Association of anti-calcitonin gene-related peptide with other monoclonal antibodies for different diseases: A multicenter, prospective, cohort study.

BACKGROUND AND PURPOSE: Although there is extensive evidence about the safety of monoclonal antibodies against calcitonin gene-related peptide (anti-CGRP mAbs) in combination with traditional drugs, scarce data are available on the safety of their combination with other mAbs. This study aimed to evaluate the 6-month effectiveness and tolerability of anti-CGRP mAbs in combination with other mAbs for different diseases. METHODS: Patients included in the Italian Headache Registry and treated concomitantly with an anti-CGRP mAb and another mAb were included. Effectiveness outcomes for migraine included reduction from baseline of monthly headache days (MHDs), Migraine Disability Assessment (MIDAS) score, Headache Impact Test-6 (HIT-6) scores, and Patients' Global Impression of Change (PGIC) scale. Adverse events (AEs) were recorded. RESULTS: Thirty-eight patients were included. In 27 patients (71.1%), the anti-CGRP mAb was added to a previously ongoing mAb. Nine patients (23.7%) discontinued one of the two mAbs before the end of treatment (seven discontinued the anti-CGRP mAb and two the other mAb). One patient discontinued for AEs. Anti-CGRP mAbs were discontinued due to ineffectiveness (n = 5, 55.5%) and one each (11.1%) for clinical remission and lost to follow-up. MHDs significantly decreased from baseline to 3 months (p < 0.0001) and 6 months (p < 0.001), as did the MIDAS and the HIT-6 scores at 3 and 6 months (p < 0.001). For anti-CGRP mAbs, 27.4% of patients reported PGIC ≥ 5 at 3 months and 48.3% at 6 months. Mild AEs associated with introduction of a second mAb were detected in six patients (15.8%). CONCLUSIONS: In this real-world study, anti-CGRP mAbs showed safety and effectiveness when administered concomitantly with other mAbs.

Standardization and optimization of the hiPSC-based PluriLum assay for detection of embryonic and developmental toxicants.

New approach methodologies (NAMs) for predicting embryotoxicity and developmental toxicity are urgently needed for generating human relevant data, while reducing turnover time and costs, and alleviating ethical concerns related to the use of animal models. We have previously developed the PluriLum assay, a NKX2.5-reporter gene 3D model using human-induced pluripotent stem cells (hiPSCs) that are genetically modified to enable the assessment of adverse effects of chemicals on the early-stage embryo. Aiming at improving the predictive value of the PluriLum assay for future screening purposes, we sought to introduce standardization steps to the protocol, improving the overall robustness of the PluriLum assay, as well as a shortening of the assay protocol. First, we showed that the initial size of embryoid bodies (EBs) is crucial for a proper differentiation into cardiomyocytes and overall reproducibility of the assay. When the starting diameter of the EBs exceeds 500 µm, robust differentiation can be anticipated. In terms of reproducibility, exposure to the fungicide epoxiconazole at smaller initial diameters resulted in a larger variation of the derived data, compared to more reliable concentration-response curves obtained using spheroids with larger initial diameters. We further investigated the ideal length of the differentiation protocol, resulting in a shortening of the PluriLum assay by 24 h to 7 days. Following exposure to the teratogens all-trans and 13-cis retinoic acid, both cardiomyocyte contraction and measurement of NKX2.5-derived luminescence were recorded with a similar or increased sensitivity after 6 days of differentiation when compared to the original 7 days. Finally, we have introduced an efficient step for enzymatic dissociation of the EBs at assay termination. This allows for an even splitting of the individual EBs and testing of additional endpoints other than the NKX2.5-luciferase reporter, which was demonstrated in this work by the simultaneous assessment of ATP levels. In conclusion, we have introduced standardizations and streamlined the PluriLum assay protocol to improve its suitability as a NAM for screening of a large number of chemicals for developmental toxicity testing.

Investigating the applicability domain of the hiPSC-based PluriLum assay: an embryotoxicity assessment of chemicals and drugs.

