The cyclic GMP-AMP (cGAMP) synthase (cGAS) has been identified as a cytosolic double stranded DNA sensor that plays a pivotal role in the type I interferon and inflammation responses via the STING-dependent signaling pathway. In the past several years, a growing body of evidence has revealed that cGAS is also localized in the nucleus where it is associated with distinct nuclear substructures such as nucleosomes, DNA replication forks, the double-stranded breaks, and centromeres, suggesting that cGAS may have other functions in addition to its role in DNA sensing. However, while the innate immune function of cGAS is well established, the non-canonical nuclear function of cGAS remains poorly understood. Here, we review our current understanding of the complex nature of nuclear cGAS and point to open questions on the novel roles and the mechanisms of action of this protein as a key regulator of cell nuclear function, beyond its well-established role in dsDNA sensing and innate immune response.
Cell Nucleus/immunology* ; Humans ; Immunity, Innate ; Nucleotidyltransferases/immunology* ; Signal Transduction/immunology*

Bromodeoxyuridine, an analogue of thymidine, can be detected by means of monoclonal antibodies and utilized as a marker of the S-phase of the cell cycle. In vitro immunohistochemical application of the BrdU/anti-BrdU-MAb method permits a quantitative assessment of the proliferative activity of a tissue as well as the direct location of the actively replicating cells in histological sections. In this paper, a method for the detection of the labeling index of S-phase cells in normal and neoplastic tissues with in vitro BrdU labeling and standard immunohistochemical techniques using anti-BrdU-MAb and avidin-biotin peroxidase complex is described. We have employed this method in 47 human solid tumor samples, including squamous cell carcinomas of head and neck and cervix uteri, adenocarcinomas and malignant lymphomas, and also evaluated the possible application of the BrdU labeling index to estimate the cycling S-phase cells in neoplastic cell populations. In our data, the in vitro labeling index varied greatly in an individual case (3.56-29.2%) and from an area to an area within the same case. Squamous cell carcinomas of the head and neck showed higher LI than those of the cervix uteri. A case of metastatic carcinoma to the lung from ductal carcinoma of the breast had the highest LI (29.2%), in contrast to the low LI (3.6%) in the primary ductal carcinoma of breast.
Adenocarcinoma/immunology/pathology ; Antibodies, Monoclonal/*immunology ; Breast Neoplasms/immunology/pathology ; Bromodeoxyuridine/*immunology ; Carcinoma, Squamous Cell/immunology/pathology ; Cell Nucleus/immunology/*physiology ; Head and Neck Neoplasms/immunology/pathology ; Humans ; Immunohistochemistry ; *Interphase ; Lymphoma/immunology/pathology ; Neoplasms/immunology/*pathology

Toxoplasma gondii provokes rapid and sustained nuclear translocation of the signal transducer and activator of transcription 6 (STAT6) in HeLa cells. We observed activation of STAT6 as early as 2 hr after infection with T. gondii by the nuclear translocation of fluorescence expressed from exogenously transfected pDsRed2-STAT6 plasmid and by the detection of phosphotyrosine-STAT6 in Western blot. STAT6 activation occurred only by infection with live tachyzoites but not by co-culture with killed tachyzoites or soluble T. gondii extracts. STAT6 phosphorylation was inhibited by small interfering RNA of STAT6 (siSTAT6). In view of the fact that STAT6 is a central mediator of IL-4 induced gene expression, activation of STAT6 by T. gondii infection resembles that infected host cells has been stimulated by IL-4 treatment. STAT1 was affected to increase the transcription and expression by the treatment of siSTAT6. STAT6 activation was not affected by any excess SOCS's whereas that with IL-4 was inhibited by SOCS-1 and SOCS-3. T. gondii infection induced Eotaxin-3 gene expression which was reduced by IFN-gamma. These results demonstrate that T. gondii exploits host STAT6 to take away various harmful reactions by IFN-gamma. This shows, for the first time, IL-4-like action by T. gondii infection modulates microbicidal action by IFN-gamma in infected cells.
Active Transport, Cell Nucleus ; Animals ; Chemokines, CC/biosynthesis ; Hela Cells ; Humans ; Interleukin-4/*immunology ; Mice ; Mice, Inbred BALB C ; STAT6 Transcription Factor/*immunology/*metabolism ; Toxoplasma/*immunology

OBJECTIVETo evaluate the diagnostic role of nuclear expression of bcl-10 protein in extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT) type.

