Comparative Analysis of Chromatin-Delivered Biomarkers in the Monitoring of Sepsis and Septic Shock: A Pilot Study.

Sepsis management remains one of the most important challenges in modern clinical practice. Rapid progression from sepsis to septic shock is practically unpredictable, hence the critical need for sepsis biomarkers that can help clinicians in the management of patients to reduce the probability of a fatal outcome. Circulating nucleoproteins released during the inflammatory response to infection, including neutrophil extracellular traps, nucleosomes, and histones, and nuclear proteins like HMGB1, have been proposed as markers of disease progression since they are related to inflammation, oxidative stress, endothelial damage, and impairment of the coagulation response, among other pathological features. The aim of this work was to evaluate the actual potential for decision making/outcome prediction of the most commonly proposed chromatin-related biomarkers (i.e., nucleosomes, citrullinated H3, and HMGB1). To do this, we compared different ELISA measuring methods for quantifying plasma nucleoproteins in a cohort of critically ill patients diagnosed with sepsis or septic shock compared to nonseptic patients admitted to the intensive care unit (ICU), as well as to healthy subjects. Our results show that all studied biomarkers can be used to monitor sepsis progression, although they vary in their effectiveness to separate sepsis and septic shock patients. Our data suggest that HMGB1/citrullinated H3 determination in plasma is potentially the most promising clinical tool for the monitoring and stratification of septic patients.

A functional assay for serum detection of antibodies against SARS-CoV-2 nucleoprotein.

The humoral immune response to SARS-CoV-2 results in antibodies against spike (S) and nucleoprotein (N). However, whilst there are widely available neutralization assays for S antibodies, there is no assay for N-antibody activity. Here, we present a simple in vitro method called EDNA (electroporated-antibody-dependent neutralization assay) that provides a quantitative measure of N-antibody activity in unpurified serum from SARS-CoV-2 convalescents. We show that N antibodies neutralize SARS-CoV-2 intracellularly and cell-autonomously but require the cytosolic Fc receptor TRIM21. Using EDNA, we show that low N-antibody titres can be neutralizing, whilst some convalescents possess serum with high titres but weak activity. N-antibody and N-specific T-cell activity correlates within individuals, suggesting N antibodies may protect against SARS-CoV-2 by promoting antigen presentation. This work highlights the potential benefits of N-based vaccines and provides an in vitro assay to allow the antibodies they induce to be tested.

Purification of Crimean-Congo hemorrhagic fever virus nucleoprotein and its utility for serological diagnosis.

Crimean-Congo hemorrhagic fever virus (CCHFV) causes a zoonotic disease, Crimean-Congo hemorrhagic fever (CCHF) endemic in Africa, Asia, the Middle East, and Southeastern Europe. However, the prevalence of CCHF is not monitored in most of the endemic countries due to limited availability of diagnostic assays and biosafety regulations required for handling infectious CCHFV. In this study, we established a protocol to purify the recombinant CCHFV nucleoprotein (NP), which is antigenically highly conserved among multiple lineages/clades of CCHFVs and investigated its utility in an enzyme-linked immunosorbent assay (ELISA) to detect CCHFV-specific antibodies. The NP gene was cloned into the pCAGGS mammalian expression plasmid and human embryonic kidney 293 T cells were transfected with the plasmid. The expressed NP molecule was purified from the cell lysate using cesium-chloride gradient centrifugation. Purified NP was used as the antigen for the ELISA to detect anti-CCHFV IgG. Using the CCHFV NP-based ELISA, we efficiently detected CCHFV-specific IgG in anti-NP rabbit antiserum and CCHFV-infected monkey serum. When compared to the commercially available Blackbox CCHFV IgG ELISA kit, our assay showed equivalent performance in detecting CCHFV-specific IgG in human sera. These results demonstrate the usefulness of our CCHFV NP-based ELISA for seroepidemiological studies.

Systemic Lupus Erythematosus and DNA Degradation and Elimination Defects.

