Plasminogen Activator Inhibitor-1 and Vitamin D Association in the Overweight and Obese Pediatric Population.

BACKGROUND: Childhood overweight and obesity have been described by the World Health Organization as noncommunicable diseases and among the greatest public health threats since they have reached epidemic proportions. A child with obesity risks becoming an adult with obesity and developing metabolic and hemostatic disorders which are the basis for the development of coronary heart diseases. Recently, a number of clinical reports have demonstrated that both an increase in plasminogen activator inhibitor-1 (PAI-1) and a deficiency in 25OH-vitamin D3 (VD) are associated with an increase in thrombotic episodes. METHODS: PAI-1 and VD levels were measured in 259 clinically overweight and obese children aged between 2 and 18 years enrolled in the Nutritional Education Program of the Bambino Gesù Children's Hospital and Research Institute of Rome (Italy) and 80 normal-weight subjects. RESULTS: We observed increased HOMA-IR, PAI-1, and other inflammation indices associated with decreased VD levels when compared to normal-weight children. CONCLUSIONS: Our results demonstrated that overweight and obesity are correlated with higher levels of the inflammation index. Moreover, our patients show high PAI-1 and low VD levels, confirming the high thrombotic risk in our pediatric population.

Influence of Defatting and Pasteurization on Nutrients and Oxidative Stress Markers in Human Milk.

BACKGROUND: It is well known that the best nutritional option for infants is human milk, and that when breastfeeding is not possible, human milk banks are a possible alternative. However, in the case of infants with fat transport disorder like chylothorax, defatting of human milk is mandatory. RESEARCH AIM: The aim of the study was to reduce milk fat content without reducing other nutrients, increasing oxidative stress, or introducing harmful microorganisms. METHODS: In this prospective, cross-sectional, observational study, we examined the influence of defatting and pasteurization of 50 donor samples on fat, macro- and micronutrients, as well as on oxidative stress markers. RESULTS: Low-temperature centrifugation proved to be very efficient in defatting, reducing the concentration of triglycerides by 85% and cholesterol by 50%. The macronutrients (proteins, albumin, and Immunoglobulin A) did not undergo significant changes due to defatting and pasteurization procedures, while iron decreased by 36%. However, as the majority of iron is retained, this result does not remarkably change the milk composition. Furthermore, oxidative stress markers and antioxidant levels were unchanged, and the milk result was microbiologically safe. CONCLUSIONS: Cold milk centrifugation proved to be an effective technique that allows the reduction of human milk lipids. The determination of triglycerides and cholesterol can be used as an indicator of skimming. This procedure is not accompanied by substantial modifications of other components present in the milk.

Spermine oxidase induces DNA damage and sensitizes fusion negative rhabdomyosarcoma cells to irradiation.

