Stability study of dehydrocholesterols in dried spot of blood from patients with Smith–Lemli–Opitz syndrome, using filter-paper treated with butylated hydroxytoluene

Considering the clinical importance of implementing a screening method for cholesterol and dehydrocholesterols in dried blood spot, this study aims to standardize the preanalytical variability of DHC in DBS for detecting SLOS by using filter paper impregnated with BHT as an antioxidant

Sterol profiles in plasma and erythrocyte membranes in patients with Smith-Lemli-Opitz syndrome: a six-year experience

Background: This study reports our experience over the last six years in the diagnosis of Smith-Lemli-Opitz syndrome and other inborn errors of cholesterol biosynthesis. Methods: Gas chromatography/mass spectrometry was used to obtain sterol profiles in plasma and erythrocyte membranes of suspected patients. Results: Plasma sterol reference values calculated in unaffected subjects (ns276) were in agreement with those previously reported. Among patients investigated from 2005 to 2010, we report 16 patients affected by Smith-Lemli-Opitz syndrome, three of whom represent new cases and 13 of whom were follow-up patients. In this period we also identified a new case of chondrodysplasia punctata 2 X-linked. The estimated incidence obtained for Smith-Lemli-Opitz syndrome was 1:93 suspected patients (1.08%). We also studied the effect of storage on the dehydrocholesterols/ cholesterol ratio in plasma and erythrocyte membranes of patients affected by Smith-Lemli-Opitz syndrome stored at –208C for up to 22 and 20 months, respectively. A significant negative linear correlation between storage time and the dehydrocholesterols/cholesterol ratio was identified in both plasma and erythrocyte membranes. The decrease in the dehydrocholesterols/cholesterol ratio in erythrocyte membranes was at least two-fold higher than in plasma. Conclusions: The results of this study may be helpful for diagnosis and interpretation of data in patients with findings suggestive of a cholesterol biosynthesis defect

An enhanced procedure for the analysis of organic binders in Pompeian's wall paintings from Insula Occidentalis

Abstract Quantitation of paint powders of ancient wall paintings is often hindered by the calcite contamination during samples withdrawal. To overcome this problem, a new approach was explored based on the mechanical pulverization of the paint powder followed by the evaluation of its true concentration, namely binders, pigments, and decaying compounds, from the comparison of the calcite FT-IR peak area at 2510 cm−1 with that of the corresponding underlying calcite used as calibrator. After extraction of the pulverized paint powder with polar and nonpolar solvents, liquid chromatography, gas chromatography with flame ionization detection, and gas chromatography–mass spectrometry were used to estimate the free amino acids, and fatty acids profiles. Compared to our previous investigation, the results obtained showed a better yield of the extracted organic materials as mg/kg of powder and also a qualitative improvement of the lipids profile

Step-Up Approach for Sodium Butyrate Treatment in Children With Congenital Chloride Diarrhea

Objectives: Oral salt substitutive therapy is pivotal for the survival of patients with congenital chloride diarrhea (CLD), however this therapy is unable to influence the symptoms severity. Butyrate has been proposed to limit diarrhea severity in CLD. Unfortunately, the optimal dose schedule is still largely undefined. In addition, butyrate seems not to be well-tolerated by all patients, with some subjects reporting diarrhea worsening. We investigated the efficacy of a step-up therapeutic approach with sodium butyrate in patients who experienced a diarrhea worsening or an absent improvement after the direct administration of 100 mg/kg/day of sodium butyrate. Methods: The efficacy of a step-up therapeutic approach starting from 50 mg/Kg/day with a subsequent 25 mg/kg/day weekly increase up to 100 mg/kg/day of oral sodium butyrate was investigated in previously three unresponsive CLD children. Results: The step-up therapeutic approach resulted effective in limiting diarrhea severity in all our three previously unresponsive CLD patients. Conclusions: Our results suggest the efficacy of the step-up therapeutic approach in CLD children

