Alzheimer's disease risk associated with changes in Epstein-Barr virus nuclear antigen 1-specific epitope targeting antibody levels.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder influenced by age, sex, genetic factors, immune alterations, and infections. Multiple lines of evidence suggest that changes in antibody response are linked to AD pathology. METHODS: To elucidate the mechanisms underlying AD development, we investigated antibodies that target autoimmune epitopes using high-resolution epitope microarrays. Our study compared two groups: individuals with AD (n = 19) and non-demented (ND) controls (n = 19). To validate the results, we measured antibody levels in plasma samples from AD patients (n = 96), mild cognitive impairment (MCI; n = 91), and ND controls (n = 97). To further explore the invlovement of EBV, we performed epitope masking immunofluorescence microscopy analysis and tests to induce lytic replication using the B95-8 cell line. RESULTS: In this study, we analyzed high-resolution epitope-specific serum antibody levels in AD, revealing significant disparities in antibodies targeting multiple epitopes between the AD and control groups. Particularly noteworthy was the significant down-regulation of antibody (anti-DG#29) targeting an epitope of Epstein-Barr virus nuclear antigen 1 (EBNA1). This down-regulation increased AD risk in female patients (odds ratio up to 6.6), but not in male patients. Our investigation further revealed that the down-regulation of the antibody (anti-DG#29) is associated with EBV reactivation in AD, as indicated by the analysis of EBV VCA IgG or IgM levels. Additionally, our data demonstrated that the epitope region on EBNA1 for the antibody is hidden during the EBV lytic reactivation of B95-8 cells. CONCLUSION: Our findings suggest a potential relationship of EBV in the development of AD in female. Moreover, we propose that antibodies targeting the epitope (DG#29) of EBNA1 could serve as valuable indicators of AD risk in female.

Variants in the WDR44 WD40-repeat domain cause a spectrum of ciliopathy by impairing ciliogenesis initiation.

WDR44 prevents ciliogenesis initiation by regulating RAB11-dependent vesicle trafficking. Here, we describe male patients with missense and nonsense variants within the WD40 repeats (WDR) of WDR44, an X-linked gene product, who display ciliopathy-related developmental phenotypes that we can model in zebrafish. The patient phenotypic spectrum includes developmental delay/intellectual disability, hypotonia, distinct craniofacial features and variable presence of brain, renal, cardiac and musculoskeletal abnormalities. We demonstrate that WDR44 variants associated with more severe disease impair ciliogenesis initiation and ciliary signaling. Because WDR44 negatively regulates ciliogenesis, it was surprising that pathogenic missense variants showed reduced abundance, which we link to misfolding of WDR autonomous repeats and degradation by the proteasome. We discover that disease severity correlates with increased RAB11 binding, which we propose drives ciliogenesis initiation dysregulation. Finally, we discover interdomain interactions between the WDR and NH2-terminal region that contains the RAB11 binding domain (RBD) and show patient variants disrupt this association. This study provides new insights into WDR44 WDR structure and characterizes a new syndrome that could result from impaired ciliogenesis.

Mycoplasma fermentans infection induces human necrotic neuronal cell death via IFITM3-mediated amyloid-ß (1-42) deposition.

Mycoplasma fermentans is a proposed risk factor of several neurological diseases that has been detected in necrotic brain lesions of acquired immunodeficiency syndrome patients, implying brain invasiveness. However, the pathogenic roles of M. fermentans in neuronal cells have not been investigated. In this study, we found that M. fermentans can infect and replicate in human neuronal cells, inducing necrotic cell death. Necrotic neuronal cell death was accompanied by intracellular amyloid-ß (1-42) deposition, and targeted depletion of amyloid precursor protein by a short hairpin RNA (shRNA) abolished necrotic neuronal cell death. Differential gene expression analysis by RNA sequencing (RNA-seq) showed that interferon-induced transmembrane protein 3 (IFITM3) was dramatically upregulated by M. fermentans infection, and knockdown of IFITM3 abolished both amyloid-ß (1-42) deposition and necrotic cell death. A toll-like receptor 4 antagonist inhibited M. fermentans infection-mediated IFITM3 upregulation. M. fermentans infection also induced necrotic neuronal cell death in the brain organoid. Thus, neuronal cell infection by M. fermentans directly induces necrotic cell death through IFITM3-mediated amyloid-ß deposition. Our results suggest that M. fermentans is involved in neurological disease development and progression through necrotic neuronal cell death.