Introduction: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that is characterized by the production of autoantibodies specific for components of the cell nucleus and that causes damage to body tissues and organs. The pathogenesis of SLE remains unclear, with numerous studies pointing to a combination of genetic and environmental factors. A critical stage in SLE development is cell necrosis, in which undegraded chromatin and nucleoproteins are released into the blood, resulting in circulating cell-free DNA and serum nucleoproteins that trigger anti-dsDNA autoantibody production. This systematic literature review aimed to examine whether SLE stems from a DNA degradation and elimination defect. Materials and Methods: An advanced literature search was conducted in PubMed using the following keywords: [("SLE" OR "Systemic Lupus Erythematosus" OR "Lupus")] AND [("DNA" OR "DNA Degradation")] AND [("Defect Elimination")]. More articles were obtained from the references of the identified articles and basic Google searches. Twenty-five peer-reviewed articles published within the past 10 years (2007-2018) were included for review. Results: The findings of each study are summarized in Tables 1, 2. Discussion and Conclusion: The etiopathogenesis of SLE remains controversial, which limits therapeutic inventions for this disease. However, SLE is a DNA degradation and elimination disorder caused by uncleared histones and nuclear material that leak into the extracellular space and form cell-free DNA, triggering an immune response that destroys tissues and organs. Under normal conditions, apoptosis allows DNA and other nuclear material to be efficiently cleared through degradation and additional complex mechanisms such that this material does not trigger the immune system to produce nuclear autoantibodies.

Detection of Antibody and Antigen for Lassa Virus Nucleoprotein in Monkeys from Southern Nigeria.

Lassa fever is a deadly viral haemorrhagic fever caused by Lassa Virus (LASV). Rodents, especially, Mystomys natalensis, are the known reservoirs of LASV and humans are the defined hosts. Monkeys share many illnesses with humans and experimental LASV infections in monkeys are fatal but natural LASV infection of monkeys has not been reported. Serum samples obtained between August 2015 and December 2017 from 62 monkeys belonging to six species in Southern Nigeria were tested for LASV as part of an ongoing surveillance of monkeys in the region for zoonotic pathogens. Commercially available Recombinant LASV (ReLASV) Pan-Lassa enzyme-linked immunosorbent assay (ELISA) test kits (Zalgen Labs, Germantown, MD, USA) were used to detect antibodies (IgG and IgM) and antigen specific for LASV nucleoprotein in the sera. Lassa-fever-specific IgG and IgM, and antigen specific for LASV nucleoprotein were detected in 5/62, 0/62, and 1/62 samples, respectively. The presence of LASV-specific antibodies in the sera suggests natural exposure to the virus, while the presence of LASV antigen may mean that monkeys are carriers of the virus. There is a need to broaden Lassa fever surveillance to include nonhuman primates (NHPs) for their probable role in the epidemiology of the disease.HIGHLIGHTS.• Rodents are the natural reservoirs of Lassa fever virus (LASV) and humans are the defined hosts.• Experimental LASV infections in non-human primates (NHP) are fatal but natural infection of NHP with the virus have not been reported.• We detected antigen and antibody specific for LASV in free-living Monkeys from southern Nigeria which implies that monkeys in the region are naturally exposed to LASV and are probable carriers of the virus.

Protein Content of Circulating Nucleoprotein Complexes.

In the current study we have investigated the protein content of blood plasma deoxyribonucleoprotein complexes. The complexes were isolated using affinity chromatography with immobilized polyclonal anti-histone antibodies. Proteins were separated by SDS PAAGE and identified by MALDI-TOF mass-spectrometry. 111 and 56 proteins (excluding histones), respectively, were identified with a good score in deoxyribonucleoprotein complexes of healthy females and breast cancer patients. However, only four of these proteins were found in 30 % of all samples. Fourteen proteins previously described as tumor specific proteins were found in cancer patients whereas not one of them was found in healthy individuals. The data obtained demonstrate the involvement of different cellular and extracellular proteins in circulating cell-free DNA.

[Experimental modeling of nucleoprotein disposal disorders in systemic lupus erythematosus].