Lipocalina e Delayed Graft Function nel paziente trapiantato renale

INTRODUZIONE: Il rene trapiantato è esposto agli effetti dell’ischemia-riperfusione responsabili di ritardata ripresa funzionale dell’organo (delayed graft function; DGF). DGF può incidere negativamente sull’evoluzione del rene trapiantato. Nel presente studio è stato valutato il ruolo della lipocalina urinaria quale predittore di DGF. MATERIALI E METODI: Sono stati valutati pazienti sottoposti a trapianto di rene da donatore cadavere. Prelievi ematici erano effettuati immediatamente prima del trapianto. Le urine erano raccolte per le 24 ore successive al trapianto. DGF era definita dalla necessità di trattamento dialitico entro la prima settimana dal trapianto. RISULTATI: Sono stati valutati 20 pazienti. I pazienti che avevano una rapida ripresa funzionale del rene trapiantato (NO-DGF) erano 14 (70%). DGF era osservata in 6 pazienti. L’età media nei DGF era superiore (58±6 Vs 51±11, p=0.001). Nei pazienti DGF risultavano significativamente ridotta la diuresi (57±35 Vs 4150 ± 2230 ml/24h; p=0.001) e la escrezione urinaria di creatinina (191±184 Vs 683±660 mg/24h; p=0.001), misurate nel primo giorno successivo al trapianto. Non erano osservate significative differenze tra pazienti DGF e NO-DGF per la escrezione urinaria di lipocalina (1,20±2,20 Vs 2,44±4,0 mg/24h; p<0.20). In univariata, DGF risultava associata negativamente alla diuresi (r2=-0.795, p=0.001) ed alla escrezione urinaria di creatinina (r2=-0.480, p=0.037) e positivamente all’età (r2=0.446, p=0.049). In multivariata diuresi (p=0.014) ed escrezione urinaria di creatinina (p=0.039) erano associati a DFG. CONCLUSIONI: Lipocalina urinaria, misurata nel giorno successivo al trapianto renale, non è biomarcatore predittivo di DGF. I risultati del presente studio possono essere stati influenzati dal campione limitato di pazienti e dalla bassa incidenza di DGF

8-Hydroxy-2-Deoxyguanosine and 8-Iso-Prostaglandin F2α: Putative Biomarkers to assess Oxidative Stress Damage Following Robot-Assisted Radical Prostatectomy (RARP)

Objective: Prostate cancer (PCa) is the most common type of cancer. Biomarkers help researchers to understand the mechanisms of disease and refine diagnostic panels. We measured urinary 8-hydroxy-2-deoxyguanosine (8-OHdG) and 8-iso-prostaglandin F2α (8-IsoF2α) to assess oxidative stress damage in PCa patients undergoing robot-assisted radical prostatectomy (RARP). Methods: Forty PCa patients were enrolled in the study. Urine was collected before (T0) and 3 months after the RARP procedure (T1). 8-OHdG and 8-IsoF2α were measured through liquid chromatography-tandem mass spectrometry. Sex- and age-matched healthy subjects served as controls (CTRL). Results: At T0, patients exhibited significantly higher levels of 8-OHdG than CTRL (p = 0.026). At T1, 23/40 patients who completed the 3-month follow-up showed levels of 8-OHdG that were significantly lower than at T0 (p = 0.042), and comparable to those of the CTRL subjects (p = 0.683). At T0, 8-Iso-PGF2α levels were significantly higher in PCa patients than in CTRL subjects (p = 0.0002). At T1, 8-Iso-PGF2α levels were significantly lower than at T0 (p < 0.001) and were comparable to those of CTRL patients (p = 0.087). Conclusions: A liquid chromatography-tandem mass spectrometry method reveals enhanced OHdG and 8-Iso-PGF2α in the urine of PCa patients. RARP normalizes such indices of oxidative stress. Large-sized sample studies and long-term follow-ups are now needed to validate these urinary biomarkers for use in the early prevention and successful treatment of PCa

Polar and non-polar organic binder characterization in Pompeian wall paintings: comparison to a simulated painting mimicking an a secco technique.

The use of Fourier transform infrared spectromicroscopy and mass spectrometry (MS) allowed us to characterize the composition of polar and non-polar binders present in sporadic wall paint fragments taken from Pompeii's archaeological excavation. The analyses of the polar and non-polar binder components extracted from paint powder layer showed the presence of amino acids, sugars, and fatty acids but the absence of proteinaceous material. These results are consistent with a water tempera painting mixture composed of pigments, flours, gums, and oils and are in agreement with those obtained from a simulated wall paint sample made for mimicking an ancient "a secco" technique. Notably, for the first time, we report the capability to discriminate by tandem MS the presence of free amino acids in the paint layer

Immunophenotyping of peripheral blood cells allows to discriminate MIS-C and Kawasaki disease

Background: The pathogenesis of the novel described multisystem inflammatory syndrome in children (MIS-C) and Kawasaki disease (KD) is still debated as it is not clear if they are the same or different nosological entities. However, for both the diseases a rapid and unequivocal diagnosis is mandatory to start the therapy before the onset of severe complications. In this study, we aimed to evaluate the white cell populations in MIS-C and KD as potential markers to discriminate between the two diseases. Methods: We studied white cell populations by flow cytometry in 46 MIS-C and 28 KD patients in comparison to 70 age-matched healthy children. Results: MIS-C patients had a significant lymphopenia that involved both B and T populations while KD patients showed a significant neutrophilia and thrombocythemia. Granulocyte/lymphocyte ratio helped to diagnose both MIS-C and KD with a high diagnostic sensitivity, while a multivariate analysis of granulocyte and T lymphocyte number contributed to discriminate between the two diseases. Conclusions: The relevant lymphopenia observed in MIS-C patients suggests that the disease would be a post-infectious sequel of COVID-19 immunologically amplified by a massive cytokine release, while the significant neutrophilia and thrombocythemia observed in KD confirmed that the disorder has the genesis of a systemic vasculitis. The analysis of a panel of circulating cells may help to early diagnose and to discriminate between the two diseases. Supplementary information: The online version contains supplementary material available at 10.1186/s41231-022-00128-2

