The mucopolysaccharidoses (MPS) are a heterogeneous group of in-born metabolic conditions caused by genetic defects that result in the absence or severe deficiency of one of the lysosomal hydrolases responsible for the degradation of glycosaminoglycans (GAGs). Such enzyme deficiency causes accumulation of GAGs that begins in infancy and progressively worsens, often affecting several organs including the central nervous system (CNS) inducing mental retardation, progressive neurodegeneration, and premature death. Over the last years, enormous progress has been made in the treatment of many MPS types, and available treatments are efficacious for many of them. Nevertheless, treatment of MPS with CNS involvement is limited mostly because of delivery impediments related to the presence of the blood–brain barrier (BBB). This chapter presents an overview of the BBB and of the different strategies that have been developed to overcome the problem of drug transport at the BBB, assuring efficient delivery of therapeutic agents to the brain. The mucopolysaccharidoses (MPS) are a heterogeneous group of hereditary metabolic disorders caused by genetic defects that result in the absence or severe deficiency of one of the specific lysosomal enzymes involved in the degradation of mucopolysaccharides, also known as glycosaminoglycans (GAGs). Such deficiency leads to an abnormal accumulation of GAGs in various organs and tissues including the arteries, skeleton, eyes, joints, ears, skin, liver, spleen, and/or teeth. Storage may also be found in the respiratory system, blood, and bone marrow. MPS mostly affect the paediatric population and unfortunately about 70% of all affected patients present a neurological involvement with storage also affecting the central nervous system (CNS), where GAG accumulation can seriously affect neurons leading to death through apoptosis or necrosis during the advanced stages of the disease . AlthougAlthough there is no curative treatment for MPS, today several therapeutic options for MPS exist and are commercially available including enzyme replacement therapy (ERT). ERT consists of replacing the defective enzyme thorough regular intravenous infusions of a working copy of the enzyme which is then scavenged by affected cells through the mannose-6-phosphate (M6P) pathway, then endocytosed and translocated into the lysosomes where they replace the defective enzyme and restore functional activity. However, improvement in the brain is limited by the presence of the blood–brain barrier (BBB), a selectively permeable barrier between the capillaries and the brain that prevents the efficient transfer of high molecular weight therapeutic drugs from the blood to the brain parenchyma and thus hinders effective treatment of MPS with CNS involvement. The greatest challenge therefore consists of developing therapies capable of achieving efficient delivery of the recombinant enzyme in the CNS across the BBB. It must be highlighted that not only patients with MPS but, in general, more than 70% of all patients affected by lysosomal storage disorders (LSDs) suffer from different grades of CNS involvement. Research efforts are therefore today particularly focused on the development of new strategic approaches for enhancing enzyme delivery across the BBB.The BBB is a semi-permeable membranous barrier, localized at the interface between the blood and the cerebral tissue, composed of a complex system of endothelial cells, astroglia, pericytes, and perivascular mast cells Fig. . It is mThis is achieved through: 1) prevention of the paracellular diffusion of hydrophilic compounds; 2) mediation of the active transport of nutrients to the brain; 3) activation of efflux transport of hydrophobic molecules and drugs from the brain to the blood; and 4) regulation of the transendothelial migration of circulating blood cells and pathogens.This unique dynamic cellular complex is characterized in the BBB by the fact that the endothelial cells fit tightly together giving origin to a continuous endothelium which is not fenestrated, and which exhibits a relatively low endocytic activity; this differs from the other parts of the body. The paracellular aqueous diffusional pathways between the cells is also further prevented by the presence of tight junctions (TJs) among adjacent endothelial cells. Thanks to their adhesive function, TJs effectively seal microvessels and preclude the passive diffusion of polar solutes and proteins in and out of the CNS. Thanks to such restrictive angioarchitecture, the BBB finely regulates and controls the internal brain compartment by constraining any molecular traffic to be primarily across the cell (transcellular), thus deeply restricting the passage of drugs and other undesired solutes .Being the tightest endothelium in the body, the BBB also represents the main impediment to drug delivery to the brain. For this reason, CNS drug development is devoted to both CNS drug discovery and CNS drug delivery. In particular, the understanding of molecular and physiological mechanisms involved in the transport of compounds through the BBB represents an important key for brain delivery.Generally, only lipid soluble (lipophilic) molecules with a low molecular weight (under 400–600 Da) and of positive charge can cross the BBB. Other molecules require certain cell endogenous transport systems, such as carrier-mediated transport, receptor-mediated transport, or absorptive-mediated transport. Commonly, there are five basic mechanisms by which solute molecules move across membranes.Firstly, simple (or passive) diffusion is a spontaneous process depending on the magnitude of the concentration gradient. Several lipid-soluble molecules can enter the cell membrane and diffuse passively across the endothelium into the brain. There is a correlation between increased lipid solubility and the rate and extent of penetration into the brain. Blood gases and several drugs, for example anaesthetics and heroin, enter the brain in this fashion .+ or H+ gradient) or by concentration gradients established by the solutes that are being transported. SLC are therefore classified as either facilitated transporters or secondary active transporters [Secondly, the solute carriers (SLC) constitute a superfamily of membrane transport proteins that facilitate the bi-directional movement of solutes across the cell membrane. Polar molecules may be transported across the endothelial cell membrane. Differing from their ATP-binding cassette (ABC) transporter counterparts, SLC transport does not require ATP since it is driven either by electrochemical gradients (i.e. Nasporters .l-DOPA) are also transported into the brain by these transporters [Typically, the entry into the brain of major nutrients such as glucose, amino acids, nucleosides, monocarboxylates, and organic anions and cations, and the efflux of several metabolites are mediated by SLC. Some drugs  represents another significant transport mechanism at the BBB. ABC transporters have a wide affinity for a wide category of solutes, especially large, lipid-soluble molecules with a number of nitrogen and oxygen atoms in their structure. These ABC transporters use ATP hydrolysis to pump molecules across the membrane and therefore they can force the efflux of solutes against a concentration gradient. P-glycoprotein (Pgp:ABCB1) and breast cancer-related protein (BCRP:ABCG2) are the principal ABC efflux transporters in the BBB. A number of cytotoxic drugs are substrates, which confounds the treatment of brain tumours and brain metastases .Fourthly, receptor-mediated transcytosis is a class of transport system that utilizes the vesicular transport system of the endothelial cells to transport substrates on the brain side of the barrier. Receptor-mediated transcytosis (RMT) is commonly induced by the binding of large molecules such as peptides and proteins to receptors  receptor its related protein, etc.) that are highly expressed on the endothelial cell membrane. Typically, nutrients such as iron, insulin, and leptin are transported into the brain by such an endocytic event also known as transcytosis , 9.Finally, there is diapedesis of mononuclear leukocytes. Leukocytes may penetrate the BBB by transendothelial diapedesis, migrating directly through the cytoplasm of the endothelial cells without tight junction disruption. Once in the brain they become microglia, the immune competent cells of the brain. It has been speculated that, during the brain inflammatory process, leucocytes can also move additionally through the tight junctions and infiltrate at a faster rate .The absence of mannose or mannose-6-phosphate receptor expression at the luminal