Uncertainty in Environmental Micropollutant Modeling.

Water pollution policies have been enacted across the globe to minimize the environmental risks posed by micropollutants (MPs). For regulative institutions to be able to ensure the realization of environmental objectives, they need information on the environmental fate of MPs. Furthermore, there is an urgent need to further improve environmental decision-making, which heavily relies on scientific data. Use of mathematical and computational modeling in environmental permit processes for water construction activities has increased. Uncertainty of input data considers several steps from sampling and analysis to physico-chemical characteristics of MP. Machine learning (ML) methods are an emerging technique in this field. ML techniques might become more crucial for MP modeling as the amount of data is constantly increasing and the emerging new ML approaches and applications are developed. It seems that both modeling strategies, traditional and ML, use quite similar methods to obtain uncertainties. Process based models cannot consider all known and relevant processes, making the comprehensive estimation of uncertainty challenging. Problems in a comprehensive uncertainty analysis within ML approach are even greater. For both approaches generic and common method seems to be more useful in a practice than those emerging from ab initio. The implementation of the modeling results, including uncertainty and the precautionary principle, should be researched more deeply to achieve a reliable estimation of the effect of an action on the chemical and ecological status of an environment without underestimating or overestimating the risk. The prevailing uncertainties need to be identified and acknowledged and if possible, reduced. This paper provides an overview of different aspects that concern the topic of uncertainty in MP modeling.

Head down tilt 15° to preserve salvageable brain tissue in acute ischemic stroke: A pre-clinical pooled analysis, with focus on cerebral hemodynamics.

Neurological outcome after ischemic stroke depends on residual salvageable brain tissue at the time of recanalization. Head down tilt 15° (HDT15) was proven effective in reducing infarct size and improving functional outcome in rats with transient middle cerebral artery occlusion (t-MCAO) by increasing cerebral perfusion within the ischemic penumbra. In this pooled analysis, individual animal-level data from three experimental series were combined in a study population of 104 t-MCAO rats (45 in HDT15 group and 59 in flat position group). Co-primary outcomes were infarct size and functional outcome at 24 h in both groups. The secondary outcome was hemodynamic change induced by HDT15 in ischemic and non-ischemic hemispheres in a subgroup of animals. Infarct size at 24 h was smaller in HDT15 group than in flat position group (absolute mean difference 31.69 mm3 , 95% CI 9.1-54.2, Cohen's d 0.56, p = 0.006). Functional outcome at 24 h was better in HDT15 group than in flat position group (median [IQR]: 13[10-16] vs. 11), with a shift in the distribution of the neurobehavioural scores in favour of HDT15. Mean cerebral perfusion in the ischemic hemisphere was higher during HDT15 than before its application (Perfusion Unit [P.U.], mean ± SD: 52.5 ± 19.52 P.U. vs. 41.25 ± 14.54 P.U., mean of differences 13.36, 95% CI 7.5-19.18, p = 0.0002). Mean cerebral perfusion in the non-ischemic hemisphere before and during HDT15 was unchanged (P.U., mean ± SD: 94.1 ± 33.8 P.U. vs. 100.25 ± 25.34 P.U., mean of differences 3.95, 95%, CI -1.9 to 9.6, p = 0.1576). This study confirmed that HDT15 improves the outcome in t-MCAO rats by promoting cerebral perfusion in the ischemic territory, without disrupting hemodynamics in non-ischemic areas.

Recurrent NF1 gene variants and their genotype/phenotype correlations in patients with Neurofibromatosis type I.