The objective of this research was to adapt the experimental model simulating the nucleoprotein disposal disorders in systemic lupus erythematosus (SLE) for further study of its extracorporeal correction, as well as to assess validity of the model by short-term experiment. Twenty to female Wistar rats were intraperitoneally injected with the chromatin-containing extract from bovine liver followed by intravenous administration of anti-DNA antibodies derived from SLE patients. After these procedures plasma concentrations of anti-dsDNA, circulating immune complexes and DNA became sharply increased, together with distinct elevation of leukocytes. On the contrary, changes in erythrocytes, platelets, total protein concentration, creatinine, asparagine and alanine aminotransferase activities, as well as blood coagulation time were changed insignificantly. Using direct immunofluorescence of cryosections, we detected human IgG deposition in rat kidneys treated in accordance with the simulation protocol. Thus, our model reproduces essential DNA disposal disorders in SLE without any animal death or the life-threatening changes in examined markers during short-term experiment.

[Extracorporeal treatment of nucleoprotein disposal disorders using the systemic lupus erythematosus model: evaluation of efficacy and safety in a short-term simulated experiment].

Efficacy and safety of the extracorporeal blood perfusion through DNase I- and C1q-containing magnetic beads have been evaluated using the experimental model simulating the nucleoprotein disposal disorders in systemic lupus erythematosus (SLE). The study was performed using 20 rats in which the essential impairments of nucleoprotein catabolism typical for SLE were modeled. The animals were randomized into the experimental group and the placebo perfusion control group. Rats of the experimental group were characterized by the statistically significant reduction of increased levels of circulating immune complexes and plasma DNA as well as diminished levels of plasma creatinine and kidney IgG deposition as compared with placebo controls. During short-term experiment there were neither animal deaths nor substantial blood cell destruction and hepatotoxicity signs.

[Protein Identification of Blood Nucleoprotein Complexes].

Circulating nucleoprotein complexes were isolated-from blood plasma by affinity chromatography using immobilized polyclonal anti-histone antibodies. It was found, that the main part of DNA from histone-contained nucleoprotein complexes have size 170-180 b.p., in blood of breast cancer patients DNA with size 170-180 b.p. and DNA more then 6 k.b.p. are presented in equal quantity. Proteins from circulating nucleoprotein complexes were identified using MALDI-TOF mass-spectrometry. It was shown that nucleoprotein complexes from blood of breast cancer patients contain tumor-specific proteins, such as LDOC1L, ADP/ATP translocase 3 and Lamellipodin. These data indicate, that a part of circulating extracellular DNA have tumor origin.

5' tRNA halves are present as abundant complexes in serum, concentrated in blood cells, and modulated by aging and calorie restriction.

BACKGROUND: Small RNAs complex with proteins to mediate a variety of functions in animals and plants. Some small RNAs, particularly miRNAs, circulate in mammalian blood and may carry out a signaling function by entering target cells and modulating gene expression. The subject of this study is a set of circulating 30-33 nt RNAs that are processed derivatives of the 5' ends of a small subset of tRNA genes, and closely resemble cellular tRNA derivatives (tRFs, tiRNAs, half-tRNAs, 5' tRNA halves) previously shown to inhibit translation initiation in response to stress in cultured cells. RESULTS: In sequencing small RNAs extracted from mouse serum, we identified abundant 5' tRNA halves derived from a small subset of tRNAs, implying that they are produced by tRNA type-specific biogenesis and/or release. The 5' tRNA halves are not in exosomes or microvesicles, but circulate as particles of 100-300 kDa. The size of these particles suggest that the 5' tRNA halves are a component of a macromolecular complex; this is supported by the loss of 5' tRNA halves from serum or plasma treated with EDTA, a chelating agent, but their retention in plasma anticoagulated with heparin or citrate. A survey of somatic tissues reveals that 5' tRNA halves are concentrated within blood cells and hematopoietic tissues, but scant in other tissues, suggesting that they may be produced by blood cells. Serum levels of specific subtypes of 5' tRNA halves change markedly with age, either up or down, and these changes can be prevented by calorie restriction. CONCLUSIONS: We demonstrate that 5' tRNA halves circulate in the blood in a stable form, most likely as part of a nucleoprotein complex, and their serum levels are subject to regulation by age and calorie restriction. They may be produced by blood cells, but their cellular targets are not yet known. The characteristics of these circulating molecules, and their known function in suppression of translation initiation, suggest that they are a novel form of signaling molecule.