cell membrane of the BBB, the absence of a BBB transport system for acid hydrolases, and the high molecular weight of the compounds render any paracellular or transcellular diffusion of systematic infused therapeutic enzymes across the BBB almost non-existent. This results in the incapacity of enzymes infused by ERT to cross the barrier and enter the CNS in any significant amount.It has been estimated that more than 90% of all small-molecule drugs and nearly 100% of all larger therapeutics are not able to overcome the BBB . Huge reThese strategic therapeutic approaches for bypassing the BBB can be broadly classified into one or more of the following categories: invasive techniques and non-invasive or miscellaneous techniques Fig. .This technique consists of the use of noxious agents, hyperosmotic solutions, or ultrasounds  to shrink the brain’s endothelial cells by breaking down tight junctions, allowing various molecules to pass into the cerebral tissue. Unfortunately, this technique has several limitations; it is in fact non-patient friendly, and can compromise the integrity and the physiological functions of the BBB leading to potential accumulation of unwanted blood components, neurotoxic, xenobiotics, and exogenous agents, thus causing injury to the CNS.These strategies consist of the injection or intraventricular infusion of therapeutic proteins directly into the cerebrospinal fluid (CSF).The advantages of these methods over systemic ERT are that they allow delivery to the brain of a higher amount of enzymes and, consequently, it is not necessary to use massive concentrations of therapeutic drugs. Furthermore, these strategies overcome the problems related to the short half-life of drugs in the blood, avoiding the ones related to systemic exposure and toxicity . IntrathData from animal models of MPS I, II, and IIIA, and also of other LSDs such as infantile neuronal ceroid lipofuscinosis and Niemann-Pick A, indicate that ERT through intrathecal injection is able to distribute the recombinant enzyme throughout the CNS where it can penetrate the brain tissue promoting the clearance of accumulated material within the lysosomes , 13. In Non-invasive techniques mainly consist of pharmacological strategies capable of modifying drugs to facilitate transport across the BBB. The main non-invasive techniques are discussed below.Since lipid solubility is a strategic factor in passive diffusion into the BBB, this technique consists of chemically transforming water-soluble molecules into lipid-soluble molecules capable of crossing the BBB. This is performed by adding lipid groups or functional groups to the polar ends of drug molecules .This technique consists of chemically modifying a small-molecule therapeutic drug to allow it to use the endogenous transport/carrier systems, mimicking the structure of the related specific endogenous molecules . The glucose transporter type 1 (GLUT1), the large neutral amino-acid transporter type 1 (LAT1), the cationic amino-acid transporter type 1 (CAT1), the monocarboxylic acid transporter type 1 (MCT1), and the equilibrative nucleoside transporter 1 (ENT1) are some of the most commonly endogenous carrier-mediated BBB transporters used as carrier systems employed for drug delivery .This technique consists of the pharmacological inhibition of selected efflux transporters that, being expressed by the cerebrovascular endothelium, prevent blood-to-brain drug uptake. Examples are P-glycoprotein, breast cancer resistance protein (BCRP) in humans, Bcrp in rodents, and multidrug resistance proteins , etc. .This method consists of using molecules such as endogenous ligands or monoclonal antibodies that, acting as molecular ‘Trojan horses’, bind exofacial epitopes on BBB receptor-mediated transport systems, triggering internalization of the receptor and of the attached drug. After internalization, the two components separate and take distinct paths, with the receptor travelling back to the membrane and the therapeutic drug freely diffusing into the brain parenchyma. This technique is being used to ferry drugs, proteins, and non-viral gene medicines across the BBB .This technique is being used for the transport across the BBB of those therapeutic compounds that are transportable only at a very low rate. This method consists of making a chimeric peptide by covalently coupling an otherwise non-transportable drug to a BBB-transportable peptide vector  by a disulphide bond. Such a chimeric peptide is then endocytosed by the capillary endothelial cells through receptor-mediated transcytosis and transported to the brain where, thanks to the presence of brain disulphide reductases, the pharmacologically active therapeutic peptide can be cleaved from the peptide vector. BBB peptide receptor systems include those for insulin, insulin-like growth factor, transferrin, and leptin. The brain contains the necessary disulphide reductases for rapid cleavage of the chimeric peptide .This technique consists of re-engineering the biologic drug as an IgG fusion protein, thus making brain penetration possible. The IgG domain, being a monoclonal antibody (mAb), binds an endogenous BBB receptor such as the transferrin receptor (TfR). The complex, referred to as TfRmAb, acts as a molecular Trojan horse and delivers the attached drug compound into the brain via receptor-mediated transport on the endogenous BBB TfR . The mAbThese methods consist of developing pro-drug compounds  that are therapeutically inactive agents capable of crossing the BBB and entering into the brain parenchyma. Once reaching the target site they undergo enzymatic and/or chemical transformations and modify their structure achieving a biologically active form able to exert the desired pharmacological effect .This approach is mainly based on the use of nanosized technology for drug release in the brain. This delivery system uses a wide variety of nanoscale drug delivery platforms mainly including lipid- and polymer-based nanoparticles (NPs) that assure a controlled and improved release of their cargo by protecting loaded drugs from being metabolized . The maiThis technique consists of transferring recombinant DNA with therapeutic function directly into the cells of specific organs. It represents a promising solution for those neurodegenerative conditions where the neuropathology is spread throughout the entire brain and therefore a global CNS gene delivery is required for an effective treatment . Two typThe technique consists of directly injecting viral gene transfer into the brain parenchyma or ventricular system. Studies in certain small (rodent) and large (canine and feline) animal models of LSDs have shown functional improvement and reduction in lysosomal storage . VariousThis approach constitutes a potential alternative method for therapeutic brain targeting and is based on the direct transport into the CSF enabled by the presence of the neural pathways connecting the nasal mucosa and the brain . Such a l-iduronidase (IDUA) encoding adeno-associated virus serotype 9 (AAV9) vector results in enzyme diffusion into deeper areas of the brain and related reduction of tissue GAG storage materials in the brain [Lipid-based NPs have been studied for intranasal drug delivery and this non-invasive strategy approach has been used to assess the efficacy of treatment of neurological manifestation in MPS I disease. Laboratory experiments in mice showed that intranasal administration of an α-he brain . Intranahe brain .Treatment of MPS is mostly challenging because delivery of therapeutic drug molecules to the brain is frequently obstructed by the presence of the BBB which constitutes the main obstacle for the treatment of those forms of MPS characterized by neurological involvement. It is clear that nowadays there is an urgent need to direct research efforts to overcome the BBB and to develop new therapeutic strategies capable of successfully targeting the drug to the brain compartment. Although there are some interesting outcomes at present for brain-targeted drug delivery by mean of clinical (invasive) or technological (non-invasive) approaches using innovative drug delivery systems for crossing the BBB, these are still not well defined. Further studies are needed to better understand the BBB transport systems, to assess brain drug pharmacokinetics, and to improve the delivery and distribution of the drugs into specific areas of the brain. Nevertheless, it seems quite reasonable to think that the BBB is no longer an impenetrable barrier. This raises significant hopes, not just for patients affected by MPS but for all those suffering from LSDs and other conditions characterized by brain pathology.