Neurofibromatosis type I, a genetic condition due to pathogenic variants in the NF1 gene, is burdened by a high rate of complications, including neoplasms, which increase morbidity and mortality for the disease. We retrospectively re-evaluated the NF1 gene variants found in the period 2000-2019 and we studied for genotype/phenotype correlations of disease complications and neoplasms 34 variants, which were shared by at least two unrelated families (range 2-11) for a total 141 of probands and 21 relatives affected by Neurofibromatosis type I. Recurrent variants could be ascribed to the most common mutational mechanisms (C to T transition, microsatellite slippage, non-homologous recombination). In genotype/phenotype correlations, the variants p.Arg440*, p.Tyr489Cys, and p.Arg1947*, together with the gross gene deletions, displayed the highest rates of complications. When considering neoplasms, carriers of variants falling in the extradomain region at the 5' end of NF1 had a lower age-related cancer frequency than the rest of the gene sequence, showing a borderline significance (p = 0.045), which was not conserved after correction with covariates. We conclude that (1) hotspots in NF1 occur via different mutational mechanisms, (2) several variants are associated with high rates of complications and cancers, and (3) there is an initial evidence toward a lower cancer risk for carriers of variants in the 5' end of the NF1 gene although not significant at the multivariate analysis.

How I do it: anterior interhemispheric approach to tuberculum sellae meningiomas.

BACKGROUND: Tuberculum sellae meningiomas are deep-seated tumors difficult to access, located in close relation with important neurovascular structures. While the transsphenoidal approach is linked to specific complications, the different reported transcranial approaches are associated with advantages and drawbacks due to the respective angle of attack, with some areas adequately exposed and others partially hidden. METHOD: We report the technical aspects of the anterior interhemispheric approach we practice. CONCLUSION: This approach has the advantage of providing full control over all the vasculo-nervous structures involved and of allowing access to the medial aspect of both optic canals tangentially to the dorsum sellae.

Free-hand stereotactic ventricular catheter insertion technique based on radio-anatomical landmarks. How I do it.

BACKGROUND: Accurate ventricular catheter (VC) placement plays an important role in reducing the risk of ventriculoperitoneal shunt failure. Free-hand VC insertion is associated with a significant misplacement rate. Consequently, several expensive alternative methods that are unfortunately not available worldwide have been used. To overcome these limitations, we developed a simple surgical technique based on radio-anatomical landmarks aimed at reducing VC's misplacements. METHOD: We reproduce the preoperative imaging on the patient's head using common anatomical landmarks. This allows defining stereotactic VC coordinates to be followed during the surgical procedure. CONCLUSION: This simple and cost-effective method improves VC insertion accuracy.

Freehand stereotactic ventricular catheter insertion for ventriculoperitoneal shunts based on individualized radio-anatomical landmarks.

INTRODUCTION: The accurate placement of the ventricular catheter (VC) is critical in reducing the incidence of proximal failure of ventriculoperitoneal shunts (VPSs). The standard freehand technique is based on validated external anatomical landmarks but remains associated with a relatively high rate of VC malposition. Already proposed alternative methods have all their specific limitations. Herein, we evaluate the accuracy of our adapted freehand technique based on an individualized radio-anatomical approach. Reproducing the preoperative imaging on the patient's head using common anatomical landmarks allows to define stereotactic VC coordinates to be followed at surgery. MATERIAL AND METHODS: Fifty-five consecutive patients treated with 56 VPS between 11/2005 and 02/2020 fulfilled the inclusion criteria of this retrospective study. Burr hole coordinates, VC trajectory, and length were determined in all cases on preoperative computed tomography (CT) scan and were accurately reported on patients' head. The primary endpoint was to evaluate VC placement accuracy. The secondary endpoint was to evaluate the rate and nature of postoperative VC-related complications. RESULTS: Our new technique was applicable in all patients and no VC-related complications were observed. Postoperative imaging showed VC optimally placed in 85.7% and sub-optimally placed in 14.3% of cases. In all procedures, all the holes on the VC tip were found in the ventricular system. CONCLUSIONS: This simple individualized technique improves the freehand VC placement in VPS surgery, making its accuracy comparable to that of more sophisticated and expensive techniques. Further randomized controlled studies are required to compare our results with those of the other available techniques.

Occupational health and safety of road haulage company employees.

