Cytidine deamination-induced perpetual immunity to SAR-CoV-2 infection is a potential new therapeutic target.

As the world is racing to develop perpetual immunity to the SARS-CoV-2 virus. The emergence of new viral strains, together with vaccination and reinfections, are all contributing to a long-term immunity against the deadly virus that has taken over the world since its introduction to humans in late December 2019. The discovery that more than 95 percent of people who recovered from COVID-19 had long-lasting immunity and that asymptomatic people have a different immune response to SARS-CoV-2 than symptomatic people has shifted attention to how our immune system initiates such diverse responses. These findings have provided reason to believe that SARS-CoV-2 days are numbered. Hundreds of research papers have been published on the causes of long-lasting immune responses and variations in the numbers of different immune cell types in COVID 19 survivors, but the main reason of these differences has still not been adequately identified. In this article, we focus on the activation-induced cytidine deaminase (AID), which initiates molecular processes that allow our immune system to generate antibodies against SARS-CoV-2. To establish lasting immunity to SARS-CoV-2, we suggest that AID could be the key to unlocking it.

Genome-Wide Mapping of DNA Methylation 5mC by Methylated DNA Immunoprecipitation (MeDIP)-Sequencing.

Methylated DNA immunoprecipitation is a large scale purification technique. It enables the isolation of methylated DNA fragments for subsequent locus-specific or genome-wide analysis. Here we describe an immunoprecipitation protocol using a monoclonal mouse anti 5-methyl-cytidine antibody followed by next-generation sequencing (MeDIP-Seq).

N4-acetylcytidine is required for sustained NLRP3 inflammasome activation via HMGB1 pathway in microglia.

Persistent inflammasome activation contributes to chronic, low grade inflammation. However, it is unclear how the inflammasome activation is sustained after initiation. Here we reported that N4-acetylcytidine (N4A), a nucleoside metabolite, activated microglia and sustained NLRP3 inflammasome activation by inducing HMGB1 signaling. Released HMGB1 through N4A activated NFκB and induced NLRP3 expression. HMGB1 silencing abolished N4A-stimulated NFκB activation, NLRP3 and persistent HMGB1 expression. In addition, inhibiting NLRP3 expression by RNAi abrogated N4A-mediated HMGB1 expression. Lack of NLRP3 inflammasome adaptor named apoptosis-associated speck-like protein containing a CARD (ASC) abrogated N4A-induced HMGB1 expression, NFκB activation, and NLRP3 expression. Taken together, our results reveal a novel role of N4A in activation of NLRP3 inflamasome via HMGB1 feedback.

Label-free DNA methylation analysis using opto-fluidic ring resonators.

The opto-fluidic ring resonator (OFRR) is a sensitive label-free optical biosensor that is uniquely well suited for photonic and fluidic integration. For the first time we have explored the utility of this novel instrument for the analysis of methylation in oligonucleotides using the MBD-2 (methyl binding) protein as the capture molecule. This application has strong relevance to cancer research and future clinical tools through the study of methylation patterns in important gene promoters. In this work we quantitatively characterized the OFRR's response to artificially methylated ssDNA and dsDNA as a function of the number of methylated cytosines and DNA concentration. The effect of hemi- versus fully methylated oligonucleotides was also investigated. Additionally, anti 5-methylcytidine antibody was also used as the capture molecule and compared with MBD-2. It is found that the antibody has stronger affinity for ssDNA, whereas MBD-2 is much better at binding dsDNA.

Titrimetric immunohistochemical evaluation of DNA hypomethylation in uterine tumours.

BACKGROUND: Global DNA hypomethylation is a well established feature of many common cancers. AIMS: To establish a simple semi-quantitative, titrimetric immunohistochemical method in order to exploit this trait for prognostic purposes, in uterine cancers. METHODS: A monoclonal antibody against 5-methylcytidine was used for immunohistochemical staining of methylated DNA in tumour cells. The degree of methylated DNA in the tumour tissue was visually compared and matched to that of normal tissues stained by serial decreasing concentrations of antibody to 5-methylcytidine. RESULTS: Using this method a significant correlation was found between the histological stage and the reduction in DNA methylation in uterine adenocarcinoma (n = 39) and uterine squamous cell carcinoma (n = 23). CONCLUSIONS: A simple titrimetric immunohistochemical method has been developed for quantitative evaluation of ligands. This method should be further employed in follow-up studies, in order to establish the prognostic value of DNA hypomethylation in uterine cancer.