To meet the growing demand for developmental toxicity assessment of chemicals, New Approach Methodologies (NAMs) are needed. Previously, we developed two 3D in vitro assays based on human-induced pluripotent stem cells (hiPSC) and cardiomyocyte differentiation: the PluriBeat assay, based on assessment of beating differentiated embryoid bodies, and the PluriLum assay, a reporter gene assay based on the expression of the early cardiac marker NKX2.5; both promising assays for predicting embryotoxic effects of chemicals and drugs. In this work, we aimed to further describe the predictive power of the PluriLum assay and compare its sensitivity with PluriBeat and similar human stem cell-based assays developed by others. For this purpose, we assessed the toxicity of a panel of ten chemicals from different chemical classes, consisting of the known developmental toxicants 5-fluorouracil, all-trans retinoic acid and valproic acid, as well as the negative control compounds ascorbic acid and folic acid. In addition, the fungicides epoxiconazole and prochloraz, and three perfluoroalkyl substances (PFAS), PFOS, PFOA and GenX were tested. Generally, the PluriLum assay displayed higher sensitivity when compared to the PluriBeat assay. For several compounds the luminescence readout of the PluriLum assay showed effects not detected by the PluriBeat assay, including two PFAS compounds and the two fungicides. Overall, we find that the PluriLum assay has the potential to provide a fast and objective detection of developmental toxicants and has a level of sensitivity that is comparable to or higher than other in vitro assays also based on human stem cells and cardiomyocyte differentiation for assessment of developmental toxicity.

Genetic Diversity and Pathogenicity of the Fusarium Species Complex on Soybean in Serbia.

Using morphological and cultural characteristics for identification, 36 Fusarium isolates were recovered from diseased roots, stems, and seeds of soybean from several localities throughout Vojvodina Province, Serbia. Based on molecular characterization, 12 Fusarium species were identified: F. acuminatum, F. avenaceum, F. commune, F. equiseti, F. graminearum, F. incarnatum, F. oxysporum, F. proliferatum, F. solani, F. sporotrichioides, F. subglutinans, and F. tricinctum. The elongation factor 1-α-based phylogeny grouped the isolates into 12 well-supported clades, but polymorphisms among sequences in some clades suggested the use of the species complex concept: (i) F. incarnatum-equiseti species complex (FIESC)-F. incarnatum and F. equiseti; (ii) F. oxysporum species complex (FOSC)-F. oxysporum; (iii) F. solani species complex (FSSC)-F. solani; and (iv) F. acuminatum/F. avenaceum/F. tricinctum species complex (FAATSC)-F. acuminatum, F. avenaceum, and F. tricinctum. Pathogenicity tests showed that the most aggressive species causing soybean seed rot were F. sporotrichioides, F. graminearum, FIESC, and F. avenaceum. Furthermore, F. subglutinans, FSSC, and F. proliferatum showed a high percentage of pathogenicity on soybean seeds (80 to 100%), whereas variability in pathogenicity occurred within isolates of F. tricinctum. FOSC, F. commune, and F. acuminatum had the lowest pathogenicity. To our knowledge, this is the first study of the characterization of Fusarium species on soybean in Serbia. This study provides valuable information about the composition of Fusarium species and pathogenicity that will be used in further research on soybean resistance to Fusarium-based diseases.

Toxicity Mechanisms of Gadolinium and Gadolinium-Based Contrast Agents-A Review.

Gadolinium-based contrast agents (GBCAs) have been used for more than 30 years to improve magnetic resonance imaging, a crucial tool for medical diagnosis and treatment monitoring across multiple clinical settings. Studies have shown that exposure to GBCAs is associated with gadolinium release and tissue deposition that may cause short- and long-term toxicity in several organs, including the kidney, the main excretion organ of most GBCAs. Considering the increasing prevalence of chronic kidney disease worldwide and that most of the complications following GBCA exposure are associated with renal dysfunction, the mechanisms underlying GBCA toxicity, especially renal toxicity, are particularly important. A better understanding of the gadolinium mechanisms of toxicity may contribute to clarify the safety and/or potential risks associated with the use of GBCAs. In this work, a review of the recent literature concerning gadolinium and GBCA mechanisms of toxicity was performed.