BACKGROUND: This paper follows up on a project that was launched in 2008 and contributed to the development of the new Italian Society of Occupational Medicine (SIML) guidelines for the road haulage industry. OBJECTIVE: To reach a better understanding of occupational illness amongst truck drivers, in order to define appropriate health monitoring protocols and promote a healthy life-style. METHODS: We assessed 673 male drivers (mean age 43.85 years, SD 9.56; mean working seniority 27.28 years, SD 10.59), employed by 46 different companies. The drivers, who were gradually recruited in the study over the years, had a maximum of 8 re-assessments each, for a total of 2608 examinations. We applied a survey protocol consisting in a medical examination, questionnaires for the most common risks and instrumental and laboratory tests in compliance with SIML guidelines. RESULTS: We identified a total of 44 work-related diseases: 22 cases of noise-induced hearing loss (NIHL) and 22 cases of lumbar degenerative disc disease. As regards metabolic disorders, we observed 28 cases of diabetes mellitus, in most cases (71.4%) as a first diagnosis or under poor therapeutic control. We observed poorly-controlled hypertension in 103 drivers, the majority of whom (54%) were diagnosed for the first time. Over 30% of the workers in our study were obese and approximately 40% were tobacco smokers. We identified just 9 individuals (1.3%) with a positive toxicological screening for use of recreational drugs. Our data confirm a high prevalence of occupational illness amongst truck drivers. Cardiovascular and metabolic conditions require close monitoring.

Brain ultrasound rehearsal before surgery: A pilot cadaver study.

It has been shown that brain ultrasonography (US) is an efficient tool for improving three-dimensional (3D) spatial orientation during neurosurgical interventions. However, it necessitates specific training as it is highly operator-dependent. To date, neurosurgeons have relied solely on intraoperative practice to improve their mastery of brain US; this has obvious limitations. Herein, we consider whether a study of brain US on human cadavers could enable a training platform for neurosurgeons and residents to be developed. Standard two-dimensional (2D) brain US was performed on two human cadavers (one fresh-frozen and one Thiel-prepared) through left frontoparietal, left frontal, right temporal, and left parietal craniotomies. US workflow and image quality were assessed in both preparations. It was possible to assess US in both cadaver preparations; however, the specimen prepared with Thiel-fixation performed better, with superior image quality and specimen usability at room temperature. US images were obtainable through all surgical corridors with the main intracranial anatomical landmarks easily identifiable. US of cadaveric brains is feasible and delivers good quality results. This technique could allow neurosurgeons to develop the expertise required for a successful clinical application preoperatively. Clin. Anat. 30:1017-1023, 2017. © 2017 Wiley Periodicals, Inc.

CART is overexpressed in human type 2 diabetic islets and inhibits glucagon secretion and increases insulin secretion.

AIMS/HYPOTHESIS: Insufficient insulin release and hyperglucagonaemia are culprits in type 2 diabetes. Cocaine- and amphetamine-regulated transcript (CART, encoded by Cartpt) affects islet hormone secretion and beta cell survival in vitro in rats, and Cart (-/-) mice have diminished insulin secretion. We aimed to test if CART is differentially regulated in human type 2 diabetic islets and if CART affects insulin and glucagon secretion in vitro in humans and in vivo in mice. METHODS: CART expression was assessed in human type 2 diabetic and non-diabetic control pancreases and rodent models of diabetes. Insulin and glucagon secretion was examined in isolated islets and in vivo in mice. Ca(2+) oscillation patterns and exocytosis were studied in mouse islets. RESULTS: We report an important role of CART in human islet function and glucose homeostasis in mice. CART was found to be expressed in human alpha and beta cells and in a subpopulation of mouse beta cells. Notably, CART expression was several fold higher in islets of type 2 diabetic humans and rodents. CART increased insulin secretion in vivo in mice and in human and mouse islets. Furthermore, CART increased beta cell exocytosis, altered the glucose-induced Ca(2+) signalling pattern in mouse islets from fast to slow oscillations and improved synchronisation of the oscillations between different islet regions. Finally, CART reduced glucagon secretion in human and mouse islets, as well as in vivo in mice via diminished alpha cell exocytosis. CONCLUSIONS/INTERPRETATION: We conclude that CART is a regulator of glucose homeostasis and could play an important role in the pathophysiology of type 2 diabetes. Based on the ability of CART to increase insulin secretion and reduce glucagon secretion, CART-based agents could be a therapeutic modality in type 2 diabetes.

[Not Available]. / Diabetes mellitus in critical jobs.