Detection of cancer clones in human colorectal adenoma as revealed by increased DNA instability and other bio-markers.

An immunohistochemical differential staining of cancerous cells with anti-cytidine antibody after denaturation of nuclear DNA by acid hydrolysis with 2N HCl at 30 degrees C for 20 min (DNA-instability test) has been used as a marker for malignancy. The test was applied to bioptic tissues of human colorectal polyps assessed histopathologically as hyperplastic polyp (11 cases), tubular adenoma of mild (68 cases), moderate (102 cases), and severe (46 cases) dysplasia, and adenocarcinoma (30 cases). The serial sections of the same tissues were also subjected to immunohistochemical staining for Ki67, p53, DNA-fragmentation factor 45 (DFF45) and vascular endothelial growth factor (VEGF). The DNA-instability test was positive in 30 (100%) adenocarcinoma cases, 46 (100%) severe dysplasia adenoma cases, 36 (35.29%) moderate dysplasia adenoma cases, and 8 (11.76%) mild dysplasia adenoma cases, indicating malignancy. All hyperplastic polyps were negative to the DNA-instability test. Furthermore, the percentage of glands positive in the DNA-instability test steadily increased in going from mild (10%), to moderate (35%), to severe (100%) dysplasia, and adenocarcinoma (100%). All other biological markers tested in the present study showed significantly higher values in those adenoma glands that were positive to the DNA-instability test, irrespective of the dysplasia grade, as compared to the markers in the adenoma glands that were negative to DNA instability testing. Furthermore, the former values were comparable to those in adenocarcinoma. The results indicate that cancer cell clones are already present at the adenoma stages showing positivity to DNA instability testing, enhanced proliferative activity, p53 mutation and induction of DFF45 and VEGF, at a time when the degree of morphological atypia are not yet large enough for them to be identified as cancer. These factors promote cancer cell proliferation, produce heterogeneous subclones due to DNA instability, enhance their survival by escaping apoptosis, and provide abundant nutrients by neovascularization during the early-stage progression of colorectal cancer.

Poly-guanosine motifs costimulate antigen-reactive CD8 T cells while bacterial CpG-DNA affect T-cell activation via antigen-presenting cell-derived cytokines.

Pathogen-derived pattern recognition ligands like lipopolysaccharide (LPS) and bacterial cytidine-guanosine (CpG)-DNA not only activate dendritic cells and macrophages but are also mitogenic for B cells. Less clear are the claimed effects of CpG-DNA on T cells, which range from direct activation, costimulation, or indirect transient activation via antigen-presenting cell (APC)-derived interferon type I (IFN type I). Here we demonstrate that CpG-DNA sequence specifically triggers macrophages to produce IFN type I, interleukin (IL)-12, IL-6 and tumour necrosis factor (TNF), but lacks the ability to directly costimulate T cells. Strikingly, poly-guanosine (poly-G) extensions to CpG-containing oligonucleotides (ODN) abolished the macrophage stimulatory potential yet generated T-cell costimulatory activities. In fact, independently of CpG-motifs, poly-G-ODN displayed the ability to costimulate T cells. Costimulation was operative on CD8 T cells but not CD4 T cells. Poly-G-mediated costimulation resulted in IL-2-driven T-cell proliferation and induced cytolytic T cells. Overall the data imply that poly-G motifs costimulate antigen reactive CD8 T cells, while CpG-DNA motifs fail to do so but may affect T-cell activation via APC derived cytokines such as IFN type I.

Monoclonal antibody affinities of structurally related modified nucleosides.

A scale of relative affinities of a series of 2'-deoxycytidine and cytidine (CD) derivatives was established based on the data of cross-reactivities of these compounds as well as the displacements obtained from a competitive ELISA. No correlation could be established between the nucleosides modifying structures and the affinities. This can be explained by the possibilities of the modifying structures of intra- and intermolecular nonimmunospecific interactions owing to their degree of functionalization.

