Generation of induced Pluripotent Stem Cells as disease modelling of NLSDM

Neutral Lipid Storage Disease with Myopathy (NLSDM) is a rare defect of triacylglycerol metabolism, characterized by the abnormal storage of neutral lipid in organelles known as lipid droplets (LDs). The main clinical features are progressive myopathy and cardiomyopathy. The onset of NLSDM is caused by autosomal recessive mutations in the PNPLA2 gene, which encodes adipose triglyceride lipase (ATGL). Despite its name, this enzyme is present in a wide variety of cell types and catalyzes the first step in triacylglycerol lipolysis and the release of fatty acids. Here, we report the derivation of NLSDM-induced pluripotent stem cells (NLSDM-iPSCs) from fibroblasts of two patients carrying different PNPLA2 mutations. The first patient was homozygous for the c.541delAC, while the second was homozygous for the c.662G>C mutation in the PNPLA2 gene. We verified that the two types of NLSDM-iPSCs possessed properties of embryonic-like stem cells and could differentiate into the three germ layers in vitro. Immunofluorescence analysis revealed that iPSCs had an abnormal accumulation of triglycerides in LDs, the hallmark of NLSDM. Furthermore, NLSDM-iPSCs were deficient in long chain fatty acid lipolysis, when subjected to a pulse chase experiment with oleic acid. Collectively, these results demonstrate that NLSDM-iPSCs are a promising in vitro model to investigate disease mechanisms and screen drug compounds for NLSDM, a rare disease with few therapeutic options

Combined Docking and Quantum Chemical Study on CYP-mediated Metabolism of Estrogens in Man

Long-term exposure to estrogens seriously increases the incidence of various diseases including breast cancer. Experimental studies indicate that cytochrome P450 (CYP) enzymes catalyze the bioactivation of estrogens to catechols, which can exert their harmful effects via various routes. It has been shown that the 4-hydroxylation pathway of estrogens is the most malign, while 2-hydroxylation is considered a benign pathway. It is also known experimentally that with increasing unsaturation of ring B of estrogens the prevalence of the 4-hydroxylation pathway significantly increases. In this study, we used a combination of structural analysis, docking, and quantum chemical calculations at the B3LYP/6-311+G* level to investigate the factors that influence the regioselectivity of estrogen metabolism in man. We studied the structure of human estrogen metabolizing enzymes (CYP1A1, CYP1A2, CYP1B1, and CYP3A4) in complex with estrone using docking and investigated the susceptibility of estrone, equilin, and equilenin (which only differ in the unsaturation of ring B) to undergo 2- and 4-hydroxylation using several models of CYP enzymes (Compound I, methoxy, and phenoxy radical). We found that even the simplest models could account for the experimental difference between the 2- and 4- hydroxylation pathways and thus might be used for fast screening purposes. We also show that reactivity indices, specifically in this case the radical and nucleophilic condensed Fukui functions, also correctly predict the likeliness of estrogen derivatives to undergo 2- or 4-hydroxylation

Androgen metabolism and inhibition of interleukin-1 synthesis in primary cultured human synovial macrophages

The presence of androgen receptors on synovial macrophages in human normal and rheumatoid synovial tissues has been described previously. It is now reported that primary cultured human macrophages obtained from normal and rheumatoid synovia express functional androgen receptors. We have investigated the capacity of cultured macrophages to metabolize androgens and have found that these cells were capable of metabolizing testosterone to the bioactive metabolite dihydrotestosterone. Therefore, macrophages contain the key enzymes of steroidogenesis, in particular the 5α-treductase. Furthermore, interleukin-1β production by primary cultured rheumatoid macrophages was analysed, following exposure to physiological concentrations of testosterone (10−8 M). A significant decrease of IL-1β levels in conditioned media after 24 h (p < 0.05) was observed. It is concluded that androgens may act directly on human macrophages and may interfere with some of their functions via receptor-dependent mechanisms

Interlaboratory comparison of four in vitro assays for assessing androgenic and antiandrogenic activity of environmental chemicals.