METHODSOne hundred and forty cases of MALT lymphoma were collected from Cancer Hospital of Fudan University (including 38 cases from stomach, 35 cases from ocular adnexa, 16 cases from intestine, 15 cases from skin, 15 cases from salivary gland, 14 cases from lung, 3 cases from thyroid and 4 cases from other sites). Ten cases of reactive follicular hyperplasia of tonsil, 5 cases of reactive lymphoid hyperplasia of orbit and 143 cases of non-Hodgkin's lymphoma other than MALT lymphoma (including 20 cases of NK/T cell lymphoma, 20 cases of follicular lymphomas, 20 cases of anaplastic large cell lymphomas, 20 cases of nodal diffuse large cell B-cell lymphoma (DLBCL), 10 cases of gastric diffuse large B-cell lymphoma, 13 cases of nodal marginal zone B-cell lymphoma, 12 cases of mantle cell lymphoma, 11 cases of splenic marginal zone B-cell lymphoma, 6 cases of angioimmunoblastic T-cell lymphoma, 6 cases of peripheral T-cell lymphoma, not otherwise specified, 3 cases of small lymphocytic lymphoma, 1 case of lymphoplasmacytic lymphoma and 1 case of plasmacytoma were used as controls. Immunohistochemical study for bcl-10, as well as dual staining with CD20, was performed by EnVision method in paraffin sections.

RESULTSIn reactive follicular hyperplasia of tonsil, bcl-10 was moderately or strongly expressed in the cytoplasm of germinal center B cells, while the mantle cells were negative and the marginal zone cells and paracortical T cells showed weak staining. In the 5 cases of reactive lymphoid hyperplasia of orbit, 2 were bcl-10-negative and the remaining 3 expressed bcl-10 in the cytoplasm of germinal center B cells. As for non-MALT lymphomas, 3 gastric DLBCL showed nuclear expression. The remaining cases showed variable cytoplasmic staining. In some cases of lymphoma, bcl-10 was expressed in tumor cells but not in reactive lymphoid cells. On the other hand, 92.1% (129/140) of MALT lymphoma were bcl-10 positive. Among those cases, 54.3% (76/140) showed cytoplasmic positivity and 37.9% (53/140) showed nuclear positivity. The nuclear positivity rate of bcl-10 in different anatomic sites was different. The staining was most intense in MALT lymphoma of ocular adnexa. Dual staining with CD20 showed that the bcl-10-positive cells were also CD20-positive, though the number of bcl-10-positive cells were less than that of CD20-positive cells.

CONCLUSIONSBcl-10 expression in lymphoid hyperplasia is a universal phenomenon. Cytoplasmic expression of bcl-10 is seen in many different kinds of non-Hodgkin's lymphoma and reactive lymphoid conditions. In some cases of lymphoma, bcl-10 is expressed in tumor cells but not in reactive lymphoid cells, suggesting a possible role of abnormal bcl-10 expression in tumorgenesis. Nuclear expression of bcl-10 is seen mainly in MALT lymphoma, especially when occurring in ocular adnexa and lung. This is in contrast to loss of bcl-10 expression in residual germinal center cells.

Adaptor Proteins, Signal Transducing ; genetics ; Antigens, CD20 ; immunology ; B-Cell CLL-Lymphoma 10 Protein ; Cell Nucleus ; genetics ; Cytoplasm ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Lymphocytes ; pathology ; Lymphoma, B-Cell, Marginal Zone ; genetics ; immunology ; pathology ; Palatine Tonsil ; pathology ; Pseudolymphoma ; genetics

Monocyte chemoattractant protein-1 (MCP1) plays a key role in monocyte/macrophage infiltration to the sub-endothelial space of the blood vessel wall, which is a critical initial step in atherosclerosis. In this study, we examined the intracellular signaling pathway of IL-1beta-induced MCP1 expression using various chemical inhibitors. The pretreatment of a phosphatidylcholine (PC)-specific PLC (PC-PLC) inhibitor (D609), PKC inhibitors, or an NF-kappaB inhibitor completely suppressed the IL-1beta-induced MCP1 expression through blocking NF-kappaB translocation to the nucleus. Pretreatment with inhibitors of tyrosine kinase or PLD partially suppressed MCP1 expression and failed to block nuclear NF-kappaB translocation. These results suggest that IL-1beta induces MCP1 expression through activation of NF-kappaB via the PC-PLC/PKC signaling pathway.
Active Transport, Cell Nucleus/drug effects ; Aorta/pathology ; Atherosclerosis/immunology/metabolism ; Bridged Compounds/pharmacology ; Cell Nucleus/*metabolism ; Cells, Cultured ; Chemokine CCL2/*biosynthesis ; Estrenes/pharmacology ; Genistein/pharmacology ; Humans ; Interleukin-1beta/metabolism ; Myocytes, Smooth Muscle/drug effects/immunology/*metabolism/pathology ; NF-kappa B/*metabolism ; Phospholipases/antagonists & inhibitors ; Protein-Tyrosine Kinases/antagonists & inhibitors ; Pyrrolidinones/pharmacology ; Recombinant Proteins/metabolism ; Signal Transduction/*drug effects ; Thiones/pharmacology