Epitope complementarity and idiotypic interactions: a study of idiotypic-like interactions between anti-cytidine and anti-guanosine A/J mouse monoclonal antibodies--I. Characterization of these interactions.

Idiotypic-like interactions between mAbs directed against cytidine (Cyd) or guanosine (Guo) nucleosides were characterized. These mAbs, Cyd-1 (IgG2b, kappa), Guo-1 (IgG1, kappa) and Guo-2 (IgG1, kappa) were derived from splenocytes of A/J mice immunized with Cyd-KLH or Guo-KLH and recognized the nucleoside base moieties involved in hydrogen bonding. The interactions between Guo-1 or Guo-2 and Cyd-1 involved cross-reactive or distinct-but-neighboring paratope-associated idiotopes. These interactions were characterized by KD values of 4.6 x 10(-6) and 1.8 x 10(-6)M, respectively. The three anti-nucleoside mAbs exhibited Ab2 beta properties and manifested epibody (Ab2 epsilon) activity towards ssDNA. We compared these idiotypic-like reactivities with the anti-idiotypic activity of an intentionally induced IgG1, kappa anti-idiotype mAb prepared with splenocytes from A/J mice immunized with Cyd-1. This Ab2 antibody which bound to Cyd-1 with a KD of 1.1 x 10(-9) M, manifested an Ab2 gamma activity, i.e. it recognized a paratope-associated idiotope on Cyd-1 without exhibiting Ab2 beta properties. In addition, the anti-(Cyd-1) completely inhibited (Cyd-1)-(Guo-1) and (Cyd-1)-(Guo-2) interactions.

Brequinar sodium inhibits interleukin-6-induced differentiation of a human B-cell line into IgM-secreting plasma cells.

Brequinar sodium (BQR) has been shown recently to be a potent immunosuppressive agent. This property has been attributed to the capacity of BQR to inhibit de novo pyrimidine nucleoside biosynthesis and consequently, to blockade the synthesis both of DNA and RNA. The influence of this new immunosuppressant on lymphocyte function has not been fully characterized. To determine the potential efficacy of BQR for the control of antibody-mediated graft rejection, which is of particular significance in the context of xenotransplantation, we have examined the influence of the drug on interleukin-6-dependent IgM production by the human B-cell line, SKW 6.4. At concentrations up to 10 micrograms/ml, BQR did not affect concanavalin A (Con A)-induced human peripheral blood lymphocyte proliferation or IL-6 production by blood mononuclear leucocytes. In contrast, the drug was very effective in inhibiting IL-6-stimulated IgM production by SKW 6.4 cells, with an optimal inhibitory concentration of 0.3 microgram/ml. As expected, addition of exogenous uridine (0.1 mM), the precursor of uridine triphosphate (UTP), reversed the inhibitory effect of BQR on antibody production, while cytidine (0.1 mM) potentiated the inhibitory activity of the drug. It was further demonstrated that the inhibition of IgM production was unrelated to DNA synthesis, indicating that BQR may affect IL-6 signal transduction and IgM production in SKW 6.4 cells independent of any effect on cell proliferation.

Molecular geometry of antigen binding by a monoclonal antibody against 5-methylcytidine.

1. The specificity of a monoclonal IgG1 raised against a 5-methylcytidine-keyhole limpet hemocyanin conjugate was investigated by inhibition experiments with soluble competing antigens. 2. A competitive enzyme immunoassay has been set up, with the antigen immobilized on polystyrene microtitration wells. 3. The analysis of the cross-reaction profile allowed the topography of the antigen-antibody interaction to be described. 4. The binding properties of the monoclonal antibody are discussed in terms of both analytical applications and working limitations in the immunochemical study of gene methylation.

Probing the surface of Z-DNA with anti-nucleoside antibodies.

