Unconventional activation of PRKDC by TNF-α: deciphering its crucial role in Th1-mediated inflammation beyond DNA repair as part of the DNA-PK complex.

Background: The DNA-dependent protein kinase (DNA-PK) complex comprises a catalytic (PRKDC) and two requisite DNA-binding (Ku70/Ku80) subunits. The role of the complex in repairing double-stranded DNA breaks (DSBs) is established, but its role in inflammation, as a complex or individual subunits, remains elusive. While only ~ 1% of PRKDC is necessary for DNA repair, we reported that partial inhibition blocks asthma in mice without causing SCID.

Progenitor identification and SARS-CoV-2 infection in long-term human distal lung organoid cultures.

The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange and is affected by disorders including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. Investigations of these localized pathologies have been hindered by a lack of 3D in vitro human distal lung culture systems. Further, human distal lung stem cell identification has been impaired by quiescence, anatomic divergence from mouse and lack of lineage tracing and clonogenic culture.

Organoid Modeling of the Tumor Immune Microenvironment.

In vitro cancer cultures, including three-dimensional organoids, typically contain exclusively neoplastic epithelium but require artificial reconstitution to recapitulate the tumor microenvironment (TME). The co-culture of primary tumor epithelia with endogenous, syngeneic tumor-infiltrating lymphocytes (TILs) as a cohesive unit has been particularly elusive. Here, an air-liquid interface (ALI) method propagated patient-derived organoids (PDOs) from >100 human biopsies or mouse tumors in syngeneic immunocompetent hosts as tumor epithelia with native embedded immune cells (T, B, NK, macrophages).

Deficient Activity of the Nuclease MRE11A Induces T Cell Aging and Promotes Arthritogenic Effector Functions in Patients with Rheumatoid Arthritis.

Immune aging manifests with a combination of failing adaptive immunity and insufficiently restrained inflammation. In patients with rheumatoid arthritis (RA), T cell aging occurs prematurely, but the mechanisms involved and their contribution to tissue-destructive inflammation remain unclear. We found that RA CD4 T cells showed signs of aging during their primary immune responses and differentiated into tissue-invasive, proinflammatory effector cells.

NADPH oxidase deficiency underlies dysfunction of aged CD8+ Tregs.

Immune aging results in progressive loss of both protective immunity and T cell-mediated suppression, thereby conferring susceptibility to a combination of immunodeficiency and chronic inflammatory disease. Here, we determined that older individuals fail to generate immunosuppressive CD8+CCR7+ Tregs, a defect that is even more pronounced in the age-related vasculitic syndrome giant cell arteritis. In young, healthy individuals, CD8+CCR7+ Tregs are localized in T cell zones of secondary lymphoid organs, suppress activation and expansion of CD4 T cells by inhibiting the phosphorylation of membrane-proximal signaling molecules, and effectively inhibit proliferative expansion of CD4 T cells in vitro and in vivo.

Age-Associated Failure To Adjust Type I IFN Receptor Signaling Thresholds after T Cell Activation.

With increasing age, naive CD4 T cells acquire intrinsic defects that compromise their ability to respond and differentiate. Type I IFNs, pervasive constituents of the environment in which adaptive immune responses occur, are known to regulate T cell differentiation and survival. Activated naive CD4 T cells from older individuals have reduced responses to type I IFN, a defect that develops during activation and that is not observed in quiescent naive CD4 T cells.

DNA-dependent protein kinase inhibition blocks asthma in mice and modulates human endothelial and CD4⁺ T-cell function without causing severe combined immunodeficiency.

Background: We reported that DNA-dependent protein kinase (DNA-PK) is critical for the expression of nuclear factor κB-dependent genes in TNF-α-treated glioblastoma cells, suggesting an involvement in inflammatory diseases.

Objective: We sought to investigate the role of DNA-PK in asthma.

Methods: Cell culture and ovalbumin (OVA)- or house dust mite-based murine asthma models were used in this study.

