantly higher than the value of 11.1 observed for nonsmokers (p = 0.001 by the Wilcoxon rank sum test). Maternal smoking had no significant effect on overall frequencies of SCEs in maternal blood cultured with 20 microM BUdR either with or without ANF or when the differential between cells cultured with and without ANF was considered. Use of caffeinated beverages was associated with increased SCE values for maternal lymphocytes cultured with 20 microM BUdR (Tau beta = 0.36, p = 0.02 for the Kendall's Rank Correlation), but no such association was seen with 100 microM BUdR. For cord blood lymphocytes, however, neither smoking nor caffeine use were associated with SCE values obtained by any of the assay conditions used. The findings suggest that results of human monitoring studies using SCEs could differ depending on the concentration of BUdR used in cultures.