OBJECTIVETo investigate the tumorigenic mechanisms of human leukemia cell line HL60-n in nude mice.

METHODSDifferent clone strains of HL60-n cells were established by limited dilution and their biological features were compared with parental HL-60 cells.

RESULTSThe colony yields in soft agar, especially the large colony yields of the high tumorigenic clone strains HL60-n/A, HL60-n/B were significantly higher than that of the HL-60 cells (P < 0.01). There was no significant difference between the low tumorigenic clone strains HL60-n/E, HL60-n/F and the HL-60 cells. Ultrastructurally, the nucleus was highly abnormal, the euchromatic element of nuclear chromatin increased, the heterochromatin sparse, and the microfilaments in cytoplasm increased and disarranged in the high tumorigenic cells as compared with HL-60 cells. Cell cycle analysis by flow cytometer showed higher S phase fractions in the high tumorigenic cells. The killing activities of NK cells to the high tumorigenic clone strains were significant lower than to the contrast (P < 0.01). The histopathological features produced by the low tumorigenic leukemia cells showed that there were many inflammatory cells infiltrated, the majority of them were lymphocytes, and many tumor cells were killed especially in vessel abundant areas. By contrast, there were few inflammatory cells infiltrated in the tumors produced by the high tumorigenic cell strains.

CONCLUSIONThe mechanism of the high tumorigenic activity of the HL60-n cell line involved higher colony yields in soft agar, higher S phase fraction, decreased susceptibility to NK cell killing, and the inhibition of the host immunity.

Animals ; Carcinogenicity Tests ; Cell Division ; physiology ; Cell Nucleus ; pathology ; ultrastructure ; Disease Models, Animal ; HL-60 Cells ; Humans ; Killer Cells, Natural ; cytology ; immunology ; Leukemia ; pathology ; physiopathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; pathology ; Tumor Stem Cell Assay

OBJECTIVEInflammatory reaction and injury in immature lungs are associated with activation of nuclear factor-kappa B (NF-kappaB) to trigger proinflammatory cytokine release, but the mechanism thereof is not fully understood. The present study was conducted to understand possible relationship between expression of NF-kappaB and its inhibitor and severity and outcome of neonates with hyaline membrane disease (HMD).

METHODSSerial samples of bronchoalveolar lavage fluid (BALF) were obtained during mechanical ventilation from 31 preterm infants with HMD. These infants were divided into two groups: survivors group [n = 22, birth weight (1500 +/- 320) g and gestational age (31.2 +/- 1.8) weeks] and nonsurvivors group [birth weight (1340 +/- 280) g, gestational age (30.8 +/- 2.1) weeks]. Nineteen preterm infants [birth weight (1470 +/- 280) g, gestational age (30.6 +/- 1.9) weeks] without respiratory disorders were enrolled as control subjects. Alveolar macrophages (AM) were isolated by differential adherence. AM was cultured and treated with lipopolysaccharide (LPS) for 1 hr. Then, nuclear extracts of AM were analyzed by electrophoretic mobility shift assay (EMSA) for NF-kappaB expression. NF-kappaB inhibitor (IkappaB-alpha protein) in cytoplasmic extracts was detected by using Western blotting and IL-1beta and IL-8 in BALF by enzyme-linked immunosorbent assay (ELISA).