Age-Related Changes in Clinical and Analytical Variables in Chronic Hemodialyzed Patients.

Worldwide, the number of elderly individuals receiving chronic hemodialysis is rising. The aim of our study was to evaluate several clinical and analytical biomarkers in chronically dialyzed patients and analyze how they change with age. A cross-sectional study was performed by evaluating 289 end-stage renal disease patients undergoing dialysis. We evaluated the hemogram, adipokines, the lipid profile, and several markers related to inflammation, endothelial function/fibrinolysis, nutrition, iron metabolism, and cardiac and renal fibrosis. Clinical data and dialysis efficacy parameters were obtained from all patients. The relationships between studied biomarkers and age were assessed by a statistical comparison between younger (adults with age < 65 years) and older (age ≥ 65 years) patients and by performing regression analysis. Participants presented a mean age of 68.7 years (±13.6), with 66.8% (n = 193) being classified as older. Compared to younger patients, older patients presented the following: (a) significantly lower values of diastolic blood pressure (DBP) and ultrafiltration volume; (b) lower levels of phosphorus, uric acid, creatinine, and albumin; and (c) higher circulating concentrations of tissue-type plasminogen activator (tPA), D-dimer, interleukin-6, leptin, N-terminal pro B-type natriuretic peptide, and tissue inhibitor of metalloproteinase-1. In the multiple linear regression analysis, DBP values, tPA, phosphorus, and D-dimer levels were independently associated with the age of patients (standardized betas: -0.407, 0.272, -0.230, and 0.197, respectively; p < 0.001 for all), demonstrating relevant changes in biomarkers with increasing age at cardiovascular and nutritional levels. These findings seem to result from crosstalk mechanisms between aging and chronic kidney disease.

Second-line therapy in pancreatic ductal adenocarcinoma (PDAC) patients with germline BRCA1-2 pathogenic variants (gBRCA1-2pv).

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) harbouring germline BRCA1-2 pathogenic variants (gBRCA1-2pv) is a distinct nosological entity. Information on second-line therapy (2LT) outcome in this setting is lacking. METHODS: Data of gBRCA1-2pv metastatic PDAC patients treated with chemotherapy were collected. A primary analysis of 2LT RECIST response, median progression-free survival (mPFS2) and overall survival (mOS2), was performed. A secondary analysis addressed the impact of timing of platinum introduction on the outcome of patients receiving at least a first-line combination chemotherapy (1LT). RESULTS: Eighty-four gBRCA1-2pv metastatic PDAC patients were enrolled. The primary analysis, including 43 patients, highlighted a significant improvement of mPFS2 and a doubled response rate, in the platinum-based 2LT subgroup as compared to the platinum-free (8.8 versus 3.7 months, p = 0.013). Seventy-seven patients were included in the secondary analysis. Median PFS1 of 3- and 4-drug platinum-based 1LT significantly outperformed both platinum-free combinations and platinum-based doublets (11.4 versus 6.4 versus 7.9 months, p = 0.01). Albeit still immature, data on mOS paralleled those on mPFS. CONCLUSIONS: This study highlighted the beneficial role of platinum agents in gBRCA1-2pv PDAC patients also in second-line treatment setting. However, our data suggest that early use of 3- and 4-drug platinum-based chemotherapy combinations provides a survival outcome advantage.

HNF4α, SP1 and c-myc are master regulators of CNS autoimmunity.