Rhabdomyosarcoma (RMS) is a pediatric myogenic soft tissue sarcoma that includes fusion-positive (FP) and fusion-negative (FN) molecular subtypes. FP-RMS expresses PAX3-FOXO1 fusion protein and often shows dismal prognosis. FN-RMS shows cytogenetic abnormalities and frequently harbors RAS pathway mutations. Despite the multimodal heavy chemo and radiation therapeutic regimens, high risk metastatic/recurrent FN-RMS shows a 5-year survival less than 30% due to poor sensitivity to chemo-radiotherapy. Therefore, the identification of novel targets is needed. Polyamines (PAs) such as putrescine (PUT), spermidine (SPD) and spermine (SPM) are low-molecular-mass highly charged molecules whose intracellular levels are strictly modulated by specific enzymes. Among the latter, spermine oxidase (SMOX) regulates polyamine catabolism oxidizing SPM to SPD, which impacts cellular processes such as apoptosis and DNA damage response. Here we report that low SMOX levels are associated with a worse outcome in FN-RMS, but not in FP-RMS, patients. Consistently, SMOX expression is downregulated in FN-RMS cell lines as compared to normal myoblasts. Moreover, SMOX transcript levels are reduced FN-RMS cells differentiation, being indirectly downregulated by the muscle transcription factor MYOD. Noteworthy, forced expression of SMOX in two cell lines derived from high-risk FN-RMS: 1) reduces SPM and upregulates SPD levels; 2) induces G0/G1 cell cycle arrest followed by apoptosis; 3) impairs anchorage-independent and tumor spheroids growth; 4) inhibits cell migration; 5) increases γH2AX levels and foci formation indicative of DNA damage. In addition, forced expression of SMOX and irradiation synergize at activating ATM and DNA-PKCs, and at inducing γH2AX expression and foci formation, which suggests an enhancement in DNA damage response. Irradiated SMOX-overexpressing FN-RMS cells also show significant decrease in both colony formation capacity and spheroids growth with respect to single approaches. Thus, our results unveil a role for SMOX as inhibitor of tumorigenicity of FN-RMS cells in vitro. In conclusion, our in vitro results suggest that SMOX induction could be a potential combinatorial approach to sensitize FN-RMS to ionizing radiation and deserve further in-depth studies.

Contribution of vitamin D3 and thiols status to the outcome of COVID-19 disease in Italian pediatric and adult patients.

The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARSCoV-2), was declared a global pandemic by the World Health Organization (WHO) on March 2020, causing unprecedented disease with million deaths across the globe, mostly adults. Indeed, children accounted for only a few percent of cases. Italy was the first Western country struck by the COVID-19 epidemic. Increasing age, which is one of the principal risk factors for COVID-19 mortality, is associated with declined glutathione (GSH) levels. Over the last decade, several studies demonstrated that both vitamin D (VD) and GSH have immunomodulatory properties. To verify the association between VD, GSH and the outcome of COVID-19 disease, we conducted a multicenter retrospective study in 35 children and 128 adult patients with COVID-19. Our study demonstrated a hypovitaminosis D in COVID-19 patients, suggesting a possible role of low VD status in increasing the risk of COVID-19 infection and subsequent hospitalization. In addition, we find a thiol disturbance with a GSH depletion associated to the disease severity. In children, who fortunately survived, both VD and GSH levels at admission were higher than in adults, suggesting that lower VD and thiols levels upon admission may be a modifiable risk factor for adverse outcomes and mortality in hospitalized patients with COVID-19.

Study of the Association between Thiols and Oxidative Stress Markers in Children with Obesity.

Obesity has reached epidemic proportions, and the World Health Organization defined childhood overweight and obesity as a noncommunicable disease that represents the most serious public health challenges of the twenty-first century. Oxidative stress, defined as an imbalance between oxidants and antioxidants causing an impairment of the redox signals, is linked to the development of metabolic diseases. In addition, reactive oxygen species generated during metabolic disorder could increase inflammation, causing the development of insulin resistance, diabetes, and cardiovascular disease. We analyze serum levels of cysteine (Cys), cysteinyl-glycine (Cys-Gly), homocysteine (Hcy), and glutathione (GSH), and other markers of oxidative stress, such as thiobarbituric acid reactive substances (T-BARS), 8-isoprostane, and protein carbonyl in our children with obesity. Total antioxidant status was also determined. We found lower GSH and Cys-Gly levels, and higher Hcy and oxidative stress markers levels. We also found a positive correlation between Body Mass Index (BMI), Cys, GSH, and Hcy levels, between insulin and Cys levels, and between BMI and the homeostasis model assessment-estimated insulin resistance (HOMA-IR) with 8-isoprostane levels. Finally, we found a correlation between age and GSH and Cys levels. The deficiency of GSH could be restored by dietary supplementation with GSH precursors, supplying an inexpensive approach to oppose oxidative stress, thus avoiding obesity complications.