A sensitive ELISPOT assay for detection of CD8+ T lymphocytes specific for HLA class I-binding peptide epitopes derived from influenza proteins in the blood of healthy donors and melanoma patients.

An enzyme-linked immunospot (ELISPOT) assay was adapted to detect peptide-specific CD8+ T lymphocytes in peripheral blood mononuclear cells (PBMCs). In HLA-A1-, HLA-A2-, and/or HLA-A3-positive individuals, we determined the release of IFN-gamma on a single cell level in response to three different peptide epitopes derived from the influenza matrix protein and nuclear protein containing the HLA-A2.1- and HLA-A1- or HLA-A3-binding motif, respectively. Comparison of the ELISPOT assay with the standard chromium release assay revealed a close correlation between the number of peptide-specific IFN-gamma-releasing T cells in PBMCs and the level of specific cytotoxicity after 14 days of in vitro expansion. The ELISPOT assay detected T cells with specificity for the HLA-A2. 1-binding epitope derived from the matrix protein in 76% of HLA-A2-positive healthy individuals (n = 25); the median frequency was 41 in 10(6) PBMCs. We also detected peptide-specific T cells in 10 of 12 HLA-A2-positive patients with metastatic melanoma with a median frequency of 20.5 in 10(6) PBMCs. In 10 of 24 HLA-A3-positive individuals and in 2 of 14 HLA-A1-positive individuals, peptide-specific T cells for a HLA-A3- and a HLA-A1-binding epitope derived from the nucleoprotein, respectively, were present. In conclusion, the ELISPOT assay may be suitable to monitor a peptide-specific T-cell response in vaccination protocols using peptides derived from tumor or viral antigens.

Detection of measles virus genome directly from clinical samples by reverse transcriptase-polymerase chain reaction and genetic variability.

A simple and sensitive method for the detection of measles virus genome was developed, amplifying the regions encoding the nucleocapsid (N) protein and hemagglutinin (H) protein of measles virus by reverse transcriptase-polymerase chain reaction (RT-PCR). We examined a variety of measles patients: 28 patients with natural infection, 4 with measles encephalitis and 1 with subacute sclerosing panencephalitis (SSPE). In 28 patients with natural measles infection a single step PCR amplifying the N region resulted in a high detection rate for all plasma samples (28/28) within 3 days of the onset of rash and 80% (20/25) even on day 7 of the onset of rash and later. Within 3 days of the onset of rash, 24/25 (96.0%) of nasopharyngeal secretions (NPS) and 27/28 (96.4%) of peripheral blood mononuclear cells (PBMC) were positive for the N region PCR and the positivity rate of PCR decreased in NPS and PBMC after 7 days of the rash. In acute measles infection, measles genome was detected in all cell fractions, CD4, CD8, B cells, and monocytes/macrophages by the H gene nested PCR. Measles genome was also detected from cerebrospinal fluids (CSF) in patients with measles encephalitis, SSPE, and acute measles by the H gene nested PCR. PCR products of the N and H regions were sequenced and we confirmed the presence of measles genome. Based on the sequence data, chronological sequence differences were observed over the past 10 years. The sequences obtained from the SSPE patient were closely related to those of the wild viruses that were circulating at the time when the patient initially acquired measles. RT-PCR for NPS, PBMC, CSF, and plasma provides a useful method for the diagnosis of measles and molecular epidemiological study in addition to virus isolation.

[The nucleoprotein fraction of mouse serum in normal and pathological processes]. / Issledovanie nukleoproteinovoi fraktsii syvorotki krovi myshei v norme i pri patologicheskikh protsessakh.

Analysis of the blood serum of healthy and infected with malaria plasmodium mice showed a steep rise in content of linear double-stranded DNA (0.2-0.5 and 2-15 gamma/ml, respectively). Some physico-chemical properties of serum DNA and a DNA-associated glycoprotein (M approximately 40 kDa) are determined.