Hepatocyte nuclear factor 4 α (HNF4α), a transcription factor (TF) essential for embryonic development, has been recently shown to regulate the expression of inflammatory genes. To characterize HNF4a function in immunity, we measured the effect of HNF4α antagonists on immune cell responses in vitro and in vivo. HNF4α blockade reduced immune activation in vitro and disease severity in the experimental model of multiple sclerosis (MS). Network biology studies of human immune transcriptomes unraveled HNF4α together with SP1 and c-myc as master TF regulating differential expression at all MS stages. TF expression was boosted by immune cell activation, regulated by environmental MS risk factors and higher in MS immune cells compared to controls. Administration of compounds targeting TF expression or function demonstrated non-synergic, interdependent transcriptional control of CNS autoimmunity in vitro and in vivo. Collectively, we identified a coregulatory transcriptional network sustaining neuroinflammation and representing an attractive therapeutic target for MS and other inflammatory disorders.

An Exemestane Derivative, Oxymestane-D1, as a New Multi-Target Steroidal Aromatase Inhibitor for Estrogen Receptor-Positive (ER+) Breast Cancer: Effects on Sensitive and Resistant Cell Lines.

Around 70-85% of all breast cancer (BC) cases are estrogen receptor-positive (ER+). The third generation of aromatase inhibitors (AIs) is the first-line treatment option for these tumors. Despite their therapeutic success, they induce several side effects and resistance, which limits their efficacy. Thus, it is crucial to search for novel, safe and more effective anti-cancer molecules. Currently, multi-target drugs are emerging, as they present higher efficacy and lower toxicity in comparison to standard options. Considering this, this work aimed to investigate the anti-cancer properties and the multi-target potential of the compound 1α,2α-epoxy-6-methylenandrost-4-ene-3,17-dione (Oxy), also designated by Oxymestane-D1, a derivative of Exemestane, which we previously synthesized and demonstrated to be a potent AI. For this purpose, it was studied for its effects on the ER+ BC cell line that overexpresses aromatase, MCF-7aro cells, as well as on the AIs-resistant BC cell line, LTEDaro cells. Oxy reduces cell viability, impairs DNA synthesis and induces apoptosis in MCF-7aro cells. Moreover, its growth-inhibitory properties are inhibited in the presence of ERα, ERß and AR antagonists, suggesting a mechanism of action dependent on these receptors. In fact, Oxy decreased ERα expression and activation and induced AR overexpression with a pro-death effect. Complementary transactivation assays demonstrated that Oxy presents ER antagonist and AR agonist activities. In addition, Oxy also decreased the viability and caused apoptosis of LTEDaro cells. Therefore, this work highlights the discovery of a new and promising multi-target drug that, besides acting as an AI, appears to also act as an ERα antagonist and AR agonist. Thus, the multi-target action of Oxy may be a therapeutic advantage over the three AIs applied in clinic. Furthermore, this new multi-target compound has the ability to sensitize the AI-resistant BC cells, which represents another advantage over the endocrine therapy used in the clinic, since resistance is a major drawback in the clinic.

Multisensory mental imagery of fatigue: Evidence from an fMRI study.

Functional imaging experimental designs measuring fatigue, defined as a subjective lack of physical and/or mental energy characterizing a wide range of neurologic conditions, are still under development. Nineteen right-handed healthy subjects (9 M and 10 F, mean age 43.15 ± 8.34 years) were evaluated by means of functional magnetic resonance imaging (fMRI), asking them to perform explicit, first-person, mental imagery of fatigue-related multisensory sensations. Short sentences designed to assess the principal manifestations of fatigue from the Multidimensional Fatigue Symptom Inventory were presented. Participants were asked to imagine the corresponding sensations (Sensory Imagery, SI). As a control, they had to imagine the visual scenes (Visual Imagery, VI) described in short phrases. The SI task (vs. VI task) differentially activated three areas: (i) the precuneus, which is involved in first-person perspective taking; (ii) the left superior temporal sulcus, which is a multisensory integration area; and (iii) the left inferior frontal gyrus, known to be involved in mental imagery network. The SI fMRI task can be used to measure processing involved in mental imagery of fatigue-related multisensory sensations.

Inflammatory biomarkers in staging of chronic kidney disease: elevated TNFR2 levels accompanies renal function decline.

