Knockouts of CYP51A1, DHCR24, or SC5D from cholesterol synthesis reveal pathways modulated by sterol intermediates.

Sterols from cholesterol synthesis are crucial for cholesterol production, but also have individual roles difficult to assess in vivo due to essentiality of cholesterol. We developed HepG2 cell models with knockouts (KOs) for three enzymes of cholesterol synthesis, each accumulating specific sterols. Surprisingly, KOs of CYP51, DHCR24, and SC5D shared only 9% of differentially expressed genes. The most striking was the phenotype of CYP51 KO with highly elevated lanosterol and 24,25-dihydrolanosterol, significant increase in G2+M phase and enhanced cancer and cell cycle pathways. Comparisons with mouse liver Cyp51 KO data suggest 24,25-dihydrolanosterol activates similar cell proliferation pathways, possibly via elevated LEF1 and WNT/NFKB signaling. In contrast, SC5D and DHCR24 KO cells with elevated lathosterol or desmosterol proliferated slowly, with downregulated E2F, mitosis, and enriched HNF1A. These findings demonstrate that increase of lanosterol and 24,25-dihydrolanosterol, but not other sterols, promotes cell proliferation in hepatocytes.

Inflammatory and immune variables as predictors of survival in dogs with myxomatous mitral valve disease.

BACKGROUND: We aimed to investigate the association between selected inflammatory and immune variables and survival of dogs with myxomatous mitral valve disease (MMVD). We evaluated data of 62 client-owned dogs with MMVD, grouped into preclinical, stable congestive heart failure (CHF) and unstable CHF. Univariate Cox proportional hazards regression analysis was used to quantify the association of white blood cell count, concentrations and percentages of T lymphocytes and their subtypes (T helper lymphocytes, cytotoxic T lymphocytes, double positive T lymphocytes, double negative T lymphocytes) and B lymphocytes with survival. P values < 0.1 in individual groups and P values < 0.05 in the group of all patients were considered significant. Spearman correlation coefficients between significant covariates were calculated to assess the relationships among variables and with survival. RESULTS: In the preclinical group, percentage of double positive T lymphocytes was negatively associated with survival (hazard ratio (HR) = 2.328; P = 0.051). In the unstable CHF, T lymphocyte (HR = 1.613; P = 0.085), cytotoxic T lymphocyte (HR = 1.562; P = 0.048), double positive (HR = 1.751; P = 0.042), and double negative T lymphocyte (HR = 1.613; P = 0.096) concentrations were negatively associated with survival, as well as cytotoxic T lymphocyte (HR = 1.502; P = 0.007) concentration in the group of all patients. The percentage of T helper lymphocytes was positively associated with survival in the unstable CHF (HR = 0.604; P = 0.053) and in the group of all patients (HR = 0.733; P = 0.044). The concentration of cytotoxic T lymphocytes positively correlated with left atrial to aortic ratio (LA/Ao) (rho = 0.259, P = 0.037), and peak velocity of early diastolic mitral flow (rho = 0.259, P = 0.039), whereas the percentage of T helper lymphocytes negatively correlated with left atrial to aortic ratio (LA/Ao) (rho = -0.212, P = 0.090) and early to late mitral flow ratio (rho = -0.232, P = 0.072). CONCLUSIONS: Cytotoxic T lymphocytes, T helper lymphocytes, double positive and double negative T lymphocytes as well as biomarkers cardiac troponin I, N-terminal pro-B-type natriuretic peptide, C-reactive protein are implicated in the progression of MMVD.

Effects of Coenzyme Q10 Supplementation on Oxidative Stress Markers, Inflammatory Markers, Lymphocyte Subpopulations, and Clinical Status in Dogs with Myxomatous Mitral Valve Disease.

Scarce data exist on the effects of coenzyme Q10 (CoQ10) supplementation in dogs with myxomatous mitral valve disease (MMVD). The purpose of this study was to investigate the effect of CoQ10 supplementation on oxidative stress markers (glutathione peroxidase, F2-isoprostanes), markers of inflammation (tumor necrosis factor-α, TNF soluble receptor II, leucocytes, and their subtypes), lymphocyte subpopulations (T helper and cytotoxic T lymphocytes, including activated T lymphocytes, and B lymphocytes), and echocardiographic and clinical parameters in dogs with MMVD. In this randomized, controlled, double-blind, longitudinal study, 43 MMVD dogs in stages ACVIM (American College of Veterinary Internal Medicine classification) B2 and ACVIM C and D (congestive heart failure (CHF)) received water-soluble coenzyme Q10 (100 mg twice daily) or placebo for 3 months, and 12 non-supplemented healthy dogs served as controls. All parameters were measured before and after supplementation in MMVD dogs and once in healthy dogs. CoQ10 supplementation had a positive impact on neutrophil percentage, lymphocyte percentage, and lymphocyte concentration in our cohort of dogs with CHF (ACVIM C and D). Conclusion: CoQ10 as an oral supplement may have benefits in terms of decreasing inflammation in dogs with MMVD and CHF.

Peripheral blood lymphocyte subtypes in dogs with different stages of myxomatous mitral valve disease.