Introduction: Experiencing adverse life events and early disturbed patterns of interaction are crucial determinants for the development of Borderline Personality Disorder (BPD). Parental suicidal attempts can be considered a major adverse life event and a potentially traumatic experience. The aim of this study was to examine differences in parental suicidal attempts among a BPD adolescent population vs. a matched control group. We also aimed to understand if attachment styles and the number of parental suicidal attempts predicted the severity of borderline symptomatology.Methods: Our study (EURNET BPD) comprised 85 BPD adolescents and 85 matched controls. Axis II disorders were investigated using the French version of SIDP-IV. Parental suicidal behaviors were assessed during a face-to-face interview with the adolescent. Attachment style was assessed with the Relationship Questionnaire (RQ).Results: Parents of BPD adolescents made more suicidal attempts than controls . The linear regression showed that the best model explaining the severity of borderline symptomatology  was a model including the number of suicidal attempts realized by the parent  and the preoccupied attachment style of the adolescents toward their parents .Conclusion: Our results highlight the usefulness of assessing parental suicidal behavior when interviewing patients with BPD traits. This could be useful in clinical practice as an early clue to identify patients with potentially severe clinical profiles and parents needing a specific support. Further research is needed to confirm our findings. Borderline Personality Disorder (BPD) in adolescence is a severe mental disorder characterized by pervasive and persistent patterns of instability—affective, relational, of the self—and impulsivity. The condition is associated with frequent risk-taking and self-harm behaviors and many psychiatric comorbidities as well as severe psychosocial impairments, and extensive use of mental health services. Cumulative prevalence rates suggest that 1.4% of young people will meet diagnostic criteria for BPD by age 16 years, rising to 3.2% by age 22 years  were inpatients. The control sample included 85 healthy adolescents individually matched for gender, age and socio-economic status. Control subjects were excluded if they had a history of or ongoing psychiatric follow-up, and if they were positive for a DSM-IV diagnosis of personality disorder. The study was approved by our local institutional review board and a written informed consent was obtained from the adolescents and at least one of their parents.The study sample was drawn from a European research project investigating the phenomenology of BPD in adolescence [the European Research Network on Borderline Personality Disorder, EURNET BPD; see  for a fuAll subjects completed a research protocol  and a self-administered questionnaire to collect socio-demographic and psychopathological data. Axis II disorders were investigated using the French version of SIDP-IV, which is known to have good psychometric properties in adolescents and young adults . BorderlParental suicidal behaviors  were assessed during the face to face interview with the adolescent. The age of the adolescent at the time of the first suicidal attempt of the parent was also recorded.Attachment was assessed with the Relationship Questionnaire (RQ) , a validCategorical variables  were compared between groups (borderline and control sample) using chi-square analyses. To compare the mean scores of the borderline and the control sample on the number of parental suicidal attempts and on attachment styles we used a one-way anova. Bivariate associations between attachment styles and the overall severity of borderline symptomatology were examined with Pearson correlations in the borderline sample only. Finally, to test whether attachment styles and the number of parental suicidal attempts predicted the severity of borderline symptomatology we conducted a linear regression analysis with a stepwise procedure. All analyses were conducted using SPSS (25th version).N = 29) adolescents from the borderline group reported a suicidal attempt made by their parents compared to 11% (N=9) from the control sample. This difference was statistically significant   with some parents reporting up to 10 suicidal attempts. Mothers accounted for the majority of the suicidal attempts (73%). Borderline adolescents reported that the first suicidal attempt of their parents happened during their childhood and early adolescence .34%   was significantly correlated to the severity of borderline symptomatology, with fearful and preoccupied attachment styles showing a trend to a significant correlation  was a model including the number of suicidal attempts realized by the parent  and the preoccupied attachment style of the adolescents toward their parents   could differ in a BPD adolescent population vs. control. First, our study shows that parents of BPD adolescents  made more suicidal attempts than controls. These suicidal attempts happened several years earlier, when patients were still children, or in early adolescence.Second, the severity of borderline symptomatology seemed to correlate with the number of parental suicidal attempts  and preoccupied attachment style.Psychopathology is a frequent underlying cause of suicidal attempts. Severe depression, anxiety and substance abuse are commonly found among suicidal attempters . StudiesOur study is somewhat limited by the sampling method, size, and cross-sectional design. Indeed, our data comes from adolescents only, who report their perceived patterns of interaction. Yet number of parental suicidal attempts' report is a rather objective data and less sensible to bias.Our results highlight the usefulness of assessing parental suicidal behavior when interviewing patients with BPD traits. This pattern of behavior is overrepresented in this population and might correlate with psychopathological severity in the offspring. This could be useful in clinical practice as an early clue to identify patients with potentially severe clinical profiles and parents needing a specific support. Further research is needed to confirm our findings.