PDZK1 is a novel factor in breast cancer that is indirectly regulated by estrogen through IGF-1R and promotes estrogen-mediated growth.

Although a relationship between PDZK1 expression and estrogen receptor (ER)-α stimulation has been suggested, the nature of such a connection and the function of PDZK1 in breast cancer remain unknown. Human tissue microarrays (cancer tissue: 262 cores; normal tissue: 87 cores) and breast cancer cell lines were used to conduct the study. We show that PDZK1 protein expression is tightly correlated with human breast malignancy, is negatively correlated with age and had no significant correlation with ER-α expression levels.

ICAD deficiency in human colon cancer and predisposition to colon tumorigenesis: linkage to apoptosis resistance and genomic instability.

We previously showed that DNA fragmentation factor, which comprises a caspase-3-activated DNase (CAD) and its inhibitor (ICAD), may influence the rate of cell death by generating PARP-1-activating DNA breaks. Here we tested the hypothesis that ICAD-deficient colon epithelial cells exhibiting resistance to death stimuli may accumulate additional genetic modifications, leading to a tumorigenic phenotype. We show that ICAD deficiency may be associated with colon malignancy in humans.

Apoptotic DNA fragmentation may be a cooperative activity between caspase-activated deoxyribonuclease and the poly(ADP-ribose) polymerase-regulated DNAS1L3, an endoplasmic reticulum-localized endonuclease that translocates to the nucleus during apoptosis.

Caspase-activated DNase (CAD) is the most favorable candidate for chromatin degradation during apoptosis. Ca(2+)-dependent endonucleases are equally important in internucleosomal DNA fragmentation (INDF), including the PARP-1-regulated DNAS1L3. Despite the elaborate work on these endonucleases, the question of whether these enzymes cooperate during INDF was not addressed.

Cordycepin blocks lung injury-associated inflammation and promotes BRCA1-deficient breast cancer cell killing by effectively inhibiting PARP.

Cordycepin has been shown to interfere with a myriad of molecular processes from RNA elongation to kinase activity, and prevents numerous inflammatory processes in animal models. Here we show in a mouse model of LPS-induced acute lung injury that cordycepin prevents airway neutrophilia via a robust blockade of expression of several inflammatory genes, including the adhesion molecule ICAM-1 and VCAM-1, the cytokine/chemokine MCP-1, MIP-1α, MIP-2 and KC, and the chemokine receptor CXCR2. Such a blockade appears to be related to a severe reduction in TNF-α expression.

Phosphorylation of p50 NF-kappaB at a single serine residue by DNA-dependent protein kinase is critical for VCAM-1 expression upon TNF treatment.

The DNA binding activity of NF-κB is critical for VCAM-1 expression during inflammation. DNA-dependent protein kinase (DNA-PK) is thought to be involved in NF-κB activation. Here we show that DNA-PK is required for VCAM-1 expression in response to TNF.

Requirement for inducible nitric oxide synthase in chronic allergen exposure-induced pulmonary fibrosis but not inflammation.

The role of inducible NO synthase (iNOS) in allergic airway inflammation remains elusive. We tested the hypothesis that iNOS plays different roles during acute versus chronic airway inflammation. Acute and chronic mouse models of OVA-induced airway inflammation were used to conduct the study.

Poly(ADP-ribose) polymerase-1 is a determining factor in Crm1-mediated nuclear export and retention of p65 NF-kappa B upon TLR4 stimulation.

The role of NF-kappaB in the expression of inflammatory genes and its participation in the overall inflammatory process of chronic diseases and acute tissue injury are well established. We and others have demonstrated a critical involvement of poly(ADP-ribose) polymerase (PARP)-1 during inflammation, in part, through its relationship with NF-kappaB. However, the mechanism by which PARP-1 affects NF-kappaB activation has been elusive.

Opposing roles of PARP-1 in MMP-9 and TIMP-2 expression and mast cell degranulation in dyslipidemic dilated cardiomyopathy.