BACKGROUND: Data on alterations in peripheral blood lymphocyte (PBL) subtypes in dogs with myxomatous mitral valve disease (MMVD) is lacking. OBJECTIVES: To investigate PBL subtypes and their correlation with parameters of inflammation and MMVD progression markers in dogs with different stages of MMVD. ANIMALS: Seventy-eight client-owned dogs: 65 with MMVD (American College of Veterinary Internal Medicine [ACVIM] classification stages B2, C, and D) and 13 healthy controls. METHODS: Prospective cross-sectional study. Complete cardiac assessment, flow cytometry (T lymphocytes [CD3+], their subtypes [CD3+CD4+, CD3+CD8+, CD3+CD4+CD8+, CD3+CD4-CD8-], and B lymphocytes [CD45+CD21+]) and measurement of N-terminal pro B-type natriuretic peptide, cardiac troponin I, and C-reactive protein concentrations were performed. RESULTS: The percentage of CD3+CD4+ lymphocytes was significantly lower in stable ACVIM C patients (P = .01) and unstable ACVIM C and D patients (P = .003), the percentage of CD3+CD8+ lymphocytes was significantly higher in stable ACVIM C patients (P = .01) and unstable ACVIM C and D patients (P = .01), CD3+CD8+ lymphocyte concentration was significantly higher in unstable ACVIM C and D patients (P = .05), and the CD3+CD4+/CD3+CD8+ ratio was significantly lower in stable ACVIM C patients (P = .01) and unstable ACVIM C and D patients (P = .01) compared with healthy controls. CONCLUSIONS AND CLINICAL IMPORTANCE: The percentages of CD3+CD4+ and CD3+CD8+ PBL and CD4+/CD8+ ratio were altered in MMVD dogs with congestive heart failure (ACVIM C, D), but not in ACVIM B2, suggesting involvement of these PBL subtypes in the pathogenesis of congestive heart failure in dogs with MMVD.

Escherichia coli Affects Expression of Circadian Clock Genes in Human Hepatoma Cells.

Recent research has indicated that dysbiosis of the gut microbiota can lead to an altered circadian clock of the mammalian host. Herein we developed an original system that allows real-time circadian studies of human HepG2 hepatoma cells co-cultured with bacteria. The HepG2 cells with stably integrated firefly luciferase reporter under the control of PERIOD2 promoter were co-cultured with E. coli strains isolated from human fecal samples from healthy individuals. The two E. coli strains differ in the phylogenetic group and the number of ExPEC virulence-associated genes: BJ17 has only two, and BJ23 has 15 of 23 tested. In the first 24 h, the E. coli BJ17 affected the HepG2 circadian clock more than BJ23. Cosinor analysis shows a statistically significant change in the amplitude of PER1 and 2 and the phase advance of PER3. A high percentage of necrotic and apoptotic cells occurred at 72 h, while a correlation between the number of ExPEC genes and the influence on the HepG2 core clock gene expression was observed. Our study reveals that the E. coli genetic background is important for the effect on the mammalian circadian clock genes, indicating possible future use of probiotic E. coli strains to influence the host circadian clock.

Extracellular Vesicles Derived Human-miRNAs Modulate the Immune System in Type 1 Diabetes.

Extracellular vesicles with their molecular cargo can modulate target cell response and may affect the pathogenesis of diseases. The extracellular vesicles containing micro-RNAs (miRNAs), which are often studied as disease biomarkers, but rarely as mediators of the disease development. The role of extracellular vesicles derived miRNAs in type 1 diabetes is currently not well established. We observed a fraction of blood plasma extracellular vesicles positive for membrane proteins potentially associated with insulin-producing beta-cells and identified differentially expressed extracellular vesicles derived miRNAs in individuals with type 1 diabetes. These differentially expressed extracellular vesicles derived human miRNAs in participants with type 1 diabetes and participants with Langerhans islets beta-cells destruction showed the ability to activate TLR7/8 signaling cascade and increase activation as well as cytotoxicity of the effector blood immune cells with cytokine and chemokine release. Our results illustrate extracellular vesicles derived human miRNAs as modulators of the immune system in type 1 diabetes autoimmunity, providing potentially new insight into the pathogenesis of the disease, and novel molecular targets for intervention and type 1 diabetes prevention.

Multifocal gastric adenocarcinoma in a patient with LRBA deficiency.

BACKGROUND: Lipopolysaccharide-responsive, beige-like anchor protein (LRBA) deficiency is characterized by primary immunodeficiency and autoimmunity. Cancer may present another feature of LRBA deficiency. We describe a case history of a young adult with LRBA deficiency and two independent malignancies. METHODS: Family-trio whole exome sequencing with unbiased phenotype ontology approach was used for identification of causative mutations of a primary immune deficiency disorder. Additionally, we sought to identify germline mutations in genes known to be associated with two independent malignancies using a targeted approach. A cytotoxic T-lymphocyte associated protein 4 (CTLA4) expression in T lymphocytes was determined by flow cytometry. RESULTS: In the patient with clinical signs of LRBA deficiency multifocal gastric carcinoma and malignant melanoma were diagnosed and surgically treated at 19 and 27 years of age, respectively. Despite refusal of any adjuvant chemotherapy or radiotherapy, the patient demonstrated disease free survival for at least 13 years after the first cancer diagnosis. A homozygous frameshift deletion in LRBA gene (p.Glu946Ter) and two common variants in TYR gene were identified. Reduced CTLA4 expression in a subset of regulatory T lymphocytes was identified in the patient and his unaffected mother carrying a heterozygous LRBA mutation as compared to control in a dose-dependent manner. CONCLUSION: This is the first description of gastric cancer and malignant melanoma in a young adult with LRBA deficiency. The role of LRBA gene knockout in cancer development and its prognosis remains to be elucidated.