BACKGROUND: Diabetes is a disease with high social impact and it is important to consider how work may be influenced by it or whether work itself may promote or accelerate its course. OBJECTIVES: The objective was to investigate the prevalence of diabetes in four critical categories of workers. The survey involved construction workers, truck drivers, bus drivers and shift workers in the manufacturing sector. METHODS: In the years 2012-14 we investigated 2034 males workers, through personal history, physical examination, laboratory tests. The subjects with fasting glucose >125 mg/dl underwent a second control and haemoglobin A1c determination. Positive cases were referred to specialist control. Expected prevalence and standardized prevalence ratio (SPR) were calculated using official data regarding diabetes prevalence in Italy. RESULTS: The prevalence of diabetes in 608 truck drivers was 4.4% (expected 2.6%, SPR 1.69, IC95% 1.08 to 2.31); in 860 construction workers 1.9% (expected 2.1%, SPR 0.90, IC95% 0.48 to 1.33); in 378 bus drivers 2.6% (expected 3.5%, SPR 0.74, IC95% 0.29 to 1.20); in 188 shift workers 3.2% (expected 2.1%, SPR 1.52, IC95% 0.33 to 2.71). In the age range 35-59 years the prevalence in 467 truck drivers was 5.1% (expected 2.4%, SPR 2.13, IC 95% 1.29 to 2.96). DISCUSSION: Diabetes was confirmed to be highly prevalent at working ages. The study highlighted an increased diabetes prevalence among truck drivers, whereas other jobs might represent protective factors; this hypothesis, however, needs further investigation.

Cerebral collateral flow defines topography and evolution of molecular penumbra in experimental ischemic stroke.

Intracranial collaterals are dynamically recruited after arterial occlusion and are emerging as a strong determinant of tissue outcome in both human and experimental ischemic stroke. The relationship between collateral flow and ischemic penumbra remains largely unexplored in pre-clinical studies. The aim of the present study was to investigate the pattern of collateral flow with regard to penumbral tissue after transient middle cerebral artery (MCA) occlusion in rats. MCA was transiently occluded (90min) by intraluminal filament in adult male Wistar rats (n=25). Intracranial collateral flow was studied in terms of perfusion deficit and biosignal fluctuation analyses using multi-site laser Doppler monitoring. Molecular penumbra was defined by topographical mapping and quantitative signal analysis of Heat Shock Protein 70kDa (HSP70) immunohistochemistry. Functional deficit and infarct volume were assessed 24h after ischemia induction. The results show that functional performance of intracranial collaterals during MCA occlusion inversely correlated with HSP70 immunoreactive areas in both the cortex and the striatum, as well as with infarct size and functional deficit. Intracranial collateral flow was associated with reduced areas of both molecular penumbra and ischemic core and increased areas of intact tissue in rats subjected to MCA occlusion followed by reperfusion. Our findings prompt the development of collateral therapeutics to provide tissue-saving strategies in the hyper-acute phase of ischemic stroke prior to recanalization therapy.

Newcastle disease virotherapy induces long-term survival and tumor-specific immune memory in orthotopic glioma through the induction of immunogenic cell death.

The oncolytic features of several naturally oncolytic viruses have been shown on Glioblastoma Multiforme cell lines and in xenotransplant models. However, orthotopic glioma studies in immunocompetent animals are lacking. Here we investigated Newcastle disease virus (NDV) in the orthotopic, syngeneic murine GL261 model. Seven days after tumor induction, mice received NDV intratumorally. Treatment significantly prolonged median survival and 50% of animals showed long-term survival. We demonstrated immunogenic cell death (ICD) induction in GL261 cells after NDV infection, comprising calreticulin surface exposure, release of HMGB1 and increased PMEL17 cancer antigen expression. Uniquely, we found absence of secreted ATP. NDV-induced ICD occurred independently of caspase signaling and was blocked by Necrostatin-1, suggesting the contribution of necroptosis. Autophagy induction following NDV infection of GL261 cells was demonstrated as well. In vivo, elevated infiltration of IFN-γ(+) T cells was observed in NDV-treated tumors, along with reduced accumulation of myeloid derived suppressor cells. The importance of a functional adaptive immune system in this paradigm was demonstrated in immunodeficient Rag2(-/-) mice and in CD8(+) T cell depleted animals, where NDV slightly prolonged survival, but failed to induce long-term cure. Secondary tumor induction with GL261 cells or LLC cells in mice surviving long-term after NDV treatment, demonstrated the induction of a long-term, tumor-specific immunological memory response by ND virotherapy. For the first time, we describe the therapeutic activity of NDV against GL261 tumors, evidenced in an orthotopic mouse model. The therapeutic effect relies on the induction of ICD in the tumor cells, which primes adaptive antitumor immunity.