RESULTSNF-kappaB complexes were observed by EMSA, they were characterized by competition with cold oligonucleotide and p65-specific antibodies. The addition of an excess of cold oligonucleotide, corresponding to the NF-kappaB binding site, turned off the signal of the band, showing that the band was specific. An excess of an irrelevant oligonucleotide (corresponding to the SP-1) did not show any effect. The addition of an anti-p65 antibody caused the supershift of the two upper bands. After EMSA, the NF-kappaB complexes were quantified by using a ImageQuant software. NF-kappaB expression in AM at 24 hrs was higher in all the patients with HMD as compared with control subjects (survives/control, 34.1 vs 11.4 RDU, P < 0.01; nonsurvivors/control, 55.2 vs 11.4 RDU, P < 0.01). The NF-kappaB expression in AM at 72 hrs was higher than that in control subjects but not for nonsurvivors (survivors/control, 47.8 vs 25.6 RDU, P < 0.01; nonsurvivors/control, 21.8 vs 25.6, P > 0.05). The NF-kappaB expression in AM from nonsurvivors was depressed at 72 hrs as compared to 24 hrs (21.8 vs 55.2, P < 0.01), whereas the NF-kappaB expression in AM from survivors was still higher at 72 hrs than that at 24 hrs (47.8 vs 34.1, t = 4.43, P < 0.01).

CONCLUSIONAltered NF-kappaB activation in AM of BALF of neonates with HMD was observed, and it may be mediated by decreased IkappaB synthesis, increased IkappaB degradation, or both. In HMD nonsurvivors NF-kappaB translocation was hampered upon LPS activation.

Birth Weight ; Blotting, Western ; Bronchoalveolar Lavage Fluid ; cytology ; Cell Culture Techniques ; Cell Nucleus ; drug effects ; metabolism ; Cytoplasm ; drug effects ; metabolism ; Electrophoretic Mobility Shift Assay ; Enzyme-Linked Immunosorbent Assay ; Female ; Gestational Age ; Humans ; Hyaline Membrane Disease ; immunology ; therapy ; I-kappa B Proteins ; immunology ; Infant, Newborn ; Infant, Premature ; immunology ; Interleukin-1beta ; immunology ; Interleukin-8 ; immunology ; Lipopolysaccharides ; pharmacology ; Macrophages, Alveolar ; drug effects ; immunology ; Male ; NF-KappaB Inhibitor alpha ; NF-kappa B ; immunology ; Respiration, Artificial ; Severity of Illness Index ; Time Factors

Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen that can cause severe diseases in pigs and result in enormous economic losses in the worldwide swine industry. Previous studies revealed that PEDV exhibits an obvious capacity for modulating interferon (IFN) signaling or expression. The newly discovered type III IFN, which plays a crucial role in antiviral immunity, has strong antiviral activity against PEDV proliferation in IPEC-J2 cells. In this study, we aimed to investigate the effect of PEDV nucleocapsid (N) protein on type III IFN-λ. We found that the N proteins of ten PEDV strains isolated between 2013 and 2017 from different local farms shared high nucleotide identities, while the N protein of the CV777 vaccine strain formed a monophyletic branch in the phylogenetic tree. The N protein of the epidemic strain could antagonize type III IFN, but not type I or type II IFN expression induced by polyinosinic-polycytidylic acid (poly(I:C)) in IPEC-J2 cells. Subsequently, we demonstrated that the inhibition of poly(I:C)-induced IFN-λ3 production by PEDV N protein was dependent on the blocking of nuclear factor-κB (NF-κB) nuclear translocation. These findings might help increase understanding of the pathogenesis of PEDV and its mechanisms for evading the host immune response.
Active Transport, Cell Nucleus ; Animals ; Coronavirus Infections ; immunology ; veterinary ; virology ; Genes, Viral ; Host-Pathogen Interactions ; immunology ; Interferons ; antagonists & inhibitors ; biosynthesis ; genetics ; Interleukins ; antagonists & inhibitors ; biosynthesis ; genetics ; NF-kappa B ; metabolism ; Nucleocapsid Proteins ; genetics ; immunology ; physiology ; Porcine epidemic diarrhea virus ; genetics ; pathogenicity ; physiology ; Promoter Regions, Genetic ; Swine ; Swine Diseases ; immunology ; virology

AIMTo study the transfection and anti-hepatitis B virus (HBV) effect of the co-modified hepatocytes-targeting cationic liposomes encapsulating anti-HBV antisense oligonucleotides (asON) , and to investigate the transfection mechanisms of the liposomes.