Hydroxytyrosol Recovers SARS-CoV-2-PLpro-Dependent Impairment of Interferon Related Genes in Polarized Human Airway, Intestinal and Liver Epithelial Cells.

The SARS-CoV-2 pandemic has caused approximately 6.3 million deaths, mainly due to the acute respiratory distress syndrome or multi-organ failure that characterizes COVID-19 acute disease. Post-acute COVID-19 syndrome, also known as long-COVID, is a condition characterized by a complex of symptoms that affects 10-20% of the individuals who have recovered from the infection. Scientific and clinical evidence demonstrates that long-COVID can develop in both adults and children. It has been hypothesized that multi-organ effects of long-COVID could be associated with the persistence of virus RNA/proteins in host cells, but the real mechanism remains to be elucidated. Therefore, we sought to determine the effects of the exogenous expression of the papain-like protease (PLpro) domain of the non-structural protein (NSP3) of SARS-CoV-2 in polarized human airway (Calu-3), intestinal (Caco-2), and liver (HepG2) epithelial cells, and to evaluate the ability of the natural antioxidant hydroxytyrosol (HXT) in neutralizing these effects. Our results demonstrated that PLpro was able to induce a cascade of inflammatory genes and proteins (mainly associated with the interferon pathway) and increase the apoptotic rate and expression of several oxidative stress markers in all evaluated epithelial cells. Noteably, the treatment with 10 µM HXT reverted PL-pro-dependent effects almost completely. This study provides the first evidence that SARS-CoV-2 PLpro remaining in host cells after viral clearance may contribute to the pathogenetic mechanisms of long-COVID. These effects may be counteracted by natural antioxidants. Further clinical and experimental studies are necessary to confirm this hypothesis.

Changes in Total Homocysteine and Glutathione Levels After Laparoscopic Sleeve Gastrectomy in Children with Metabolic-Associated Fatty Liver Disease.

PURPOSE: Paediatric obesity is a well-known risk factor for metabolic-associated fatty liver disease (MAFLD). The aim of this study was to evaluate the effects of laparoscopic sleeve gastrectomy (LSG) on the levels of total homocysteine (tHcy) and total glutathione (tGSH) plasma levels in children with MAFLD. MATERIAL AND METHODS: Twenty-four children with severe obesity who underwent LSG were included in the study. The metabolic parameters, systemic inflammatory markers, one-carbon metabolism products, ultrasound and histological improvement were evaluated at baseline (T0M) and after 12 months from LSG (T12M). RESULTS: The patients exhibited a significant amelioration of several metabolic parameters at T12M. A significant reduction of steatosis was observed at ultrasound (from 72.7% of moderate-severe grade to 0% severe steatosis), accompanied by a statistically significant improvement of ballooning, portal and lobular inflammation and fibrosis. A statistically significant decrease of tumour necrosis factor circulating levels was also observed (T0M median = 290.3, IQR = 281.0-317.0 pg/mL; T12M median = 260.4, IQR = 240.0-279.0 pg/mL; p < 0.0001). After 12 months from LSG, a significant increase of mean plasma levels of tHcy(T0M mean = 15.7 ± 4.1 µmol/L; T12M mean = 21.1 ± 9.3 µmol/L; p = 0.0146) was also observed. The increase of tHcy showed no causal link with the improvement of MAFLD-related inflammatory, metabolic and histological pattern. CONCLUSION: LSG in children with obesity induces an improvement of MAFLD-related metabolic derangement and liver damage, but also a mild hyperhomocysteinemia that should be avoided to prevent cardiovascular risk.

Benefits and Toxicity of Disulfiram in Preclinical Models of Nephropathic Cystinosis.