° 0611259). Results were collected in an anonymous database according to the requirements of the French national committee for private freedoms. All participants, adolescents and parents, signed informed consent after receiving a full description of the study, explanation of its purpose, and information about the confidentiality of the data.This study was approved by the ethics committee of the Hôtel Dieu Hospital in Paris (authorization nAll the authors listed in the manuscript have contributed sufficiently to the project to be included as authors. AM participated in data analysis and interpretation and in writing the manuscript. MS, MC, and AP-S initiated and designed the protocol, collected data, participated in data analysis and interpretation and revising the manuscript. VD participated in data analysis and interpretation and revising the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Staphylococcus aureus (MRSA) is approximately double that of infections by susceptible pathogens, even in modern healthcare systems [Antimicrobial resistance (AMR) is a serious threat to global public health , but its systems . The delNational, regional and global action plans all point to surveillance as a crucial first step to control and prevent AMR. Surveillance data are needed to estimate the scope of the problem, guide empirical treatment, monitor trends, detect new resistance phenotypes and measure the effect of interventions. However, no single surveillance scheme can achieve all of these goals, and the optimal design of such programs has not yet been established. In 2015, the World Health Organization (WHO) presented a global action plan on AMR and launched the Global antimicrobial resistance surveillance system (GLASS) as a key component to strengthen the evidence base for further activities . During Eurosurveillance contains two papers on the establishment of the national Kor-GLASS surveillance programme in South Korea. The first paper describes the organisation, structure and costs of the programme [Neisseria gonorrhoea and Shigella spp., as these species were not readily available at the sentinel hospitals. The first year results demonstrated high rates of multidrug resistance in organisms from all specimen types. Among blood culture isolates, there was 54.3% MRSA among all Staphylococcus aureus, 29.0% vancomycin resistance in Enterococcus faecium, 34.7% cefotaxime resistance in Escherischia coli (mostly ESBL) and 27.0% cefotaxime resistance in Klebsiella pneumoniae. Resistance rates were higher in infections acquired in hospitals than in the community, and the highest rates were found in intensive care units. The prevalence of bloodstream infections (BSI) with major AMR pathogens among inpatients was calculated for comparison with foreign countries. The most prevalent mutidrug-resistant phenotypes were cefotaxime resistant E. coli , MRSA , imipenem resistant Acinetobacter baumannii  and cefotaxime resistant K. pneumoniae . The authors conclude that the results of Kor-GLASS helped plan national action in response to the high rates of drug resistance.This issue of rogramme , whereasrogramme . Kor-GLAAMR surveillance at the European level is presently organised through the European Antimicrobial Resistance Surveillance Network (EARS-Net)  and the EARS-Net and CAESAR are laboratory-based surveillance programs focused on the collection of routine antimicrobial susceptibility data. EARS-Net includes data generated in ca 1,080 clinical laboratories that serve more than 1,600 hospitals across the continent. No centralised microbiological analyses are performed, but the programme is supported by external quality assessment and tailored tools for data management [Eurosurveillance and in future publications will inform AMR containment activities at local and national levels. However, there are also limitations with the Korean model. The epidemiological validity of the data depends on the representativeness of sentinel hospitals as sampling sites within the South Korean healthcare system. The recruitment of relatively large facilities with access to adequate laboratory resources may skew the results in unpredictable ways. The collection of clinical data are described as ‘very burdensome’ for laboratory personnel, thus calling into question the sustainability of this part of the protocol. Finally, the considerable running costs may prove prohibitive, especially in low- and middle-income countries. Even with strong political and financial support, high running costs will necessarily limit the overall coverage of the system.The South Korean example holds important experiences and lessons for the future development of AMR surveillance in Europe and across the world. South Korea should be commended for its effort to establish a high-quality programme with a strong focus on harmonised and detailed microbiological analyses, as well as collection of relevant clinical data. The results presented in this issue of International AMR surveillance must build on national structures and systems that are adapted to specific features and needs of individual countries. The quest for comparability of data at regional and global levels must therefore be reconciled with the applicability of protocols and utility of data at the national level. In addition, AMR surveillance systems need to be designed according to the questions they are supposed to answer. Both EARS-Net/CAESAR and GLASS focus on the overall epidemiology of susceptible and resistant bacteria; however, they are not geared towards rapid alerts on unusual events, such as the discovery of new resistance phenotypes or emerging spread to new locations. Separate early warning systems have therefore been established at both the European Centre for Disease Prevention and Control (ECDC) and the WHO, but it is fair to say that these mechanisms are still in their early stages. Finally, there is a need to debate what the targets of surveillance programs should be and what should be answered by specific projects, using rigorous scientific methodology. It is tempting to suggest ongoing routine surveillance as the answer to any relevant question, but this is both scientifically flawed and financially unsustainable. The revolution in DNA sequencing technology has paved the way for detailed surveys of clone emergence, spread and decay, and in-depth analyses of patient determinants and outcomes can inform us in ways that routine surveillance can never do. ECDC has therefore supported targeted AMR projects outside the routine surveillance scheme ,9. The o


Acute facial nerve palsy secondary to neuroendocrine adenoma of the middle ear (NAME) is a rare disorder. There is only one case report in the literature describing similar findings. A 50-year-old man initially presented to ENT clinic with a right-sided middle ear mass and normal facial nerve function. Over the next six days, he developed House-Brackmann grade II facial paralysis. He underwent urgent surgical exploration of the tympanic cavity and excision of the middle ear mass via a post-auricular approach. Histopathological and immunohistochemical analysis revealed NAME. Three weeks after the surgery, facial nerve function returned to normal. No recurrence was found at a 3-year follow-up. Acute onset facial palsy induced by NAME is an extremely rare disorder. For a patient already affected by hearing impairment resulted from middle ear mass, facial weakness can have a significant additional detrimental impact on their wellbeing. The early complete excision of tumor is recommended not only as a curative treatment but also restoration of facial function. Neuroendocrine adenoma of the middle ear (NAME) is an uncommon benign