A neuronavigation-based method for locating the superficial temporal artery during extra-intracranial bypass surgery.

The superficial temporal artery (STA) is frequently used as donor vessel in extracranial to intracranial bypass surgery. Current techniques typically rely on a Doppler vascular probe to identify the STA trajectory prior to the skin incision; however, this step can be time consuming and difficult when the arterial course is tortuous. We tested an alternative neuronavigation-based technique for locating the STA. In this method, preoperative magnetic resonance angiography (MRA) or computed tomography angiography (CTA) was used to determine STA outlines that were then projected and traced onto the skin. The neuronavigation-based technique was applied to eight STA dissections. The accuracy of this method was evaluated by comparing the navigation-based STA course with the doppler-based one and the actual STA course intraoperatively. STA trajectory was determined before surgery by using three imaging techniques: CTA (3 cases), three-dimensional (3D) contrast-enhanced T1-weighted MRA (4 cases), and/or 3D time-of-flight MRA (5 cases). In all cases, the neuronavigation-based STA position was confirmed with the Doppler vascular probe before skin incision and corresponded to the actual vessel position intraoperatively. As long as the skin is not mobilized during preoperative image acquisition and surgery, this neuronavigation-based approach is a valid method to identify STA course. During the preoperative planning process, the STA trajectory should be analyzed from its origin at the level of the foramen spinosum to avoid mistaking nearby venous structures as the STA.

[Competency evaluation of the company physician to improve prevention efficiency in the company]. / La valorizzazione delle competenze del Medico del Lavoro per migliorare I'efficacia della prevenzione in azienda.

The activity of the occupational physician (OP) in the enterprise has as its purpose the protection of the health of workers and consists of two main areas: health surveillance and risk assessment. Every day thle OP have to take into account both the results of clinical diagnostic investigation and the outcomes of the estimation of occupational exposure to risk factors by making a right mix between what is proven by scientific evidence and professional experience. He also knows that its effectiveness is closely linked to the contribution of other figures ofprevention in the company. In the scientific literature, the authors are unanimous in considering the effective actions to reduce exposure to risk, risk behaviors, injuries, occupational illnesses, disability, absenteeism. The use of indicators of effectiveness and / or appropriateness for the enhancement of its contribution, especially as a consultant in the field ofprevention and health protection in the compmany and then with anmt active role in corporate governance of prevention, are a must and no longer postponed, as well as the development of tools that document in formal terms, its contributioni (annual health report, the definition of risk profiles). I, this paper, by analyzing the results of specific experiences in the surveillance of construction workers, drivers and bakers, the authors want to emmphasize the contribution of the OP.

Toward more accurate preclinical glioblastoma modeling: Reverse translation of clinical standard of care in a glioblastoma mouse model.

Glioblastoma (GBM) is the deadliest of all brain cancers. GBM patients receive an intensive treatment schedule consisting of surgery, radiotherapy and chemotherapy, which only modestly extends patient survival. Therefore, preclinical studies are testing novel experimental treatments. In such preclinical studies, these treatments are administered as monotherapy in the majority of cases; conversely, in patients the new treatments are always combined with the standard of care. Most likely, this difference contributes to the failure of clinical trials despite the successes of the preclinical studies. In this methodological study, we show in detail how to implement the full clinical standard of care in preclinical GBM research. Systematically testing new treatments, including cellular immunotherapies, in combination with the clinical standard of care can result in a better translation of preclinical results to the clinic and ultimately increase patient survival.

