Influence of cell culture conditions on aromatase activity in human genital skin fibroblasts

Summary Because the measurement of aromatase activity in cultured human genital skin fibroblasts has been proposed as a means of studying estrogen production in men, we investigated the influence of culture conditions on aromatase activity. Genital skin fibroblasts were seeded onto culture plates at a density of 1×10⁶ cells/plate and aromatase activity was determined over a 1-mo. period. Enzyme activity rose slowly over the first 14 d but then rose rapidly to a 10-fold higher plateau by Day 28. The rise in aromatase activity was similar whether activity was normalized for protein or for DNA content. When cells were seeded at the usual density of 1×10⁶ or at 0.25×10⁶ cells/plate, aromatase activity was consistently lower during the first 2 wk in cells plated at lower density, but thereafter the levels of enzyme activity in the two groups converged. In cells plated at the lower density, the lower activity observed in the first 2 wk was associated with a lower V _max . Preincubation of cells plated at one density with conditoned medium from cells plated at the other density did not change the relatve levels of activity in the two groups. By contrast, dihydrotestosterone (DHT) receptor binding and 5α-reductase activity were similar at all time points, despite differences in plating density. In additional experiments, the culture medium was replaced daily rather than every 3rd d, and aromatase activity was assayed on Day 7. In cells fed daily, DNA and protein content were twice that of cells fed every 3rd d. By contrast, aromatase activity declined to 30% of the in the latter group. DHT and dexamethasone receptor binding and 5α-reductase activity were similar in the two groups. In summary, factors such as plating density, culture density, and frequency of media replacement dramatically influence aromatase activity in cultured human genital skin fibroblasts. Therefore, the interpretation of aromatase activity data obtained from cultured cells in relation to physiologic or pathologic states should be viewed with appropriate caution. The work was supported in part by grants R01 DK 35339 and R01 DK 00180 from the National Institutes of Health, Bethesda, MD, and by RR 00035 from CLINFO Systems at the Johns Hopkins University School of Medicine, Baltimore, MD.     

Age-mediated gut microbiota dysbiosis promotes the loss of dendritic cells tolerance

The old age-related loss of immune tolerance inflicts a person with a wide range of autoimmune and inflammatory diseases. Dendritic cells (DCs) are the sentinels of the immune system that maintain immune tolerance through cytokines and regulatory T-cells generation. Aging disturbs the microbial composition of the gut, causing immune system dysregulation. However, the vis-à-vis role of gut dysbiosis on DCs tolerance remains highly elusive. Consequently, we studied the influence of aging on gut dysbiosis and its impact on the loss of DC tolerance. We show that DCs generated from either the aged (DC^Old) or gut-dysbiotic young (DC^Dysbiotic) but not young (DC^Young) mice exhibited loss of tolerance, as evidenced by their failure to optimally induce the generation of Tregs and control the overactivation of CD4⁺ T cells. The mechanism deciphered for the loss of DC^Old and DC^Dysbiotic tolerance was chiefly through the overactivation of NF-κB, impaired frequency of Tregs, upregulation in the level of pro-inflammatory molecules (IL-6, IL-1β, TNF-α, IL-12, IFN-γ), and decline in the anti-inflammatory moieties (IL-10, TGF-β, IL-4, IDO, arginase, NO, IRF-4, IRF-8, PDL1, BTLA4, ALDH2). Importantly, a significant decline in the frequency of the Lactobacillus genus was noticed in the gut. Replenishing the gut of old mice with the Lactobacillus plantarum reinvigorated the tolerogenic function of DCs through the rewiring of inflammatory and metabolic pathways. Thus, for the first time, we demonstrate the impact of age-related gut dysbiosis on the loss of DC tolerance. This finding may open avenues for therapeutic intervention for treating age-associated disorders with the Lactobacillus plantarum.     

A theoretical and experimental investigation of the electronic spectra and tautomerization of nucleobases

The electronic structures of all possible tautomers of uracil, thymine, cytosine, adenine and guanine have been carefully examined within the MNDO-MO frame-work. Equilibrium geometries are determined and the relative stabilities are discussed. Allowance for solvent effect on the stabilities is made by assuming a tetrahedral solvent cage with the DNA base occupying its centre. The electronic absorption spectra of the studied DNA bases, in solvents of different polarities are recorded and discussed. Assignments of the observed bands are facilitated using MNDO-CI computations. It is suggested that in solution the DNA bases are in some statistical mixtures of the most stable tautomers, and the Watson-Crick (WC) structure cannot account for the observed spectra alone.     

Synthesis of (+/−)–1′-Aza-carbocyclic-pyrimidine-2′,3′-dideoxynucleoside analogues as potential anti-HIV agents

1′-Aza-carbocyclic-2′, 3′-dideoxyuridine, 3′-deoxythymidine and 2′, 3′-dideoxycytidine were synthesized from 1-aminopyrrolidine intermediate 13 and evaluated as anti-HIV agents in MT-4 cells.     