neoplasm of the middle ear, first reported by Hyams et al. and Derlacki et al. in 1976 . These tA 50-year-old man presented to our Ear, Nose, and Throat (ENT) clinic with a three-month history of right otalgia, aural fullness, and hearing loss. He had no other significant past medical or surgical history. On otoscopic examination, a rounded red mass was observed completely filling the right external auditory canal and obscuring the view of the tympanic membrane. The manipulation of the tumor with a Jobson-Horne probe revealed it as a soft, non-tender, and non-friable mass. The results of tuning fork tests showed that Rinne’s test was negative on the right side, and Weber’s test lateralized to the right side indicating right conductive deafness. Facial nerve function was bilaterally normal. Rest of the ENT and systemic examination indicated insignificant results. Pure tone audiometry confirmed conductive deafness with an air-bone gap of 40 db in the right ear .Based on our clinical assessment, the differential diagnoses included the benign and neoplastic lesions of the external and middle ear. Regarding the extent of the lesion, the performance of a high-resolution computed tomography (CT) scan of the temporal bone indicated the opacification of the right middle ear cavity and the external auditory canal EAC, . MoreoveSix days after his initial clinic visit, the patient developed House-Brackmann grade II right-sided facial palsy with complete eye closure on minimal effort as well as mild forehead and oral asymmetry. After the admission of the patient, the intravenous steroid was administered for him. Since the symptoms of pain and acute onset facial palsy are typical symptoms of malignancy, we performed an urgent exploration of the right ear under anesthetic. Intra-operatively, there was a red fleshy mass arising from the middle ear through a large tympanic membrane perforation and filling the right EAC. The biopsy of the lesion was performed and sent for frozen section. The examined sections revealed solid nests of cells composed of small rounded nuclei with evenly dispersed chromatin, inconspicuous nucleoli, and eosinophilic cytoplasm, and there was no invasion of the mastoid cells. All these findings raised the possibility of a benign neoplasm of the middle ear. We subsequently proceeded the treatment procedure with an exploration of the tympanic cavity via a post-auricular approach. The lesion was encasing the malleus and incus; however, there was no obvious bony erosion. In order to achieve the complete excision of the tumor, the malleus and incus were removed after disarticulation at the incudostapedial joint. Furthermore, the tumor did not involve the inner ear and mastoid. After the middle ear mass was completely removed, we noticed edema and hyperemia of the facial nerve from small bony dehiscence in the fallopian canal. In this regard, type III tympanoplasty was performed with temporalis fascia graft placed on the stapes head.The microscopic examination of the tumor indicated round to cuboidal cells with fairly deformed nuclei and absent nucleoli . Immunohistochemistry was positive for cytokeratins  and synaptophysin . Based on these findings, the lesion was diagnosed as NAME. The patient was discharged by a course of oral steroids and antibiotics the day after the surgery. The discussions held about the case in the Multi-Disciplinary Team meeting and it was decided to perform workup to rule out the metastatic potential of the tumor, which was negative. At his 3-week post-operative appointment, his facial nerve function returned to normal (House-Brackmann grade I). There was no evidence of recurrence at a 3-year follow-up.The neuroendocrine neoplasms of the ear are rare neoplasms accounting for less than 2% of all primary ear tumors ,9. AmongAccording to the literature, the etiology of NAME is unclear. Middle ear mucosa is derived from the endoderm; however, skin cells with neuroendocrine features are absent in the middle ear cavity. There are two theories explaining the development of such tumors in the middle ear First theory states that an undifferentiated pluripotent endodermal stem cell may still be present in the middle ear mucosal surface. Second describes that the stroma of the middle ear derived from mesoderm and neural crest and the tumor may originate from neural crest-derived stem cell. Regardless of the theory, a metaplastic transformation would be needed within the resident cells of the middle ear for dual population established within these tumors .The neuroendocrine adenoma of the middle ear classified under middle ear glandular neoplasms. Currently, the most widely used classification for middle ear glandular tumors is based on the detection of immunohistochemical markers and presence of metastases proposed by Saliba et al. in 2009 . AccordiThe incidence of the tumor is equal in both male and female patients. The NAME usually occurs in adult patients with the reported age ranges of 20-80 years and mean age of 45 years . HoweverThe otoscopic examination of NAME usually shows retrotympanic mass. Perforation of the tympanic membrane, with the extension of mass into the external auditory canal (EAC) is a rare finding with only a few cases reported in the literature ,15. Our Immunohistochemistry plays an important role in differentiating various middle ear glandular neoplasms, including NAME. Therefore, their obtained results from different therapeutic procedures should always be interpreted along with conventional histopathologic findings.de novo or up-regulated expressions of specific tumor antigens. The advantages of this method outweigh the traditionally used special enzyme staining methods since they detect only a limited number of proteins, enzymes, and tissue structures (The immunohistochemistry technique involves the application of monoclonal as well as polyclonal antibodies to find out the tissue distribution of an antigen of interest with regard to ructures . In NAMEructures . Our casSurgical excision is the treatment of choice. The extent of the procedure is determined by the size and involvement of adjacent structures . SmallerFacial palsy secondary to benign middle ear tumors is rare, and the diagnosis of these tumors is a challenging issue given their asymptomatic features. In addition to facial schwannoma, other benign tumors of the middle ear that can cause facial weakness include glomus tumor, adenoma, and haemangioma -27. LikeAcute onset facial palsy due to NAME is extremely rare. For a patient already affected by hearing impairment resulted from middle ear mass, facial weakness can have a significant additional detrimental impact on their wellbeing. We recommend the early complete excision of the tumor, not only as a curative treatment but also restoration of facial function. In this regard, further research is required to have a better understanding of the pathology, management,and long-term outcome of NAME.