METHODSDipalmitoylphosphatidylcholine (DPPC) and 3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) were used as the lipids, beta-sitosterol-beta-D-glucoside (sito-G) and Brij 35 were used to modify the liposomes. Flow cytometry (FCM), fluorescence microscopy and enzyme-linked immunosorbent assay (ELISA) were utilized to evaluate the transfection improvement of the asON encapsulated in the liposomes in primary rat hepatocytes and the antigens inhibition activity in HepG 2.2.15 cells. The transfection mechanisms were evaluated based on the influence of wortmannin, nigericin, and asialofetuin on the antigens inhibition in HepG 2.2.15 cells by ELISA.

RESULTSThe co-modification with sito-G and Brij 35 significantly improved the transfection of the liposomes in primary rat hepatocytes and antigens inhibition effect in HepG 2.2.15 cells. Both transfection efficiency and antigens inhibition effect showed to be concentration-dependent with the asON-encapsulating liposomes. In fluorescence microscopy, the transfected cells showed strong fluorescence in primary rat hepatocytes, especially in the nuclei. Wortmannin, nigericin and asialofetuin decreased the antigens inhibition of the asON-encapsulating liposomes to different levels. Cationic liposomes modification with sito-G and Brij 35 could improve the transfection and antigens inhibition effect of the asON. The transfection mechanisms of the co-modified liposomes included endocytosis and membrane fusion. The ligand sito-G was confirmed to be able to enhance asialoglycoprotein receptor (ASGPR)-mediated endocytosis.

CONCLUSIONCo-modified hepatocytes-targeting cationic liposomes would be a specific and effective carrier to transfer asON into hepatocytes.

Androstadienes ; pharmacology ; Animals ; Asialoglycoproteins ; pharmacology ; Cell Line, Tumor ; Cell Nucleus ; metabolism ; Cell Survival ; Cells, Cultured ; Endocytosis ; drug effects ; Female ; Fetuins ; Flow Cytometry ; Hepatitis B Antigens ; metabolism ; Hepatitis B virus ; genetics ; immunology ; Hepatocytes ; cytology ; drug effects ; metabolism ; Humans ; Liposomes ; Microscopy, Fluorescence ; Nigericin ; pharmacology ; Oligonucleotides, Antisense ; chemistry ; genetics ; Polyethylene Glycols ; chemistry ; Rats ; Rats, Wistar ; Sitosterols ; chemistry ; Transfection ; methods ; alpha-Fetoproteins ; pharmacology

OBJECTIVETo investigate the effects of ursolic acid (UA) on T-cell proliferation and activation, as well as to examine its effect on nuclear factor-κB (NF-κB) signaling pathway in T cells.

METHODST-cells isolated from BALB/c mice were incubated with UA at concentrations ranging from 5-30 μmol/L in the presence of phorbol 12-myristate 13-acetate (PMA) or PMA plus ionomycin. The proliferation of T cells was measured by the MTT assay. The expressions of CD69, CD25, and CD71 on T-cell surface were analyzed using flow cytometry. The level of interleukin-2 (IL-2) in the culture supernatant of activated T cells was quantified by enzyme-linked immunosorbent assay (ELISA). The level of phosphorylated IκB-α (p-IκB-α) in total protein and p65, a subunit of NF-κB, nuclear translocation were measured by Western blot analysis.

RESULTSUA in a dose-dependent manner significantly decreased the proliferation and inhibited the surface expressions of CD69, CD25, and CD71 in murine T lymphocytes upon in vitro activation (P<0.01). Significant reduction of IL-2 production was found in activated T cells treated with UA (P<0.01). The PMA-induced increase in p-IκB-α protein was inhibited, and nuclear translocation of p65 from the cytoplasm was blocked by UA.

CONCLUSIONUA is a potent inhibitor for T cell activation and proliferation; these effects are associated with the inhibition of NF-κB signaling pathway.

Animals ; Cell Nucleus ; drug effects ; metabolism ; Cell Proliferation ; drug effects ; I-kappa B Proteins ; metabolism ; Interleukin-2 ; secretion ; Ionomycin ; pharmacology ; Lymphocyte Activation ; drug effects ; Mice ; Mice, Inbred BALB C ; NF-KappaB Inhibitor alpha ; NF-kappa B ; metabolism ; Phosphorylation ; drug effects ; Protein Transport ; drug effects ; Signal Transduction ; drug effects ; T-Lymphocytes ; cytology ; drug effects ; immunology ; secretion ; Tetradecanoylphorbol Acetate ; pharmacology ; Triterpenes ; pharmacology