Antibodies specific for cytidine (C) and guanosine (G) were used to probe the surface of two Z-DNA conformers. When tested by ELISA, anti-G reacted with poly(dG-dC).poly(dG-dC) treated with bromine water [Br-poly(dG-dC).poly(dG-dC)] but anti-C did not. A weak reaction with anti-C was detected by dot immunobinding. In contrast, anti-C reacted strongly with poly(dG-dC).poly(dG-dC) treated with N-acetoxy-2-(acetylamino)fluorene [AAF-poly(dG-dC).poly(dG-dC)]; anti-G reacted weakly, despite the fact that most G residues had not been substituted with AAF. Neither antinucleoside bound to the B conformation of poly(dG-dC).poly(dG-dC). In competition experiments, GMP was the most efficient competitor of the reaction of anti-G with Br-poly(dG-dC).poly(dG-dC); AMP and TMP were 100-fold less efficient, and CMP did not compete to a significant extent. In contrast, the reaction of anti-Z with Br-poly(dG-dC).poly(dG-dC) was not inhibited by nucleotides. Of five possible sites recognized on guanosine by anti-G antibodies (N1, C6, O6, N7, and C8), AMP and TMP share three or their equivalent and CMP only one. The binding of anti-C to AAF-poly(dG-dC).poly(dG-dC) was inhibited best by CMP; AMP was 8 times less efficient; GMP and TMP were about 35-fold less efficient than CMP. Thus, although the amino group on the C4 position of CMP appears to be immunodominant, the capacity of GMP and TMP to inhibit the reaction indicates that other sites are also recognized in AAF-poly(dG-dC).poly(dG-dC), e.g., the exposed C5 position.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of techniques to monitor for exposure to vinyl chloride: monoclonal antibodies to ethenoadenosine and ethenocytidine.

Monoclonal antibodies which specifically recognize ethenoadenosine and ethenocytidine, two of the adducts resulting from exposure to vinyl chloride, have been developed. The sensitivity and specificity of these antibodies have been determined by enzyme-linked immunosorbent assay (ELISA). The antibody to ethenoadenosine (1G4) reacts with both the ribose (50% inhibition at 600 fmol) and deoxyribose (50% inhibition at 980 fmol) form of the adduct. The antibody to ethenocytidine (6F5) also reacts with both the ribose (50% inhibition at 800 fmol) and deoxyribose (50% inhibition at 1000 fmol) form of the adduct. Neither antibody cross-reacts with non-modified DNA or the normal nucleotides. A more sensitive fluorescence ELISA was developed for antibody 1G4 with 50% inhibition at 212 fmol of ethenoadenosine and for antibody 6F5 with 50% inhibition at 192 fmol ethenocytidine. These antibodies have been used to determine the level of etheno derivatives in DNA modified in vitro with chloroacetaldehyde and in the DNA and RNA of cells treated in culture.

[Immunoenzyme method of determination of m5Cyd level; characteristics of the epitope and estimation of m5Cyd levels in the blood of leukemic mice]. / Enzimoimmunologicheskii metod opredeleniia soderzhaniia m5Cyd; kharakteristika épitopa, otsenka soderzhaniia m5Cyd v krovi leikoznykh myshei.

Antibodies specific to m5Cyd were obtained and a sensitive enzyme-linked immunosorbent assay (ELISA) for detecting m5Cyd was developed. This method was used for the characterization of m5Cyd epitope. It was shown that the 4-NH2 and 5-CH3-groups of the pyrimidine ring are of primary importance for the antigenic determinant assembly, whereas the ribosyl residue and its structure are less important. It was found also that the amount of m5Cyd in leukemic La murine serum after gamma-irradiation is markedly increased.

Establishment of a nucleoside-specific suppressor T cell line from SLE prone (NZB/NZW)F1 mice.