FAM167A is a key molecule to induce BCR-ABL-independent TKI resistance in CML via noncanonical NF-κB signaling activation.

BACKGROUND: BCR-ABL-independent drug resistance is a barrier to curative treatment of chronic myeloid leukemia (CML). However, the molecular pathways underlying BCR-ABL-independent tyrosine kinase inhibitor (TKI) resistance remain unclear. METHODS: In silico bioinformatic analysis was performed to identify the most active transcription factor and its inducer that contribute to BCR-ABL-independent TKI resistance. Tandem mass spectrometry analysis was performed to identify the receptor for the noncanonical NF-κB activator FAM167A. In vitro and in vivo mouse experiments revealed detailed molecular insights into the functional role of the FAM167A-desmoglein-1 (DSG1) axis in BCL-ABL-independent TKI resistance. CML cells derived from CML patients were analyzed using quantitative reverse transcription PCR and flow cytometry. RESULTS: We found that NF-κB had the greatest effect on differential gene expression of BCR-ABL-independent TKI-resistant CML cells. Moreover, we found that the previously uncharacterized protein FAM167A activates the noncanonical NF-κB pathway and induces BCR-ABL-independent TKI resistance. Molecular analyses revealed that FAM167A activates the noncanonical NF-κB pathway by binding to the cell adhesion protein DSG1 to upregulate NF-κB-inducing kinase (NIK) by blocking its ubiquitination. Neutralization of FAM167A in a mouse tumor model reduced noncanonical NF-κB activity and restored sensitivity of cells to TKIs. Furthermore, FAM167A and surface DSG1 levels were highly upregulated in CD34+ CML cells from patients with BCR-ABL-independent TKI-resistant disease. CONCLUSIONS: These results reveal that FAM167A acts as an essential factor for BCR-ABL-independent TKI resistance in CML by activating the noncanonical NF-κB pathway. In addition, FAM167A may serve as an important target and biomarker for BCR-ABL-independent TKI resistance.

TGF-ß Increases MFGE8 Production in Myeloid-Derived Suppressor Cells to Promote B16F10 Melanoma Metastasis.

There is growing evidence that myeloid-derived suppressor cells (MDSCs) are directly involved in all stages leading to metastasis. Many mechanisms for this effect have been proposed, but mechanisms of coregulation between tumor cells and MDSCs remain poorly understood. In this study, we demonstrate that MDSCs are a source of milk fat globule-epidermal growth factor (EGF) factor 8 (MFGE8), which is known to be involved in tumor metastasis. Interestingly, TGF-ß, an abundant cytokine in the tumor microenvironment (TME), increased MFGE8 production by MDSCs. In addition, co-culturing MDSCs with B16F10 melanoma cells increased B16F10 cell migration, while MFGE8 neutralization decreased their migration. Taken together, these findings suggest that MFGE8 is an important effector molecule through which MDSCs promote tumor metastasis, and the TME positively regulates MFGE8 production by MDSCs through TGF-ß.

Phosphorylation of p53 Serine 15 Is a Predictor of Survival for Patients with Hepatocellular Carcinoma.

Background: Hepatocellular carcinoma (HCC) is one of the most common malignant cancers with a poor prognosis. Several commonly investigated immunohistochemical markers in resected HCC have potential prognostic value, but the prognostic utility of p53 expression in HCC has remained elusive. Aim: To evaluate the prognostic value of p53 and p53 phosphorylation at serine 15 (p53 Ser15-P) in patients with HCC. Methods: Surgically resected tumors from 199 HCC patients were analyzed for p21, p53, p53 Ser15-P, and proliferating cell nuclear antigen (PCNA) expression using immunohistochemistry. Results: Stratifying by the expression of p53 Ser15-P (P = 0.016), but not by p53 (P = 0.301), revealed significantly different survival outcomes in patients with HCC. Moreover, our analysis demonstrated that patients who were PCNA-positive and p53 Ser15-P-negative had significantly worse survival outcomes (P = 0.001) than patients who were PCNA-positive and p53 Ser15-P-positive. Conclusions: P53 Ser15-P is associated with poor outcomes in patients with HCC, and this prognostic marker is useful for predicting the survival of patients with PCNA-positive HCC.

PKCθ-Mediated PDK1 Phosphorylation Enhances T Cell Activation by Increasing PDK1 Stability.