BACKGROUND: Inflammation is a common feature in the pathogenesis of chronic kidney disease (CKD), regardless of the disease cause. Our aim was to evaluate the potential of several inflammatory biomarkers in CKD diagnosis and staging. METHODS: A total of 24 healthy controls and 92 pre-dialysis CKD patients with diverse etiologies, were enrolled in this study and grouped according to their CKD stage. We analysed the circulating levels of inflammatory molecules, C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), tumor necrosis factor receptor 2 (TNFR2), pentraxin 3 (PTX3) and leptin, as well as the hemogram. We studied their association with parameters of kidney function and kidney injury, to evaluate their potential as early markers of the disease and/or of its worsening, as well as their interplay. RESULTS: Compared to controls, patients in CKD stages 1-2 presented significantly higher IL-6 and TNFR2 levels, and higher neutrophil-to-lymphocyte ratio. All inflammatory cytokines and acute-phase proteins showed a trend to increase up to stage 3, stabilizing or declining thereafter, save for TNFR2, which steadily increased from stage to stage. All inflammatory molecules, apart from PTX3, were negatively and significantly correlated with eGFR, with a remarkable value for TNFR2 (r = - 0.732, p < 0.001). CONCLUSION: TNFR2 might be useful for an early detection of CKD, as well as for disease staging/worsening. Still, the potential value of this biomarker in disease progression warrants further investigation.

Association of LTA and SOD Gene Polymorphisms with Cerebral White Matter Hyperintensities in Migraine Patients.

White matter hyperintensities (WMHs) in migraine could be related to inflammatory and antioxidant events. The aim of this study is to verify whether migraine patients with WMHs carry a genetic pro-inflammatory/pro-oxidative status. To test this hypothesis, we analyzed lymphotoxin alpha (LTA; rs2071590T and rs2844482G) and superoxide dismutase 1 (SOD1; rs2234694C) and 2 (SOD2; rs4880T) gene polymorphisms (SNPs) in 370 consecutive patients affected by episodic (EM; n = 251) and chronic (CM; n = 119) migraine and in unrelated healthy controls (n = 100). Brain magnetic resonance was available in 183/370 patients. The results obtained show that genotypes and allele frequencies for all tested SNPs did not differ between patients and controls. No association was found between single SNPs or haplotypes and sex, migraine type, cardiovascular risk factors or disorders. Conversely, the LTA rs2071590T (OR = 2.2) and the SOD1 rs2234694C (OR = 4.9) alleles were both associated with WMHs. A four-loci haplotype (TGCT haplotype: rs2071590T/rs2844482G/rs2234694C/rs4880T) was significantly more frequent in migraineurs with WMHs (7 of 38) compared to those without WMHs (4 of 134; OR = 8.7). We may, therefore, conclude by suggesting that that an imbalance between pro-inflammatory/pro-oxidative and antioxidant events in genetically predisposed individuals may influence the development of WMHs.

Sunitinib versus Pazopanib Dilemma in Renal Cell Carcinoma: New Insights into the In Vitro Metabolic Impact, Efficacy, and Safety.

Sunitinib and pazopanib are tyrosine kinase inhibitors (TKIs) used as first-line therapy for metastatic renal cell carcinoma (RCC). Although these TKIs are associated with similar survival outcomes, some differences have been reported in their safety profiles. In this work, traditional toxicological endpoints (cell viability and growth, oxidative stress, and nuclear morphology) and 1H NMR spectroscopy-based metabolomics analysis were used to provide new insights into the cytotoxicity and metabolic mechanisms underlying sunitinib and pazopanib treatments. Tumoral (Caki-1) and non-tumoral (HK-2) human renal cells were exposed to clinically relevant concentrations of sunitinib (2 µM) or pazopanib (50 µM). Sunitinib showed selectivity for cancer cells, inhibiting proliferation, and inducing apoptotic death of Caki-1 cells, whereas pazopanib had a similar cytotoxic effect in both tumoral and non-tumoral cells. 1H-NMR metabolomics unveiled a higher impact of sunitinib on the levels of intracellular metabolites of Caki-1 cells (seven dysregulated metabolites), suggesting dysregulations on amino acid, glutathione and glycerophospholipid metabolisms. In contrast, pazopanib had a higher impact on the levels of extracellular metabolites of Caki-1 cells (seven dysregulated metabolites in culture medium), unveiling alterations on amino acid and energetic metabolisms. In HK-2 cells, sunitinib caused only a minor increase in intracellular isoleucine levels, whereas pazopanib induced several alterations on the intracellular (three dysregulated metabolites) and extracellular (three dysregulated metabolites) compartments suggesting changes on amino acid, glycerophospholipid, and energy metabolisms. Our results demonstrate that these TKIs elicit distinct cellular and metabolic responses, with sunitinib showing better in vitro efficacy against target RCC cells and lesser nephrotoxic potential than pazopanib.