Nephropathic cystinosis is a rare disease caused by mutations of the CTNS gene that encodes for cystinosin, a lysosomal cystine/H+ symporter. The disease is characterized by early-onset chronic kidney failure and progressive development of extra-renal complications related to cystine accumulation in all tissues. At the cellular level, several alterations have been demonstrated, including enhanced apoptosis, altered autophagy, defective intracellular trafficking, and cell oxidation, among others. Current therapy with cysteamine only partially reverts some of these changes, highlighting the need to develop additional treatments. Among compounds that were identified in a previous drug-repositioning study, disulfiram (DSF) was selected for in vivo studies. The cystine depleting and anti-apoptotic properties of DSF were confirmed by secondary in vitro assays and after treating Ctns-/- mice with 200 mg/kg/day of DSF for 3 months. However, at this dosage, growth impairment was observed. Long-term treatment with a lower dose (100 mg/kg/day) did not inhibit growth, but failed to reduce cystine accumulation, caused premature death, and did not prevent the development of renal lesions. In addition, DSF also caused adverse effects in cystinotic zebrafish larvae. DSF toxicity was significantly more pronounced in Ctns-/- mice and zebrafish compared to wild-type animals, suggesting higher cell toxicity of DSF in cystinotic cells.

Drug Repurposing in Rare Diseases: An Integrative Study of Drug Screening and Transcriptomic Analysis in Nephropathic Cystinosis.

Diagnosis and cure for rare diseases represent a great challenge for the scientific community who often comes up against the complexity and heterogeneity of clinical picture associated to a high cost and time-consuming drug development processes. Here we show a drug repurposing strategy applied to nephropathic cystinosis, a rare inherited disorder belonging to the lysosomal storage diseases. This approach consists in combining mechanism-based and cell-based screenings, coupled with an affordable computational analysis, which could result very useful to predict therapeutic responses at both molecular and system levels. Then, we identified potential drugs and metabolic pathways relevant for the pathophysiology of nephropathic cystinosis by comparing gene-expression signature of drugs that share common mechanisms of action or that involve similar pathways with the disease gene-expression signature achieved with RNA-seq.

Cerebrospinal Fluid Levels of AFP and hCG: Validation of the Analytical Method and Application in the Diagnosis of Central Nervous System Germ Cell Tumors.

The determination of Human Chorionic Gonadotropin (hCG) and Alpha Fetoprotein (AFP) levels on serum and amniotic fluid plays a fundamental role in the diagnosis and follow-up of specific physiological or pathological conditions (e.g., pregnancy, threat of abortion or germ cell tumors). Recently, the quantification of hCG and AFP in other biological fluids has gained great attention to support the diagnosis, prognosis and follow-up of neoplastic diseases deriving from trophoblastic cells, such as germinomas. Most of the commercial kits for hCG and AFP assays are developed to be used on biological fluids such as serum/plasma and/or urine by manufacturing companies. The aim of this work was to evaluate the suitability of the analytical method certified for the use on serum, and/or amniotic fluid for the quantification of hCG and AFP in cerebrospinal fluid, carrying out an internal validation protocol. The data reported here show that the automated immunochemical method is fit for quantification of hCG and AFP in cerebrospinal fluid (CSF), allowing selective and specific diagnosis of secreting germ cell tumors. This is confirmed by the positive correlation between elevated levels of hCG or AFP and the diagnosis of brain tumors.