Frontiers Research Topic, some concepts of modern vaccinology are explored.The development and implementation of vaccines are one of the greatest Public Health achievements in human history . The search for a broadly protective vaccine capable of redirecting the immune responses from the variable immunodominant regions to conserved subdominant has identified potential candidates that can be delivered using novel vaccine platforms. Among the strategies, novel immunoinformatics tools to define consensus or ancestral sequences by phylogenetic analyses are discussed. Systems vaccinology approaches are needed to understand the mechanism of protections induced by UIVs.In light of the new technological advances in systems biology, Mujawar et al. report the development of protein interactomes for Acinetobacter baumannii, a causative agent of nosocomial infections, to identify potential vaccine candidates and virulence factors for immunological or pharmacological targeting. The identified proteins are then mapped onto the whole genome protein interactome for in silico verification to generate a short list of proteins for future in vivo validation.Characterizing the patterns of molecular interaction of proteins, using protein interaction networks or interactomes, is essential for understanding the cellular function and pathogen–host interactions. Contreras et al. present new data on tick vaccination, using a combination of the recombinant subolesin from Rhipicephalus microplus described as protective for this arthropod in combination with heat-inactivated Mycobacterium bovis and administered orally. The results of this study confirmed the efficacy of subolesin-based vaccines for the control of cattle tick infestations and expanded to oral vaccination using an immunostimulant. Ebensen et al. report the use of a combination of c-di-AMP, a STING agonist, and a promising adjuvant capable of stimulating an effective Th1/Th2 and cytotoxic immune response, with the well-known adjuvant alum. This adjuvant system was tested with a model antigen showing the induction of a balanced humoral and cellular immune responses.The development of vaccines based on subunits is a promising strategy given its excellent safety profile. However, candidates are usually poorly immunogenic and require the formulation with potent adjuvants. In their article, Zurita et al. address the need to develop more effective acellular pertussis vaccines. In eliciting tissue-resident memory CD4+ T cells, a critical effector subset involved in protection, the authors show that an outer membrane vesicle (OMV)-based vaccine is more effective than a commercial acellular vaccine, having a better ability to induce protection and immunological memory. Chimeric virus-like particles (VLPs) using the murine polyomavirus are reported to be an effective platform to deliver subunit vaccines by Pattinson et al. In proof-of-concept studies, the authors produced chimeric VLPs genetically modified for surface expression of CD8+, CD4+, or B cell epitopes derived from the Plasmodium yoelii circumsporozoite protein. The vaccine platform was efficient to induce CD8+ T cell and antibody responses, but limited CD4+ T cell responses. The potential of using Porcine Circovirus Type 2 (PCV2) Chimeric VLPs for surface expression of exogenous peptides is presented by Wang et al.. The availability of three-dimensional structural data of the PCV2 capsid protein allows the authors to use homology modeling to characterize surface displaying. The platform allows the insertion of foreign peptides without altering the virus assembly and its entrance to the host cell.In the search for novel vaccine delivery systems, several strategies have been proposed. Mekonnen et al., the limitations and strategies for using DNA vaccines against human immunodeficiency virus (HIV)-1 and hepatitis C virus (HCV) in humans are discussed. The authors review the potential of DNA vectors for elicit protective compartmentalized CD8 + T cells in the liver for HCV and the genito-rectal mucosa for HIV.DNA vaccines consist of plasmid vectors that, after immunization, allows intracellular expression of the encoded antigens. Protective efficacy is achieved by the induction of a strong humoral and cellular immune response dependent on B and T cells. In a mini review by McCaffery et al. reported the use of genetic conjugation to deliver a Plasmodium vivax sexual-stage vaccine candidate. The authors take advantage of a highly immunogenic chimeric protein that they designed targeting a blood-stage antigen to create a bifunctional vaccine by genetic linkage to the transmission-blocking vaccine candidate Pvs25. This approach addresses the need for the development of effective multi-stage malaria vaccines.Conjugation of poorly immunogenic antigens to carrier protein is the strategy used for glycoconjugate vaccines. The wealth of information provided by systems biology approaches can be integrated into product design for the development of novel vaccines. This Research Topic offered a glimpse into some strategies in modern vaccinology. Although these tools are essential for the development of effective vaccines against agents with complex host–pathogen interactions such as HIV, tuberculosis, and malaria, from the global health perspective, it is also critical to identify and deal with factors associated with persistent social disparities. It has been estimated that only 5% of all children born worldwide receive all 11 vaccines recommended by the WHO (Mantovani and Santoni, All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this article we explore filopodia of endothelial cells (ECs) in the developing central nervous system (CNS) using the Golgi method and transmission electron microscopy. Filopodia of ECs play a crucial role in the anastomosis of growing capillaries of the CNS. The leading ECs filopodia from approaching capillaries interconnect forming complex conglomerates that precede the anastomotic event. The contacting filopodia form narrow spaces between them filled with proteinaceous basal lamina material. The original narrow spaces coalesce into larger ones leading to the formation of a single one that will interconnect (anastomose) the two approaching capillaries. The four leading ECs (two for each approaching capillary) become the wall of the newly formed post-anastomotic CNS capillaries. These new CNS capillaries are very small with narrow and irregular lumina that might permit the passage of fluid but not yet of blood cells. Eventually, their lumen enlarges and permits the passage of blood cells. Anastomoses of blood capillaries occur during the development of any organ or tissue, playing significant roles in the healing of traumatic and surgical wounds and in bone fractures. Despite their clinical and biological importance and widespread occurrence, some aspects in the anastomosis of blood capillaries remain inadequately studied. An important aspect in the development of blood capillaries is the role of the filopodia decorating the leading endothelial cells (ECs) of the growing capillaries. The latter explore the surroundings, determine directional growth, search for filopodia of other growing capillaries and establish complex (conglomerate) interactions leading to their anastomosis. Searching filopodia are a universal property of moving cells  has explored several neuronal and microvascular aspects of the developing human cerebral cortex, using the classic Golgi method and the electron microscope. These studies include: (a) equidistant perforations (of ca. 500 μm) of the cortical glial external limiting membrane by meningeal  capillaries, delineating independent vascular functional territories  during development and the role of the ECs filopodia in the process, using the Golgi method and the electron microscope. The cortical gray matter was selected for its rich capillarogenesis that accompanies functional maturation of its neurons  capillary approach and recognition; (b) complex filopodia interconnections (conglomerates) of the approaching capillaries with creation of narrow extracellular spaces (fissures) between them, filled with secreted basal lamina material; and (c) progressive confluence of fissures into larger ones leading to the establishment of a single cavity that will eventually become luminal and thus interconnect (anastomose) the capillaries; and, the new post-anastomotic CNS capillaries. These events are accompanied by ECs mitoses and displacements. Four extra ECs, two from each approaching capillary, move towards the capillary head and will be major contributors in the anastomotic process, being the only ones with searching filopodia . The menIt must be understood that the Golgi method is not a method in the strictest sense of the word, but a reaction. Golgi  named hiThe Golgi reaction uses thick tissue preparations (150–170 μm) permitting three-dimensional visualization of neurons, neuroglia, axon terminals and growing capillaries as well as their morphological interrelationships. Despite technical difficulties, the Golgi reaction still is undoubtedly the best procedure to study the structure of the CNS, abundantly demonstrated by Cajal’ extensive work . For the TEM studies, one of us (LH), made new recut from old tissue blocks used