Beyond faith: Biomolecular evidence for changing urban economies in multi-faith medieval Portugal.

OBJECTIVES: During the Middle Ages, Portugal witnessed unprecedented socioeconomic and religious changes under transitioning religious political rule. The implications of changing ruling powers for urban food systems and individual diets in medieval Portugal is poorly understood. This study aimed to elucidate the dietary impact of the Islamic and Christian conquests. MATERIALS AND METHODS: Radiocarbon dating, peptide mass fingerprinting (ZooMS) and stable isotope analysis (δ13 C, δ15 N) of animal (n = 59) and human skeletal remains (n = 205) from Muslim and Christian burials were used to characterize the diet of a large historical sample from Portugal. A Bayesian stable isotope mixing model (BSIMM) was used to estimate the contribution of marine protein to human diet. RESULTS: Early medieval (8-12th century), preconquest urban Muslim populations had mean (±1SD) values of -18.8 ± 0.4 ‰ for δ13 C 10.4 ± 1 ‰ for δ15 N, indicating a predominantly terrestrial diet, while late medieval (12-14th century) postconquest Muslim and Christian populations showed a greater reliance on marine resources with mean (±1SD) values of -17.9 ± 1.3‰ for δ13 C and 11.1 ± 1.1‰ for δ15 N. BSIMM estimation supported a significant increase in the contribution of marine resources to human diet. DISCUSSION: The results provide the first biomolecular evidence for a dietary revolution that is not evidenced in contemporaneous historical accounts. We find that society transitioned from a largely agro-pastoral economy under Islamic rule to one characterized by a new focus on marine resources under later Christian rule. This economic change led to the naissance of the marine economy that went on to characterize the early-modern period in Portugal and its global expansion.

Stroke management during the coronavirus disease 2019 (COVID-19) pandemic: experience from three regions of the north east of Italy (Veneto, Friuli-Venezia-Giulia, Trentino-Alto-Adige).

BACKGROUND: Efficiency of care chain response and hospital reactivity were and are challenged for stroke acute care management during the pandemic period of coronavirus disease 2019 (COVID-19) in North-Eastern Italy (Veneto, Friuli-Venezia-Giulia, Trentino-Alto-Adige), counting 7,193,880 inhabitants (ISTAT), with consequences in acute treatment for patients with ischemic stroke. METHODS: We conducted a retrospective data collection of patients admitted to stroke units eventually treated with thrombolysis and thrombectomy, ranging from January to May 2020 from the beginning to the end of the main first pandemic period of COVID-19 in Italy. The primary endpoint was the number of patients arriving to these stroke units, and secondary endpoints were the number of thrombolysis and/or thrombectomy. Chi-square analysis was used on all patients; furthermore, patients were divided into two cohorts (pre-lockdown and lockdown periods) and the Kruskal-Wallis test was used to test differences on admission and reperfusive therapies. RESULTS: In total, 2536 patients were included in 22 centers. There was a significant decrease of admissions in April compared to January. Furthermore, we observed a significant decrease of thrombectomy during the lockdown period, while thrombolysis rate was unaffected in the same interval across all centers. CONCLUSIONS: Our study confirmed a decrease in admission rate of stroke patients in a large area of northern Italy during the lockdown period, especially during the first dramatic phase. Overall, there was no decrease in thrombolysis rate, confirming an effect of emergency care system for stroke patients. Instead, the significant decrease in thrombectomy rate during lockdown addresses some considerations of local and regional stroke networks during COVID-19 pandemic evolution.