YY1 mutations disrupt corticogenesis through a cell-type specific rewiring of cell-autonomous and non-cell-autonomous transcriptional programs.

Germline mutations of YY1 cause Gabriele-de Vries syndrome (GADEVS), a neurodevelopmental disorder featuring intellectual disability and a wide range of systemic manifestations. To dissect the cellular and molecular mechanisms underlying GADEVS, we combined large-scale imaging, single-cell multiomics and gene regulatory network reconstruction in 2D and 3D patient-derived physiopathologically relevant cell lineages. YY1 haploinsufficiency causes a pervasive alteration of cell type specific transcriptional networks, disrupting corticogenesis at the level of neural progenitors and terminally differentiated neurons, including cytoarchitectural defects reminiscent of GADEVS clinical features. Transcriptional alterations in neurons propagated to neighboring astrocytes through a major non-cell autonomous pro-inflammatory effect that grounds the rationale for modulatory interventions. Together, neurodevelopmental trajectories, synaptic formation and neuronal-astrocyte cross talk emerged as salient domains of YY1 dosage-dependent vulnerability. Mechanistically, cell-type resolved reconstruction of gene regulatory networks uncovered the regulatory interplay between YY1, NEUROG2 and ETV5 and its aberrant rewiring in GADEVS. Our findings underscore the reach of advanced in vitro models in capturing developmental antecedents of clinical features and exposing their underlying mechanisms to guide the search for targeted interventions.

[The importance of upper limb diseases in occupational medicine]. / La rilevanza delle patologie dell'arto superiore in ambito professionale.

BACKGROUND AND OBJECTIVES: In this work the authors analyse the results of the clinical evaluation of patients affected by suspected work related musculo-skeletal disorders (WMSDs), observed throughout 2008-2009 in the specific ambulatory of Occupational Medicine Division of Ospedali Riuaniti di Bergamo. The aim is to illustrate the epidemiological relevance of upper limb (UL) WMSDs. METHODS: We observed 430 patients (mean age 46,9 years, DS 9,3; mean working seniority 29 years, DS 10,4), investigating 600 disorders in diferent musculoskeletal segments. Most of the patients (66%) got to the division for a clinical consultation requested by general practitioners, 29,8% by occupational physicians, 4,2% by national insurance for occupational injuries and diseases (INAIL). RESULTS: Most of the patients (38,4%) were employed in construction industry. Among the 600 disorders investigated, 34,5% was at lumbar spine, 74,5% was at upper limb. The clinical diagnosis was already clear at the first consultation for 81,6% of subjects with low back pain and for 56,5% of patients with upper limb disorders; for the others was necessary to prescribe some instrumental exams or specialistic (neurologic, physiatric, orthopaedic) medical examination. We concluded for a diagnosis of WMSDs in 48,3% of the 600 cases: the percentage is 50,2% if we consider only disorders at lumbar spine and 52,5% among disorders at upper limb. The most frequent reason of refusing occupational aetiology, in the cases of low back pain, was the concomitant presence of other diseases at the segment; on the contrary, for the cases of upper limb disorders, was the lack of correlation between type of disease and professional exposure. DISCUSSION: All physicians demonstrate a high attention about upper limb disorders, topical subject of great epidemiological interest. General practitioners and occupational physicians have to take more advantage of diagnostic support and clinical evaluations offered by Occupational Medicine Divisions an Universities about WMSDs. In consideration of the dificulties to diagnose upper limb disorders and proving correlation with professional exposure is useful to promote specific courses for general practitioners and occupational physicians.

Preclinical studies performed in appropriate models could help identify optimal timing of combined chemotherapy and immunotherapy.

Immune checkpoint inhibitors (ICI) have been revolutionary in the field of cancer therapy. However, their success is limited to specific indications and cancer types. Recently, the combination treatment of ICI and chemotherapy has gained more attention to overcome this limitation. Unfortunately, many clinical trials testing these combinations have provided limited success. This can partly be attributed to an inadequate choice of preclinical models and the lack of scientific rationale to select the most effective immune-oncological combination. In this review, we have analyzed the existing preclinical evidence on this topic, which is only limitedly available. Furthermore, this preclinical data indicates that besides the selection of a specific drug and dose, also the sequence or order of the combination treatment influences the study outcome. Therefore, we conclude that the success of clinical combination trials could be enhanced by improving the preclinical set up, in order to identify the optimal treatment combination and schedule to enhance the anti-tumor immunity.