We evaluated and compared four in vitro assays to detect androgen agonists and antagonists in an international interlaboratory study. Laboratory 1 used a cell proliferation assay (assay 1) with human mammary carcinoma cells stably transfected with human androgen receptor. The other laboratories used reporter gene assays, two based on stably transfected human prostate carcinoma cells (assay 2) or human mammary carcinoma cells (assay 4), and the third based on transient transfection of Chinese hamster ovary cells (assay 3). Four laboratories received four coded compounds and two controls: two steroidal androgens, two antiandrogens, an androgenic control, 5alpha-dihydrotestosterone (DHT), and an antiandrogenic control, bicalutamide (ICI 176,334). All laboratories correctly detected the androgenic activity of 4-androsten-3,17-dione and 17alpha-methyltestosterone. For both compounds, the calculated androgenic potencies relative to the positive control (RAPs) remained within one order of magnitude. However, laboratory 3 calculated a 50-fold higher RAP for 4-androsten-3,17-dione. All assays detected and quantified the antiandrogenic effect of vinclozolin [median inhibitory concentration (IC50) values ranging from 1.1 times symbol 10(-7) M to 4.7 times symbol 10(-7) M]. In assays 2 and 3, vinclozolin showed partial androgenic activity at the highest concentrations tested. For vinclozolin, calculated antiandrogenic potencies relative to bicalutamide (RAAPs) differed no more than a factor of 10, and IC50 values matched those of bicalutamide. Similarly, we found antiandrogenic activity for tris-(4-chlorophenyl)methanol. RAAP values were between 0.086 and 0.37. Three assays showed cytotoxicity for this compound at or above 1 times symbol 10(-5) M. In summary, all assays proved sensitive screening tools to detect and quantify androgen receptor-mediated androgenic and antiandrogenic effects of these chemicals accurately, with coefficients of variation between 8 and 90%

Local Treatment of Triple-Negative Breast Cancer: Is Mastectomy Superior to Breast-Conserving Surgery?

Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer that lacks the expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). TNBC accounts for about 15% of breast cancers and has a poorer prognosis as compared with other subtypes of breast cancer. The more rapid onset of this cancer and its aggressiveness have often convinced breast surgeons that mastectomy could provide better oncological results. However, there is no relevant clinical trial that has assessed differences between breast-conserving surgery (BCS) and mastectomy (M) in these patients. This population-based study aimed to investigate the distinct outcomes between conservative treatment and M in a case series of 289 patients with TNBC treated over a 9-year period. This monocentric study retrospectively evaluated patients with TNBC who underwent upfront surgery at Fondazione Policlinico Agostino Gemelli IRCCS, in Rome, between 1 January 2013 and 31 December 2021. First, the patients were divided in two groups according to the surgical treatment received: BCS vs. M. Then, the patients were stratified into four risk subclasses based on combined T and N pathological staging (T1N0, T1N+, T2-4N0 and T2-4N+). The primary endpoint of the study was to evaluate locoregional disease-free survival (LR-DFS), distant disease-free survival (DDFS) and overall survival (OS) in the different subclasses. We analyzed 289 patients that underwent either breast-conserving surgery (247/289, 85.5%) or mastectomy (42/289, 14.5%). After a median follow-up of 43.2 months (49.7, 22.2–74.3), 28 patients (9.6%) developed a locoregional recurrence, 27 patients (9.0%) showed systemic recurrence and 19 patients (6.5%) died. No significant differences due to type of surgical treatment were observed in the different risk subclasses in terms of locoregional disease-free survival, distant disease-free survival and overall survival. With the limits of a retrospective, single-center study, our data seem to indicate similar efficacy in terms of locoregional control, distant metastasis and overall survival with the use of upfront breast-conserving surgery as compared with radical surgery in the treatment of TNBC. Therefore, TNBC should not be considered to be a contraindication for breast conservation