Focal adhesion kinase inhibitor TAE226 combined with Sorafenib slows down hepatocellular carcinoma by multiple epigenetic effects.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common and lethal malignant tumours worldwide. Sorafenib (SOR) is one of the most effective single-drug systemic therapy against advanced HCC, but the identification of novel combination regimens for a continued improvement in overall survival is a big challenge. Recent studies highlighted the crucial role of focal adhesion kinase (FAK) in HCC growth. The aim of this study was to investigate the antitumor effects of three different FAK inhibitors (FAKi), alone or in combination with SOR, using in vitro and in vivo models of HCC. METHODS: The effect of PND1186, PF431396, TAE226 on cell viability was compared to SOR. Among them TAE226, emerging as the most effective FAKi, was tested alone or in combination with SOR using 2D/3D human HCC cell line cultures and HCC xenograft murine models. The mechanisms of action were assessed by gene/protein expression and imaging approaches, combined with high-throughput methods. RESULTS: TAE226 was the more effective FAKi to be combined with SOR against HCC. Combined TAE226 and SOR treatment reduced HCC growth both in vitro and in vivo by affecting tumour-promoting gene expression and inducing epigenetic changes via dysregulation of FAK nuclear interactome. We characterized a novel nuclear functional interaction between FAK and the NuRD complex. TAE226-mediated FAK depletion and SOR-promoted MAPK down-modulation caused a decrease in the nuclear amount of HDAC1/2 and a consequent increase of the histone H3 lysine 27 acetylation, thus counteracting histone H3 lysine 27 trimethylation. CONCLUSIONS: Altogether, our findings provide the first evidence that TAE226 combined with SOR efficiently reduces HCC growth in vitro and in vivo. Also, our data highlight that deep analysis of FAK nuclear interactome may lead to the identification of new promising targets for HCC therapy.

Detection of iron deficiency in children with Down syndrome.

PURPOSE: Current American Academy of Pediatrics guidelines for children with Down syndrome (DS) recommend a complete blood count (CBC) at birth and hemoglobin annually to screen for iron deficiency (ID) and ID anemia (IDA) in low-risk children. We aimed to determine if macrocytosis masks the diagnosis of ID/IDA and to evaluate the utility of biochemical and red blood cell indices for detecting ID/IDA in DS. METHODS: We reviewed data from 856 individuals from five DS specialty clinics. Data included hemoglobin, mean corpuscular volume, red cell distribution width (RDW), percent transferrin saturation (TS), ferritin, and c-reactive protein. Receiver operating characteristic curves were calculated. RESULTS: Macrocytosis was found in 32% of the sample. If hemoglobin alone was used for screening, all individuals with IDA would have been identified, but ID would have been missed in all subjects. RDW had the highest discriminability of any single test for ID/IDA. The combination of RDW with ferritin or TS led to 100% sensitivity, and RDW combined with ferritin showed the highest discriminability for ID/IDA. CONCLUSION: We provide evidence to support that a CBC and ferritin be obtained routinely for children over 1 year old with DS rather than hemoglobin alone for detection of ID.

A novel disorder involving dyshematopoiesis, inflammation, and HLH due to aberrant CDC42 function.

Hemophagocytic lymphohistiocytosis (HLH) is characterized by immune dysregulation due to inadequate restraint of overactivated immune cells and is associated with a variable clinical spectrum having overlap with more common pathophysiologies. HLH is difficult to diagnose and can be part of inflammatory syndromes. Here, we identify a novel hematological/autoinflammatory condition (NOCARH syndrome) in four unrelated patients with superimposable features, including neonatal-onset cytopenia with dyshematopoiesis, autoinflammation, rash, and HLH. Patients shared the same de novo CDC42 mutation (Chr1:22417990C>T, p.R186C) and altered hematopoietic compartment, immune dysregulation, and inflammation. CDC42 mutations had been associated with syndromic neurodevelopmental disorders. In vitro and in vivo assays documented unique effects of p.R186C on CDC42 localization and function, correlating with the distinctiveness of the trait. Emapalumab was critical to the survival of one patient, who underwent successful bone marrow transplantation. Early recognition of the disorder and establishment of treatment followed by bone marrow transplant are important to survival.

Targeting oxidative stress improves disease outcomes in a rat model of acquired epilepsy.