Combined ipilimumab and nivolumab first‐line and after BRAF‐targeted therapy in advanced melanoma

The combination of ipilimumab and nivolumab is a highly active systemic therapy for metastatic melanoma but can cause significant toxicity. We explore the safety and efficacy of this treatment in routine clinical practice, particularly in the setting of serine/threonine‐protein kinase B‐Raf (BRAF)‐targeted therapy. Consecutive patients with unresectable stage IIIC/IV melanoma commenced on ipilimumab and nivolumab across 10 tertiary melanoma institutions in Australia were identified retrospectively. Data collected included demographics, response and survival outcomes. A total of 152 patients were included for analysis, 39% were treatment‐naïve and 22% failed first‐line BRAF/MEK inhibitors. Treatment‐related adverse events occurred in 67% of patients, grade 3–5 in 38%. The overall objective response rate was 41%, 57% in treatment‐naïve and 21% in BRAF/MEK failure patients. Median progression‐free survival was 4.0 months (95% CI, 3.0–6.0) in the whole cohort, 11.0 months (95% CI, 6.0‐NR) in treatment‐naïve and 2.0 months (95% CI, 1.4–4.6) in BRAF/MEK failure patients. The combination of ipilimumab and nivolumab can be used safely and effectively in a real‐world population. While first‐line efficacy appears comparable to trial populations, BRAF‐mutant patients failing prior BRAF/MEK inhibitors show less response.     

Secondary (iso)BAs cooperate with endogenous ligands to activate FXR under physiological and pathological conditions

IsoBAs, stereoisomers of primary and secondary BAs, are found in feces and plasma of human individuals. BA signaling via the nuclear receptor FXR is crucial for regulation of hepatic and intestinal physiology/pathophysiology. Aim: Investigate the ability of BA-stereoisomers to bind and modulate FXR under physiological/pathological conditions. Methods: Expression-profiling, luciferase-assays, fluorescence-based coactivator-association assays, administration of (iso)-BAs to WT and cholestatic mice. Results: Compared to CDCA/isoCDCA, administration of DCA/isoDCA, UDCA/isoUDCA only slightly increased mRNA expression of FXR target genes; the induction was more evident looking at pre-mRNAs. Notably, almost 50% of isoBAs were metabolized to 3-oxo-BAs within 4 h in cell-based assays, making it difficult to study their actions. FRET-based real-time monitoring of FXR activity revealed that isoCDCA>CDCA stimulated FXR, and isoDCA and isoUDCA allowed fully activated FXR to be re-stimulated by a second dose of GW4064. In vivo co-administration of a single dose of isoBAs followed by GW4064 cooperatively activated FXR, as did feeding of UDCA in a background of endogenous FXR ligands. However, in animals with biliary obstruction and concomitant loss of intestinal BAs, UDCA was unable to increase intestinal Fgf15. In contrast, mice with an impaired enterohepatic circulation of BAs (Asbt?/?, Ostα?/?), administration of UDCA was still able to induce ileal Fgf15 and repress hepatic BA-synthesis, arguing that UDCA is only effective in the presence of endogenous FXR ligands. Conclusion: Secondary (iso)BAs cooperatively activate FXR in the presence of endogenous BAs, which is important to consider in diseases linked to disturbances in BA enterohepatic cycling.     

Novel chalcone/aryl carboximidamide hybrids as potent anti-inflammatory via inhibition of prostaglandin E2 and inducible NO synthase activities: design,synthesis, molecular docking studies and ADMET prediction

Two series of chalcone/aryl carboximidamide hybrids 4a–f and 6a–f were synthesised and evaluated for their inhibitory activity against iNOS and PGE2. The most potent derivatives were further checked for their in vivo anti-inflammatory activity utilising carrageenan-induced rat paw oedema model. Compounds 4c, 4d, 6c and 6d were proved to be the most effective inhibitors of PGE2, LPS-induced NO production, iNOS activity. Moreover, 4c, 4d, 6c and 6d showed significant oedema inhibition ranging from 62.21% to 78.51%, compared to indomethacin (56.27 ± 2.14%) and celecoxib (12.32%). Additionally, 4c, 6a and 6e displayed good COX2 inhibitory activity while 4c, 6a and 6c exhibited the highest 5LOX inhibitory activity. Compounds 4c, 4d, 6c and 6d fit nicely into the pocket of iNOS protein (PDB ID: 1r35) via the important amino acid residues. Prediction of physicochemical parameters exhibited that 4c, 4d, 6c and 6d had acceptable physicochemical parameters and drug-likeness. The results indicated that chalcone/aryl carboximidamides 4c, 4d, 6c and 6d, in particular 4d and 6d, could be used as promising lead candidates as potent anti-inflammatory agents.