Introduction: Previously, we demonstrated that inhibition of poly(ADP-ribose) polymerase (PARP) exerts protective effects against high-fat (HF) diet-induced atherogenesis in part by increasing tissue inhibitor of metalloproteinase (TIMP)-2 expression. Given that characteristics of dilated cardiomyopathy closely associate with atherosclerosis and are mediated by an imbalance between matrix metalloproteinases (MMPs) and TIMPs, we hypothesized that PARP-1 gene deletion may protect against HF-induced cardiac hypertrophy and dilatations by altering TIMP-2/MMPs balance in favor of a maintenance of tissue homeostasis.

Methods And Results: Hemodynamic parameters determined by echocardiography were similar in ApoE(-/-) mice and PARP-1-deficient ApoE(-/-) mice (DKO) fed a regular diet (RD).

High-fat diet induces lung remodeling in ApoE-deficient mice: an association with an increase in circulatory and lung inflammatory factors.

Hypercholesterolemia is increasingly considered the basis for not only cardiovascular pathologies but also several complications affecting other organs such as lungs. In this study, we examined the effect of hypercholesterolemia on lung integrity using a mouse model (ApoE(-/-)) of high-fat (HF) diet-induced atherosclerosis. A 12-week HF diet regimen induced systemic production of TNF-alpha, IFN-gamma, GMC-SF, RANTES, IL-1alpha, IL-2 and IL-12 with TNF-alpha as the predominant cytokine in ApoE(-/-) mice.

Novel tumor-associated antigen of hepatocellular carcinoma defined by monoclonal antibody E4-65.

A monoclonal antibody, E4-65, produced by immunizing mice with SMMC-7721 cells, a human hepatocellular carcinoma (HCC) cell line, was used to identify and characterize an unreported HCC-associated antigen. Indirect immunofluorescence studies showed that E4-65 antibody reacted with five out of eight HCC cell lines, but not with 10 non-HCC tumor cell lines or a normal liver cell line. Using immunohistochemical examination, E4-65 antigen was detected on the cell membranes and in the cytoplasm of human liver tumor tissues, but was not found in most other tumors, or normal adult or fetal tissues, except for a weakly positive reaction in tissues of the digestive system.

Pretreatment with insulin enhances anticancer functions of 5-fluorouracil in human esophageal and colonic cancer cells.

Aim: To investigate the effects of insulin on enhancing 5-fluorouracil (5-FU) anticancer functions and its mechanisms in the human esophageal cancer cell line (Eca 109) and human colonic cancer cell line (Ls-174-t).

Methods: The effect of insulin/5-FU combination treatment on the growth of Eca 109 and Ls-174-t cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. After insulin treatment or insulin/5-FU treatment, cell cycle distribution of both cell lines was analyzed by flow cytometry.

Downregulation of NFAT5 by RNA interference reduces monoclonal antibody productivity of hybridoma cells.

Hybridoma cells display an increase in antibody productivity following exposure to hypertonic conditions. However, the underlying mechanism is not well understood. In the present study, we hypothesize that the nuclear factor of activated T cells 5 (NFAT5)/tonicity enhancer binding protein (TonEBP) functions to increase the antibody productivity of hybridoma cells.

Hyaluronan promotes tumor lymphangiogenesis and intralymphantic tumor growth in xenografts.

Hyaluronan (HA), a high molecular weight glycosaminoglycan in the extracellular matrix, has been implicated in the promotion of malignant phenotypes, including tumor angiogenesis. However, little is known about the effect of HA on tumor-associated lymphangiogenesis. In this study, mouse hepatocellular carcinoma Hca-F cells combined with or without HA were injected subcutaneously into C3H/Hej mice, then angiogenesis and lymphangiogenesis of implanted tumors were examined by immunostaining for platelet-endothelial cell adhesion molecule-1 and lymphatic vascular endothelial hyaluronan receptor-1 respectively.