From street to lab: in vitro hepatotoxicity of buphedrone, butylone and 3,4-DMMC.

Synthetic cathinones are among the most popular new psychoactive substances, being abused for their stimulant properties, which are similar to those of amphetamine and 3,4-methylenedioxymethamphetamine (MDMA). Considering that the liver is a likely target for cathinones-induced toxicity, and for their metabolic activation/detoxification, we aimed to determine the hepatotoxicity of three commonly abused synthetic cathinones: butylone, α-methylamino-butyrophenone (buphedrone) and 3,4-dimethylmethcathinone (3,4-DMMC). We characterized their cytotoxic profile in primary rat hepatocytes (PRH) and in the HepaRG and HepG2 cell lines. PRH was the most sensitive cell model, showing the lowest EC50 values for all three substances (0.158 mM for 3,4-DMMC; 1.21 mM for butylone; 1.57 mM for buphedrone). Co-exposure of PRH to the synthetic cathinones and CYP450 inhibitors (selective and non-selective) proved that hepatic metabolism reduced the toxicity of buphedrone but increased that of butylone and 3,4-DMMC. All compounds were able to increase oxidative stress, disrupting mitochondrial homeostasis and inducing apoptotic and necrotic features, while also increasing the occurrence of acidic vesicular organelles in PRH, compatible with autophagic activation. In conclusion, butylone, buphedrone and 3,4-DMMC have hepatotoxic potential, and their toxicity lies in the interference with a number of homeostatic processes, while being influenced by their metabolic fate.

NTRK Gene Fusion Detection in Atypical Spitz Tumors.

Atypical Spitz tumors (AST) deviate from stereotypical Spitz nevi for one or more atypical features and are now regarded as an intermediate category of melanocytic tumors with uncertain malignant potential. Activating NTRK1/NTRK3 fusions elicit oncogenic events in Spitz lesions and are targetable with kinase inhibitors. However, their prevalence among ASTs and the optimal approach for their detection is yet to be determined. A series of 180 ASTs were screened with pan-TRK immunohistochemistry and the presence of NTRK fusions was confirmed using FISH, two different RNA-based NGS panels for solid tumors, and a specific real time RT-PCR panel. Overall, 26 ASTs showed pan-TRK immunostaining. NTRK1 fusions were detected in 15 of these cases showing cytoplasmic immunoreaction, whereas NTRK3 was detected in one case showing nuclear immunoreaction. Molecular tests resulted all positive in only two ASTs (included the NTRK3 translocated), RNA-based NGS and real time RT-PCR were both positive in three cases, and FISH and real time RT-PCR in another two cases. In seven ASTs NTRK1 fusions were detected only by FISH and in two cases only by real time RT-PCR. The frequency of NTRK fusions in ASTs is 9%, with a clear prevalence of NTRK1 compared to NTRK3 alterations. Pan-TRK immunohistochemistry is an excellent screening test. Confirmation of NTRK fusions may require the use of different molecular techniques.

Cytokines and Growth Factors as Predictors of Response to Medical Treatment in Diabetic Macular Edema.

Diabetic macular edema (DME) is the most common cause of visual loss in patients with diabetes. Antivascular endothelial growth factors (anti-VEGF) agents are first-line therapy for DME. Nevertheless, up to 60% of patients (depending on the anti-VEGF drug used) have an inadequate response to anti-VEGF treatment. Several cytokines are increased in aqueous humor of patients with DME. Differences in response to treatment may be related to baseline cytokine levels. Intravitreal corticosteroids may be used as an alternative to anti-VEGF agents. Steroids have a different pharmacological profile and act on different pathophysiologic mechanisms. Their effect on aqueous humor cytokines is different from the effect of anti-VEGF therapy. This review highlights the major cytokines involved in DME and evaluates whether baseline cytokine levels could be predictors of response to treatment in DME. SIGNIFICANCE STATEMENT: Antivascular endothelial growth factor (anti-VEGF) agents are efficient as diabetic macular edema (DME) treatment. However, in some cases, DME fails to respond to anti-VEGF intravitreal injections. Changes in cytokine levels after treatment supported the idea that other cytokines than VEGF are implicated in DME pathogenesis and could be predictors of response to anti-VEGF treatment or corticosteroids allowing targeted and individualized therapy guided by cytokine levels.