Radiation dose-escalation and dose-fractionation modulate the immune microenvironment, cancer stem cells and vasculature in experimental high-grade gliomas.

BACKGROUND: In the context of high-grade gliomas (HGGs), very little evidence is available concerning the optimal radiotherapy (RT) schedule to be used in radioimmunotherapy combinations. This studied was aimed at shedding new light in this field by analyzing the effects of RT dose escalation and dose fractionation on the tumor microenvironment of experimental HGGs. METHODS: Neurospheres (NS) CT-2A HGG-bearing C57BL/6 mice were treated with stereotactic RT. For dose-escalation experiments, mice received 2, 4 or 8 Gy as single administrations. For dose-fractionation experiments, mice received 4 Gy as a single fraction or multiple (1.33x3 Gy) fractions. The impact of the RT schedule on murine survival and tumor immunity was evaluated. Modifications of glioma stem cells (GSCs), tumor vasculature and tumor cell replication were also assessed. RESULTS: RT dose-escalation was associated with an improved immune profile, with higher CD8+ T cells and CD8+ T cells/regulatory T cells (Tregs) ratio (P=0.0003 and P=0.0022, respectively) and lower total tumor associated microglia/macrophages (TAMs), M2 TAMs and monocytic myeloid derived suppressor cells (mMDSCs) (P=0.0011, P=0.0024 and P<0.0001, respectively). The progressive increase of RT dosages prolonged survival (P<0.0001) and reduced tumor vasculature (P=0.069), tumor cell proliferation (P<0.0001) and the amount of GSCs (P=0.0132 or lower). Compared to the unfractionated regimen, RT dose-fractionation negatively affected tumor immunity by inducing higher total TAMs, M2 TAMs and mMDSCs (P=0.0051, P=0.0036 and P=0.0436, respectively). Fractionation also induced a shorter survival (P=0.0078), a higher amount of GSCs (P=0.0015 or lower) and a higher degree of tumor cell proliferation (P=0.0003). CONCLUSIONS: This study demonstrates that RT dosage and fractionation significantly influence survival, tumor immunity and GSCs in experimental HGGs. These findings should be taken into account when aiming at designing more synergistic and effective radio-immunotherapy combinations.

Reassessment of the NF1 variants of unknown significance found during the 20-year activity of a genetics diagnostic laboratory.

The finding of variants of uncertain significance (VUS) in the activity of a diagnostic genetic laboratory is a common issue, which is however provisional and needs to be periodically re-evaluated, due to the continuous advancements in our knowledge of the genetic diseases. Neurofibromatosis type 1, caused by the occurrence of heterozygous pathogenic NF1 variants, is a good model for studying the evolution of VUS, due to the widespread use of genetic testing for the disease, the constant enrichment of the international databases with NF1 variants and the full adult penetrance of the disease, which makes genotyping the parents a crucial step in the diagnostic workflow. The present study retrospectively reviewed and reinterpreted the genetic test results of NF1 in a diagnostic genetic laboratory in the period from January 1, 2000 to December 31, 2020. All the VUS were reinterpreted using the 2015 consensus standards and guidelines for the interpretation. Out of 589 NF1 genetic tests which were performed in the period, a total of 85 VUS were found and reinterpreted in 72 cases (84.7%): 21 (29.2%) were reclassified as benign/likely benign, whereas 51 (70.8%) were recoded as pathogenic/likely pathogenic with a significant trend distribution (Chi square test for trend p = 0.005). Synonymous VUS have mainly been reclassified as class 1 and 2 (7/8, 87.5%), whereas missense variants have been attributed to class 4 and 5 in 38 out of the 58 cases (65.5%). These findings underline an improvement in the classification of variants over time, suggesting that a reinterpretation of the genetic tests should be routinely performed to support the physicians in the clinical diagnosis of genetic diseases.