in a previous study of the mouse olfactory nerve  composition of the polypoid head of growing CNS capillaries nature and; (b) nature and composition of filopodial interactions (conglomerates) between approaching capillaries; and (c) the structure of new post-anastomotic. These vascular features had not yet been investigated appropriately.The bulging (polypoid) head of growing CNS capillaries is composed of two types of ECs. Some ECs are standard components of the capillary wall .The leading filopodia of approaching capillaries establish complex interactions (conglomerates) among themselves. It is difficult to recognize these conglomerates as vascular structures. The presence of tight junctions (recognized only in high power views) among the contacting filopodia identifies them as vascular structures. Also, the presence of tight junctions among the filopodia suggests a different ECs origin, as will be those from the two approaching capillaries. Some of the filopodia conglomerates can even enclose the nucleus of one of the participant ECs. Probably, the progressive accumulation of basal lamina among the filopodia might contribute to the enlargement of the spaces between them and eventually their transformation into luminal spaces. The conglomerates have some additional peripheral filopodia that do not participate in the process.The new post-anastomotic CNS capillaries are very small, ranging from 2.5 to 3.5 μm in diameter. Some may still have more than one lumen reflecting their multiple origins , bottom.in vivo techniques will be needed to extend the findings summarized in this article.In summary, filopodia of ECs seem to play a major role in the formation of CNS capillaries during development. These structures can be visualized in 3D with the Golgi method and at higher resolution with the electron microscope. Future studies with The animal studies were reviewed and approved by the Institution Ethics Committee when the original hamsters and mouse studies were done.MM-P processed the tissue for the Golgi method, analyzed the tissue, and wrote the article. LH processed the EM preparations and sections, and took the transmission electron microscopic microphotographs.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the developing organism, complex molecular programs orchestrate the generation of cells in adequate numbers, drive them to migrate along the correct pathways towards appropriate territories, eliminate superfluous cells, and induce terminal differentiation of survivors into the appropriate cell-types. Despite strict controls constraining developmental processes, malignancies can emerge in still immature organisms. This is the case of neuroblastoma (NB), a highly heterogeneous disease, predominantly affecting children before the age of 5 years. Highly metastatic forms represent half of the cases and are diagnosed when disseminated foci are detectable. NB arise from a transient population of embryonic cells, the neural crest (NC), and especially NC committed to the establishment of the sympatho-adrenal tissues. The NC is generated at the dorsal edge of the neural tube (NT) of the vertebrate embryo, under the action of NC specifier gene programs. NC cells (NCCs) undergo an epithelial to mesenchymal transition, and engage on a remarkable journey in the developing embryo, contributing to a plethora of cell-types and tissues. Various NCC sub-populations and derived lineages adopt specific migratory behaviors, moving individually as well as collectively, exploiting the different embryonic substrates they encounter along their path. Here we discuss how the specific features of NCC in development are re-iterated during NB metastatic behaviors. Neuroblastoma (NB) is a devastating pediatric malignancy that accounts for 15% of cancer-related deaths in childhood Maris, . NB has While the scientific and medical communities have made intense efforts to better understand this complex set of malignancies and to propose new therapeutic strategies, the long-term overall survival rates remain under 50% and fall below 10% in the case of relapse Maris, . The latAnatomical localization of primary NBs, markers typically expressed by NB cells, and data obtained from mouse genetic models of the disease provided evidence that NB is a malignancy of the sympathetic nervous system, deriving from the sympatho-adrenal neural crest (NC) trunk sub-lineage , all along the rostro-caudal axis  and Schwann cells , that line the ventral roots of the spinal cord, chromaffin cells of the AM and melanocytes. The potential of trunk NCCs to differentiate into specific derivatives is not only dependent on their initial genetic multipotency but is also shaped by the successive microenvironments they interact with during their migration, by specific cues emanating from them, and by the physical structures they encounter upon migration , migration towards target tissues and finally differentiation into specific derivatives  guidance information is provided by the successively crossed microenvironments; and (2) autonomous control occurs by the NCC population itself . via a distinct mechanism. Draxin was proposed to inhibit the migration of trunk NCCs towards the dorsal path by blocking the cytoskeletal rearrangements underlying their polarization  expression, but also on NB cell growth and angiogenesis suggesting that it has a general effect on the tumorigenic process  heterozygous mutant mice show myelination defects, consistent with a loss of myelinating Schwann cells  that mostly reside in the BM. Recent research has provided an integrated and complex picture of the BM microenvironment, also called “stem cell niche,” and of its multiple and dynamic contributions to the regulation of HSC differentiation and functions , along blood vessels within the BM and also in the fetal liver. These observations strongly suggested that NCCs could themselves migrate to the BM by reaching the aorta and the blood vessels connected to the niche.Trunk NCCs pursuing a ventral pathway stop their migration in close contact with the DA endothelium. A series of studies have concluded that some of the HSCs residing in the BM, the lineage founders of the adult hematopoietic system, originate from the hemogenic ventral endothelium of the DA  of origin are not mutually exclusive and share major common points that reside in the extreme multipotency of these embryonic lineages and in the adaptive migratory mechanisms that they adopt over their developmental history. These particular properties echo the The cellular and molecular mechanisms underlying these changes are not known yet, but recent data exploring the basis of NB heterogeneity shed light on potential regulations that could sustain such adaptive modifications of NB cells in the successive environments they meet. Indeed, the existence of an intra-tumor heterogeneity in NB has been clearly documented and fits with long-term clinical data reporting the existence of different types of NB cell lines distinguished by their morphology and molecular markers. NB cell lines were historically classified as “N type” for neuroblastic; “S type,” for non-neuronal Schwann cell-like; or “I type” for morphologically Intermediate  NB is a highly particular and enigmatic form of disseminated NB that occurs only in neonates, usually before 1 year of age. Tumor foci can be numerous and are typically detected in the liver, under the skin, and/or in the BM but without skeletal involvement. Masses considered to be primary tumors are located in sympatho-adrenal derivatives  the very early occurrence of stage 4S NB in infants; (2) the anatomical location of tumor foci; and (3) the clinical observation that sympatho-adrenal tumors are similar in size to non-sympathetic tumor foci.in vitro and in vivo experimental models. Indeed, obtaining cell lines from these clinical samples has failed for many years. Attempts to create genetically-engineered or xenograft-based animal models have also been unsuccessful or insufficiently documented. These failures can be explained by the vulnerability of low stages NB, once removed from their original microenvironment, and by the fact that causal tumorigenic mechanisms including their precise cellular origin remain uncharacterized.Difficulties to conduct functional studies on low grade NBs mainly reside in the absence of via the typical metastatic scheme, undergoing secondary dissemination to these sites.The avian NB model that we recently set up could bring new insights both for NB stage 4S and localized cases. We have modeled the formation of localized NBs by performing xenografts of stage 1–2 NB patient tumor biopsies. As stage 4 NB, NB cells from localized tumors follow an NCC-like ventral pathway leading to the formation of dense, proliferating sympatho-adrenal tumors  makes a crucial link between the extreme heterogeneity of the pathology and the complex cellular and molecular mechanisms that drive NC-related NB cell(s) of origin towards their target tissue . Our incCD-B and VC wrote the manuscript and designed the figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Currently, clinicians rely mostly on population-level treatment effects from RCTs, usually considering the treatment's benefits. This study proposes a process, focused on