Inhaler training and asthma knowledge are associated with a higher proportion of patients with correct inhaler technique in young but not in elderly asthmatic patients.

Objective: Incorrect inhaler usage is frequent, particularly in elderly asthmatic patients. This study aimed at comparing inhaler technique errors and their determinants, as well inhaler technique self-perception versus real performance, between elderly and non-elderly asthmatics. Methods: Cross-sectional assessment of 92 elderly and 100 non-elderly asthmatics followed at specialty clinics. A standardized questionnaire was applied and inhaler technique demonstration was requested. Errors were assessed using checklists based on manufacturers' instructions and inhaler technique was graded as correct, acceptable or incorrect. Chi-Square Test and Fischer's Exact Test were used for comparative analysis of nominal variables. A p value equal to or less than 0.05 was considered statistically significant. Results: Inhaler technique was correct in a minority of elderly and young patients, without significant differences between study groups. Only 11.1% of the elderly who classified their inhaler as easy and 12.7% who stated their technique was correct had no errors. Previous regular inhaler training was associated with better actual performance in young but not in elderly patients. Conclusion: Our study showed that in spite of regular follow up at specialized outpatient clinics, inhaler devices are associated with a high frequency of errors in elderly and non-elderly asthmatics. In addition, most patients tend to overestimate their technique as correct. Finally, previous, frequent training was associated with a significantly higher percentage of patients showing correct or acceptable technique but only in non-elderly asthmatics, which suggests that elderly asthmatics may need specifically tailored inhaler education programs.

Emerging club drugs: 5-(2-aminopropyl)benzofuran (5-APB) is more toxic than its isomer 6-(2-aminopropyl)benzofuran (6-APB) in hepatocyte cellular models.

New phenylethylamine derivatives are among the most commonly abused new psychoactive substances. They are synthesized and marketed in lieu of classical amphetaminic stimulants, with no previous safety testing. Our study aimed to determine the in vitro hepatotoxicity of two benzofurans [6-(2-aminopropyl)benzofuran (6-APB) and 5-(2-aminopropyl)benzofuran (5-APB)] that have been misused as 'legal highs'. Cellular viability was assessed through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, following 24-h drug exposure of human hepatoma HepaRG cells (EC50 2.62 mM 5-APB; 6.02 mM 6-APB), HepG2 cells (EC50 3.79 mM 5-APB; 8.18 mM 6-APB) and primary rat hepatocytes (EC50 964 µM 5-APB; 1.94 mM 6-APB). Co-incubation of primary hepatocytes, the most sensitive in vitro model, with CYP450 inhibitors revealed a role of metabolism, in particular by CYP3A4, in the toxic effects of both benzofurans. Also, 6-APB and 5-APB concentration-dependently enhanced oxidative stress (significantly increased reactive species and oxidized glutathione, and decreased reduced glutathione levels) and unsettled mitochondrial homeostasis, with disruption of mitochondrial membrane potential and decline of intracellular ATP. Evaluation of cell death mechanisms showed increased caspase-8, -9, and -3 activation, and nuclear morphological changes consistent with apoptosis; at concentrations higher than 2 mM, however, necrosis prevailed. Concentration-dependent formation of acidic vesicular organelles typical of autophagy was also observed for both drugs. Overall, 5-APB displayed higher hepatotoxicity than its 6-isomer. Our findings provide new insights into the potential hepatotoxicity of these so-called 'safe drugs' and highlight the putative risks associated with their use as psychostimulants.