When apoptotic cells are not cleared in an efficient and timely manner, they progress to secondary necrosis and lose their membrane integrity. This results in a leakage of immunostimulatory, danger associated molecular patterns (DAMPs), similar to accidental (or primary) necrosis. However, primary necrosis is a sudden event with an inadvertent release of almost unmodified DAMPs. Secondary necrotic cells, in contrast, have gone through various modifications during the process of apoptosis. Recent research revealed that the molecules released from the cytoplasm or exposed on the cell surface differ between primary necrosis, secondary necrosis, and regulated necrosis such as necroptosis. This review gives an overview of these differences and focusses their effects on the immune response. The implications to human physiology and diseases are manifold and will be discussed in the context of cancer, neurodegenerative disorders and autoimmunity.
The lifespan of a neutrophil is short and limited by programmed cell death, followed by efferocytosis. When
ObjectiveA prospective study to investigate the pattern of pro- and anti-inflammatory cytokine responses in neonates with surgical necrotizing enterocolitis (NEC) and identify those cytokines being the most promising for future research.MethodsA panel of 11 different cytokines were measured in 9 infants with proven NEC and compared with 18 age-matched healthy neonates.ResultsThe serum concentrations of the interleukins (IL)-6, IL-8, and IL-10 were significantly (32–fold to 56-fold) higher in NEC infants compared with controls. In contrast, IL-5, IFN gamma, IL-4 and IL-2 showed slightly (1.4-fold to 5.9-fold) lower levels in the NEC samples. However, these cytokines showed a very low absolute concentration in infants with NEC and in controls. The sum of the serum concentrations of IL-6, IL-8 and IL-10 was able to clearly separate infants with NEC from control samples. IL-1 beta and TNF-alpha showed no statistically different levels. The serum levels of TNF-beta and IL-12p70 were below the detection limit in more than 50% of all samples per group.ConclusionIn spite of strong local inflammation only three out of eleven cytokines (IL-6, IL-8, and IL-10) showed strongly increased serum levels indicating an important role of them in the pathogenesis of NEC. At least two of these three cytokines were elevated in every single NEC patient. Thus, longitudinal monitoring of combined IL-8, IL-6, and IL-10 levels could reveal their potency in being clinical relevant markers in NEC.
For the preparation of proteins for proteome analysis, precipitation is frequently used to concentrate proteins and to remove interfering compounds. Various methods for protein precipitation are applied, which rely on different chemical principles. This study compares the changes in the protein composition of human blood platelet extracts after precipitation with ethanol (EtOH) or trichloroacetic acid (TCA). Both methods yielded the same amount of proteins from the platelet preparations. However, the EtOH-precipitated samples had to be dialyzed because of the considerable salt content. To characterize single platelet proteins, samples were analyzed by two-dimensional fluorescence differential gel electrophoresis. More than 90% of all the spots were equally present in the EtOH- and TCA-precipitated samples. However, both precipitation methods showed a smaller correlation with nonprecipitated samples (EtOH 74.9%, TCA 79.2%). Several proteins were either reduced or relatively enriched in the precipitated samples. The proteins varied randomly in molecular weight and isoelectric point. This study shows that protein precipitation leads to specific changes in the protein composition of proteomics samples. This depends more on the specific structure of the protein than on the precipitating agent used in the experiment.
Photolysis of [3H]tetracycline in the presence of Escherichia coli ribosomes results in an approximately 1:1 ratio of labelling ribosomal proteins and RNAs. In this work we characterize crosslinks to both 16S and 23S RNAs. Previously, the main target of photoincorporation of [3H]tetracycline into ribosomal proteins was shown to be S7, which is also part of the one strong binding site of tetracycline on the 30S subunit. The crosslinks on 23S RNA map exclusively to the central loop of domain V (G2505, G2576 and G2608) which is part of the peptidyl transferase region. However, experiments performed with chimeric ribosomal subunits demonstrate that peptidyltransferase activity is not affected by tetracycline crosslinked solely to the 50S subunits. Three different positions are labelled on the 16S RNA, G693, G1300 and G1338. The positions of these crosslinked nucleotides correlate well with footprints on the 16S RNA produced either by tRNA or the protein S7. This suggests that the nucleotides are labelled by tetracycline bound to the strong binding site on the 30S subunit. In addition, our results demonstrate that the well known inhibition of tRNA binding to the A-site is solely due to tetracycline crosslinked to 30S subunits and furthermore suggest that interactions of the antibiotic with 16S RNA might be involved in its mode of action.
Fever has been associated with shortened duration and improved survival in infectious disease. The mechanism of this beneficial response is still poorly understood. The heat-inducible 70-kDa heat shock protein (Hsp70) has been associated with protection of leukocytes against the cytotoxicity of inflammatory mediators and with improved survival of severe infections. This study characterizes the induction of Hsp70 by feverlike temperatures in human leukocytes in vitro and in vivo. Using flow cytometry, Hsp70 expression was determined in whole blood samples. This approach eliminated cell isolation procedures that would greatly affect the results. Heat treatment of whole blood in vitro for 2 hours at different temperatures revealed that Hsp70 expression depends on temperature and cell type; up to 41 degrees C, Hsp70 increased only slightly in lymphocytes and polymorphonuclear leukocytes. However, in monocytes a strong induction was already seen at 39 degrees C, and Hsp70 levels at 41 degrees C were 10-fold higher than in the 37 degrees C control. To be as close as possible to the physiological situation during fever, we immersed healthy volunteers in a hot water bath, inducing whole body hyperthermia (39 degrees C), and measured leukocyte Hsp70 expression. Hsp70 was induced in all leukocytes with comparable but less pronounced cell type-specific variations as observed in vitro. Thus, a systemic increase of body temperature as triggered by fever stimulates Hsp70 expression in peripheral leukocytes, especially in monocytes. This fever-induced Hsp70 expression may protect monocytes when confronted with cytotoxic inflammatory mediators, thereby improving the course of the disease.
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