MIS-C: A COVID-19-associated condition between hypoimmunity and hyperimmunity

: Multisystem inflammatory syndrome in children (MIS-C) is a rare, severe complication of COVID-19. A better knowledge of immunological, cellular, and genetic characteristics of MIS-C could help better understand the pathogenesis of the disease and contribute to identifying specific diagnostic biomarkers and develop targeted therapies. We studied 37 MIS-C children at hospital admission and 24 healthy controls analyzing serum cytokines (IFN-α, IFN-β, IFN-γ, IL-6, IL-10, IL-17A, IL-12p70 and TNF), lymphocyte populations by flow cytometry and 386 genes related to autoimmune diseases, autoinflammation and primary immunodeficiencies by NGS. MIS-C patients showed a significant increase of serum IFNγ (despite a significant reduction of activated Th1) and ILs, even if with a great heterogeneity among patients, revealing different pathways involved in MIS-C pathogenesis and suggesting that serum cytokines at admission may help to select the inflammatory pathways to target in each patient. Flow cytometry demonstrated a relevant reduction of T populations while the percentage of B cell was increased in agreement with an autoimmune pathogenesis of MIS-C. Genetic analysis identified variants in 34 genes and 83.3% of patients had at least one gene variant. Among these, 9 were mutated in more patients. Most genes are related to autoimmune diseases like ATM, NCF1, MCM4, FCN3, and DOCK8 or to autoinflammatory diseases associated to the release of IFNγ like PRF1, NOD2, and MEF. Thus, an incomplete clearance of the Sars-CoV2 during the acute phase may induce tissue damage and self-antigen exposure and genetic variants can predispose to hyper-reactive immune dysregulation events of MIS-C-syndrome. Type II IFN activation and cytokine responses (mainly IL-6 and IL-10) may cause a cytokine storm in some patients with a more severe acute phase of the disease, lymphopenia and multisystemic organ involvement. The timely identification of such patients with an immunocytometric panel might be critical for targeted therapeutic management

7-dehydrocholesterol and its oxidative compounds: stability study and their effects on melanoma cell lines

The 7-dehydrocholesterol (7-DHC), the precursor of cholesterol biosynthesis, is highly reactive and easily modifiable to produce 7-DHC oxidative compounds. Evidences in vitro demonstrated that the instability of 7-DHC in solution and in liposomes is due to its susceptibility to peroxidation. In addition, 7-DHC is also converted in vitamin D3 by photo-induced synthesis which occurs in the skin. 7-DHC is present at relatively high concentrations in skin where it is exposed to exogenous radical sources and oxygen. Recently, it has been reported that 7-DHC oxidative compounds can have deleterious effects on cellular functionality and viability. Ultraviolet radiation is the main cause of skin cancers, and melanoma is the most serious form of tumor. Today, there is no therapy for advanced-stage melanoma and its metastasis due to their high resistance to various anticancer therapies. In this study, we evaluated the effect on melanoma cell lines of 7-DHC as such, and for this aim much care to minimize 7-DHC modifications was used. Therefore, we evaluated the 7-DHC stability in the vehicle suspension used to transfer this compound from the culture medium into the cells. We also measured the intracellular levels of 7-DHC and its oxidative compounds after different treatment times. The stability study showed no significant changes of 7-DHC levels from baseline values in the suspension up to 90 days of storage at 4 °C. We found that from 12 to 72 hours of treatment 82-86% of 7-DHC entered the cells, and the levels of 7-DHC-derived compounds were not significant. Simultaneously, ROS production was significantly increased already after 2 hours. After 24 hours and up to 72 hours, 7-DHC treated melanoma cells showed a reduction of cell growth and viability. The cytotoxic effect of 7-DHC was associated with the increase of Bax levels, the decrease of Bcl-2/Bax ratio, the reduction of mitochondrial membrane potential, the increase of apoptosis inducing factor (AIF) levels, the unchanged caspase-3 activity, and uncleavage of PARP-1. These findings could explain the mechanism through which 7-DHC exerts its cytotoxic effect. The results of this study show that the 7-DHC has a potential pro-apoptotic on melanoma cell lines, shed light on the possible mechanisms through which this molecule exerts its cytotoxic effects and, at same time, may give new insights in the therapeutic perspective of cancer