A long-term cultured suppressor T cell line (GTS-124) was established from an autoimmune mouse strain, (NZB X NZW)F1, by a two-part procedure: a) B/W F1 mice were made tolerant to guanosine (G) by administration of a tolerogen, the G-modified copolymer of D-glutamic acid and D-lysine (G-D-GL); and b) the spleen cells obtained from tolerant mice were repeatedly stimulated with mitomycin C-treated G-modified syngeneic spleen cells. The GTS-124 cells suppressed the secondary in vitro response to G-keyhole limpet hemocyanin (G-KLH) but did not suppress the response to unrelated antigens, sheep erythrocytes (SRBC), or trinitrophenyl-KLH (TNP-KLH). The expression of Thy-1 antigen on the cell surface of GTS-124 was demonstrated by flow cytometry. Growth of GTS-124 cells was dependent on IL 2. To determine whether GTS-124 cells could suppress the response to nucleosides other than G, KLH coupled with four nucleosides (adenosine [A], G, cytidine [C], and thymine riboside [T]) collectively (AGCT-KLH) was first used as the antigen in the assay system. The PFC response to the individual nucleosides (anti-A, -G, -C, and -T PFC) were effectively inhibited by GTS-124 cells, suggesting that the GTS-124 cells mediated cross-suppression toward all four nucleosides. A more stringent cross-suppression test was conducted by using only the T moiety bound to KLH (T-KLH) as antigen. The results showed that GTS-124 cells were capable of suppressing the T-specific response. The cross-suppression could be seen after repeated selection on a G-BSA-coated dish. These results provide direct evidence that the suppressor T cells induced by in vitro stimulation with G-modified self can indeed suppress the response to nucleosides other than G.

Specificity of anti-nucleoside antibodies in systemic lupus erythematosus.

The titer of IgG antinucleoside antibodies in the sera of 162 individuals was determined by an enzyme-linked immunosorbent assay. The nucleosides used in the assay were adenosine, cytidine, guanosine, and thymine-riboside conjugated to human serum albumin. The specificity of IgG antinucleoside antibodies was indicated by appropriate reduction in antibody binding after solid-phase adsorptions of antibody with specific immobilized nucleoside conjugates. Disease-associated increases in serum IgG antibodies to cytidine and guanosine but not to adenosine or thymine-riboside occurred in patients with systemic lupus erythematosus (SLE). The epitope density of nucleosides in the conjugates and differences in the sensitivity of each nucleoside assay were not responsible for disease-associated IgG antinucleoside antibody responses. These findings support a possible pathogenic role for cytidine and guanosine as antigens or crossreactive antigenic determinants in some patients with SLE.

In vitro nucleoside specific immune response by lymphocytes from systemic lupus erythematosus.

The in vitro immune response of systemic lupus erythematosus (SLE) lymphocytes to nucleosides conjugated to keyhole limpet hemocyanin (KLH) (A,G,C,T-KLH) was investigated. The nucleosides were chosen not only because they are a part of nucleic acid antigen and involved in autoimmunity, but also because nucleoside covalently bound to either soluble IgG or cells had been shown to induce unresponsiveness in mice. A significant proliferation index was induced in SLE lymphocytes, as compared with normal or rheumatoid arthritis (RA) lymphocytes in vitro [in (A,G,C,T)-KLH, 1 microgram/ml; stimulation index = M +/- SE, SLE 2.10 +/- 0.26, RA 1.06 +/- 0.14, normal 1.12 +/- 0.12 P less than 0.05]. Lymphocytes from SLE patients responded specifically to low doses of (A,G,C,T)-KLH and not to the protein carrier KLH alone. A solid-phase radioimmunoassay was developed to detect nucleoside-specific antibody. SLE lymphocytes spontaneously produced high levels of anti-A,G,C,T antibody. This was further increased by antigenic stimulation, but not with pokeweed mitogen (PWM) stimulation. In contrast normal lymphocytes failed to produce anti-A,G,C,T antibody either spontaneously or in response to antigen. However, normal lymphocytes produced antibody after stimulation with PWM. More importantly, anti-A,G,C,T antibody production by SLE lymphocytes was suppressed by preincubation with A,G,C,T-IgG (A,G,C,T-HGG). The antigen-specific unresponsiveness caused by A,G,C,T-HGG was demonstrated by the observation that preincubation with A,G,C,T-HGG did not affect the production of anti-dinitrophenyl antibody response. The ability to manipulate the altered response of SLE lymphocytes to nucleic acid antigens may have therapeutic implications in these patients.