PDK1 is essential for T cell receptor (TCR)-mediated activation of NF-κB, and PDK1-induced phosphorylation of PKCθ is important for TCR-induced NF-κB activation. However, inverse regulation of PDK1 by PKCθ during T cell activation has not been investigated. In this study, we found that PKCθ is involved in human PDK1 phosphorylation and that its kinase activity is crucial for human PDK1 phosphorylation. Mass spectrometry analysis of wild-type PKCθ or of kinase-inactive form of PKCθ revealed that PKCθ induced phosphorylation of human PDK1 at Ser-64. This PKCθ-induced PDK1 phosphorylation positively regulated T cell activation and TCR-induced NF-κB activation. Moreover, phosphorylation of human PDK1 at Ser-64 increased the stability of human PDK1 protein. These results suggest that Ser-64 is an important phosphorylation site that is part of a positive feedback loop for human PDK1-PKCθ-mediated T cell activation.

Myeloid-Derived Suppressor Cells Are Controlled by Regulatory T Cells via TGF-ß during Murine Colitis.

Myeloid-derived suppressor cells (MDSCs) are well known regulators of regulatory T cells (Treg cells); however, the direct regulation of MDSCs by Treg cells has not been well characterized. We find that colitis caused by functional deficiency of Treg cells leads to altered expansion and reduced function of MDSCs. During differentiation of MDSCs in vitro from bone marrow cells, Treg cells enhanced MDSC function and controlled their differentiation through a mechanism involving transforming growth factor-ß (TGF-ß). TGF-ß-deficient Treg cells were not able to regulate MDSC function in an experimentally induced model of colitis. Finally, we evaluated the therapeutic effect of TGF-ß-mediated in-vitro-differentiated MDSCs on colitis. Adoptive transfer of MDSCs that differentiated with TGF-ß led to better colitis prevention than the transfer of MDSCs that differentiated without TGF-ß. Our results demonstrate an interaction between Treg cells and MDSCs that contributes to the regulation of MDSC proliferation and the acquisition of immunosuppressive functions.

Cereblon negatively regulates TLR4 signaling through the attenuation of ubiquitination of TRAF6.

Cereblon (CRBN) is a substrate receptor protein for the CRL4A E3 ubiquitin ligase complex. In this study, we report on a new regulatory role of CRBN in TLR4 signaling. CRBN overexpression leads to suppression of NF-κB activation and production of pro-inflammatory cytokines including IL-6 and IL-1ß in response to TLR4 stimulation. Biochemical studies revealed interactions between CRBN and TAK1, and TRAF6 proteins. The interaction between CRBN and TAK1 did not affect the association of the TAB1 and TAB2 proteins, which have pivotal roles in the activation of TAK1, whereas the CRBN-TRAF6 interaction critically affected ubiquitination of TRAF6 and TAB2. Binding mapping results revealed that CRBN interacts with the Zinc finger domain of TRAF6, which contains the ubiquitination site of TRAF6, leading to attenuation of ubiquitination of TRAF6 and TAB2. Functional studies revealed that CRBN-knockdown THP-1 cells show enhanced NF-κB activation and p65- or p50-DNA binding activities, leading to up-regulation of NF-κB-dependent gene expression and increased pro-inflammatory cytokine levels in response to TLR4 stimulation. Furthermore, Crbn(-/-) mice exhibit decreased survival in response to LPS challenge, accompanied with marked enhancement of pro-inflammatory cytokines, such as TNF-α and IL-6. Taken together, our data demonstrate that CRBN negatively regulates TLR4 signaling via attenuation of TRAF6 and TAB2 ubiquitination.

Secretion of IL-1ß from imatinib-resistant chronic myeloid leukemia cells contributes to BCR-ABL mutation-independent imatinib resistance.

Some cases of chronic myelogenous leukemia are resistant to tyrosine kinase inhibitors (TKIs) independently of mutation in BCR-ABL, but the detailed mechanism underlying this resistance has not yet been elucidated. In this study, we generated a TKI-resistant CML cell line, K562R, that lacks a mutation in BCR-ABL. Interleukin-1ß (IL-1ß) was more highly expressed in K562R than in the parental cell line K562S, and higher levels of IL-1ß contributed to the imatinib resistance of K562R. In addition, IL-1ß secreted from K562R cells affected stromal cell production of CXCL11, which in turn promoted migration of K562R cells into the stroma. Thus, elevated IL-1ß production from TKI-resistant K562R cells may contribute to TKI resistance by increasing cell viability and promoting cell migration.