[Analysis of the trypsin hydrolysate of a 40-kDa protein, found in the nucleoprotein of serum. Identification of it as alpha(1)-acidic glycoprotein (orosomucoid)]. / Analiz produktov tripticheskogo gidrolizata 40-kDa belka, vkhodiashchego v sostav nukleoproteinovoi fraktsii syvorotki krovi. Identifikatsiia ego v kachestve alpha(1)-kislogo glikoproteina (orozomukoida).

From the tryptic hydrolysis of 40-kDa protein from the mouse blood serum thirty-four peptides were isolated by HPLC, of which complete and partial amino acid sequence was established for twenty-eight and six, respectively. On the basis of these data the protein is identified as the blood serum alpha 1-acid glycoprotein.

Chicken erythrocyte polynucleosomes which are soluble at physiological ionic strength and contain linker histones are highly enriched in beta-globin gene sequences.

Mature chicken erythrocyte polynucleosomes which are soluble at physiological ionic strength are enriched in beta-globin DNA sequences. Vitellogenin chromatin, which is not expressed in this tissue, is found in aggregation prone, salt insoluble chromatin. There is a direct correlation between the size of soluble fragments and the degree of globin gene enrichment, with the largest fragments being most highly enriched. The highly globin enriched (about 50 fold) polynucleosomes contain significantly elevated levels of acetylated histones H4, H2A.Z, and H2B, and ubiquitinated (prefix "u") histones H2A and H2B (with a significant relative increase of uH2B over uH2A). These polynucleosomes were complexed with histones H1 and H5 but at a lower level than that found in unfractionated chromatin.

Electron-microscopical evidence that ribosomal chromatin of human circulating lymphocytes is devoid of histones.

We have studied the distribution of histones in the nucleolus of human circulating lymphocytes in situ, using thin sections, either treated with antibodies against the core histones revealed by colloidal gold, or stained with the acrolein-silver methenamine technique for basic proteins. Gold particles were not found in the fibrillar centre, nor were silver-stained structures visible in this nucleolar component. Since the fibrillar centre contains the bulk of the ribosomal chromatin which is in a completely extended, non-nucleosomal configuration, our results indicate that this chromatin is devoid of histones.

Transformation and growth related changes in levels of nuclear and cytoplasmic proteins antigenically related to mammalian beta-galactoside-binding lectin.

Immunofluorescence and immunoblotting experiments, using a monoclonal antibody to the 13 kDa mammalian beta-galactoside-binding lectin have shown that human lymphocytes contain nuclear and cytoplasmic proteins of apparent molecular masses of 130, 80, 65 and 13 kDa that are antigenically related to the lectin and whose levels and patterns of expression change in association with transformation, or after stimulation with mitogens. These observations, together with the finding that the myeloid cell line K562 is also rich in the 130 kDa component, whereas the mature granulocytes of normal donors and of patients with chronic myeloid leukaemia are lacking in all of the immunoreactive forms, raise the possibility that this family of lectin-related proteins may be components of growth regulatory systems that are variously elicited in the transformed and stimulated cells.

[Cytochemical study of lymphocyte nucleoproteins in rheumatoid arthritis patients]. / Tsitokhimichno izsledvane na nukleoproteini v limfotsitite na bolni s revmatoiden artrit.

The purpose of the study is to investigate and juxtapose the cytochemical characteristics of ribonucleoproteins (RNP) and deoxyribonucleoproteins (DNP) in the lymphocytes of patients with rheumatoid arthritis (RA), of healthy lymphocytes and lymphocytes blast-transformed by phytohemagglutinins. Significant differences in the staining of lymphocytes for nucleoproteins were established in all 54 patients with RA studied. The unidirectional changes in the morphological form of the staining for RNP and DNP in blast-transformed lymphocytes and lymphocytes in RA is, most likely, in connection with the transition of the latter into active functional state and their participation in the forming of the immune inflammation. No significant difference was established in the dependence on the RA form--sero-positive or sero-negative. Regardless of the negative serological samples in the second form, there were some changes in the lymphocytes--carriers of immune reactions in organism.