Epilepsy therapy is based on antiseizure drugs that treat the symptom, seizures, rather than the disease and are ineffective in up to 30% of patients. There are no treatments for modifying the disease-preventing seizure onset, reducing severity or improving prognosis. Among the potential molecular targets for attaining these unmet therapeutic needs, we focused on oxidative stress since it is a pathophysiological process commonly occurring in experimental epileptogenesis and observed in human epilepsy. Using a rat model of acquired epilepsy induced by electrical status epilepticus, we show that oxidative stress occurs in both neurons and astrocytes during epileptogenesis, as assessed by measuring biochemical and histological markers. This evidence was validated in the hippocampus of humans who died following status epilepticus. Oxidative stress was reduced in animals undergoing epileptogenesis by a transient treatment with N-acetylcysteine and sulforaphane, which act to increase glutathione levels through complementary mechanisms. These antioxidant drugs are already used in humans for other therapeutic indications. This drug combination transiently administered for 2 weeks during epileptogenesis inhibited oxidative stress more efficiently than either drug alone. The drug combination significantly delayed the onset of epilepsy, blocked disease progression between 2 and 5 months post-status epilepticus and drastically reduced the frequency of spontaneous seizures measured at 5 months without modifying the average seizure duration or the incidence of epilepsy in animals. Treatment also decreased hippocampal neuron loss and rescued cognitive deficits. Oxidative stress during epileptogenesis was associated with de novo brain and blood generation of high mobility group box 1 (HMGB1), a neuroinflammatory molecule implicated in seizure mechanisms. Drug-induced reduction of oxidative stress prevented HMGB1 generation, thus highlighting a potential novel mechanism contributing to therapeutic effects. Our data show that targeting oxidative stress with clinically used drugs for a limited time window starting early after injury significantly improves long-term disease outcomes. This intervention may be considered for patients exposed to potential epileptogenic insults.

Glutathione transferase P silencing promotes neuronal differentiation of retinal R28 cells.

Glutathione transferases (GSTs) play an important role in retinal pathophysiology. Within this family, the GSTP isoform is known as an endogenous regulator of cell survival and proliferation pathways and of cellular responses to oxidative stress. In the present study we silenced GSTP in R28 cells, a retinal precursor cell line with markers of both glial and neuronal origin, and obtained stable clones which were viable and, unexpectedly, characterized by a more neuronal phenotype. The degree of neuronal differentiation was inversely correlated with GSTP residual expression levels. The clone with the lowest expression of GSTP showed metabolic reprogramming, a more favorable redox status and, despite its neuronal phenotype, a sensitivity to glutamate and 4-hydroxynonenal toxicity comparable to that of control cells. Altogether, our evidence shows that near full depletion of GSTP in retinal precursor cells, triggers neuronal differentiation and prosurvival metabolic changes.

Intrinsic Bone Defects in Cystinotic Mice.

Cystinosis is a rare lysosomal storage disorder caused by loss-of-function mutations of the CTNS gene, encoding cystinosin, a symporter that mediates cystine efflux from lysosomes. Approximately 95% of patients with cystinosis display renal Fanconi syndrome, short stature, osteopenia, and rickets. In this study, we investigated whether the absence of cystinosin primarily affects bone remodeling activity, apart from the influences of the Fanconi syndrome on bone mineral metabolism. Using micro-computed tomography and histomorphometric and bone serum biomarker analysis, we evaluated the bone phenotype of 1-month-old Ctns-/- knockout (KO) male mice without tubulopathy. An in vitro study was performed to characterize the effects of cystinosin deficiency on osteoblasts and osteoclasts. Micro-computed tomography analysis showed a reduction of trabecular bone volume, bone mineral density, and number and thickness in KO mice compared with wild-type animals; histomorphometric analysis revealed a reduction of osteoblast and osteoclast parameters in tibiae of cystinotic mice. Decreased levels of serum procollagen type 1 amino-terminal propeptide and tartrate-resistant acid phosphatase in KO mice confirmed reduced bone remodeling activity. In vitro experiments showed an impairment of Ctns-/- osteoblasts and osteoclasts. In conclusion, cystinosin deficiency primarily affects bone cells, leading to a bone loss phenotype of KO mice, independent from renal failure.