practical usability, for translating RCT data into personalized treatment recommendations that weighs benefits against harms and integrates subjective perceptions of relative severity. Intensive blood pressure treatment (IBPT) was selected as the test case to demonstrate the suggested process, which was divided into three phases: (1) Prediction models were developed using the Systolic Blood-Pressure Intervention Trial (SPRINT) data for benefits and adverse events of IBPT. The models were externally validated using retrospective Clalit Health Services (CHS) data; (2) Predicted risk reductions and increases from these models were used to create a yes/no IBPT recommendation by calculating a severity-weighted benefit-to-harm ratio; (3) Analysis outputs were summarized in a decision support tool. Based on the individual benefit-to-harm ratios, 62 and 84% of the SPRINT and CHS populations, respectively, would theoretically be recommended IBPT. The original SPRINT trial results of significant decrease in cardiovascular outcomes following IBPT persisted only in the group that received a “yes-treatment” recommendation by the suggested process, while the rate of serious adverse events was slightly higher in the "no-treatment" recommendation group. This process can be used to translate RCT data into individualized recommendations by identifying patients for whom the treatment’s benefits outweigh the harms, while considering subjective views of perceived severity of the different outcomes. The proposed approach emphasizes clinical practicality by mimicking physicians’ clinical decision-making process and integrating all recommendation outputs into a usable decision support tool. While evidence-based medicine is well established with the integration of clinical trial research into medical knowledge and guidelines, there is increasing acknowledgement of the shortcomings of these data for individualized treatment decisions.10 or a composite of several outcomes,12 which are often not reflective of the multiple separate effects of many interventions. Many risk prediction models do not account for the trade-offs between potential benefits and harms (adverse events),10 and the few that do take harms into account have used a single negative treatment consequence or an overall estimation of negative effects.14 Furthermore, patient engagement and preferences are largely not actively incorporated into this weighing of benefits and harms.15Risk models have been developed to calculate patients’ individual risks in a variety of medical fields, but most models are focused on a specific outcome16 The presentation of clinically useful outputs from analytically rigorous prediction models is critical for both physician appraisal and in the ability to engage in conversation with patients.18 The potential exists to enhance physician-patient treatment dialog through clinical decision support tools,7 and is something that is desired by physicians.19The uptake of decision support systems and risk calculators in clinical practice is also limited since not enough focus is placed on presenting them in a transparent and clinically-usable format that physicians can use and trust.In this study, we present an approach focused on practical usability that translates clinical trial results into individualized treatment recommendations by considering both personalized benefits and harms, and integrating them with subjective perceptions of relative severity. We also aimed to design a clinically-relevant decision support tool concept that presents individualized risk estimates, which includes a built-in ability to enter patient or physician preferences, and presents the final treatment recommendation. The suggested process is demonstrated using data from a clinical trial that examined intensive systolic blood pressure treatment.The SPRINT population included 9360 individuals and the CHS population consisted of 88,374 individuals. A total of 1.2% of the SPRINT participants and 1.4% of the CHS members had one or more missing predictor values that were imputed. The baseline characteristics of the SPRINT and CHS populations are detailed in Table The models included an average of 6.8 predictor variables. Further details on the variables included in each prediction model and their coefficients are in the supplemental material  and consisted of a higher percent of males  than the non-intensive treatment recommendation group   Table . The int1) Table .Table 2EThe tool’s interface is presented in Fig. This study presents an approach and clinical decision support tool concept that helps to address the challenge of determining individual treatment recommendations from trial evidence. Whereas previous studies have dealt separately with various aspects of this challenge, the current study developed a comprehensive practice-focused analytic process that formalized and quantified what physicians do intuitively in treatment decision-making—consider potential benefits, harms, and subjective preferences. In addition, this study suggests a way to reflect these outputs transparently via a clinical decision support tool.20 because many clinical trials, including the SPRINT study, conclude that treatment is significantly better than the alternative for the average trial patient; but this does not account for benefit-harm trade-offs for individual patients.Increased attention has shifted the focus of treatment decision-making toward individualizing decisions,We demonstrate that in the SPRINT test case, the recommendation for intensive treatment may not be beneficial for all trial participants, but potentially only for 62% of them. It seems that the proposed process can distinguish those patients for whom the benefits of intensive blood pressure treatment outweighed potential harms from those for whom the harms outweighed the benefit . Furthermore, the individuals who would be recommended intensive treatment based on the proposed decision support tool would typically benefit more and suffer fewer negative consequences than the overall average reported in the SPRINT results.21 However, previous studies developing individualized prediction models for cardiovascular events have predominantly used a single or composite outcome or relied on population-level estimates to evaluate harms while estimating individualized benefits.21 Most of these studies conclude with a formalized prediction model as the final output, and almost none of them address the way in which clinicians will understand and use the analytic outputs in practice. There are decision support tools to demonstrate the effects of cardiovascular risk factors, which have created attractive interfaces to illustrate both the baseline risk and treatment effects, but they only focus on single or composite outcomes, and do not consider the adverse events.24 One of the stated reasons for this is that the weight of adverse events may be subjective.10 Our proposed interface has overcome this challenge by formalizing the combination of objective measures of risk with subjective views of severity.Similar to the current study, prior research has accounted for benefit harm trade-offs of intensive blood pressure treatment.25 the outcome specific severity rankings in our approach may be more intuitive and easily communicated to patients. We found that physicians had difficulty in assigning a severity score to a composite of different outcomes, while separating the outcomes allowed for consideration of each outcome separately. The development of models both for multiple individual positive and negative consequences helped to overcome the limitation of the net benefit method that can only account for a single positive and a single negative outcome. This approach allows for the integration of subjective perception of risk into the recommendation even when the composite of either the positive or negative outcomes include events of very different nature. The multiple risks could then be weighed on both sides of the scale, thereby allowing physicians to reach a final yes/no treatment decision that is aligned with “primum non nocere”.Our decision to develop risk prediction models for eight separate outcomes that could be ranked for severity (rather than composite outcomes) was guided by the need to identify building blocks of patient-specific information that can be easily assimilated by clinicians and explained to patients. In contrast to using a net benefit method, in which the physician needs to determine an exchange rate between the positive and negative outcomes,12 There were some discrepancies in the calibration results within the CHS population, with some overestimation of risk and some underestimation of risk, potentially due to an inability to identify all events within the CHS EHR data. The similar discriminatory performance across the two study populations suggests that these models are transferrable between populations, albeit, with the need to recalibrate when applied to new populations.26 The proposed process and tool in the current study yielded a recommendation for intensive blood pressure lowering in 62% of the SPRINT population, compared to 84% in the CHS population. This difference was expected since the first population represented individuals selected for a clinical trial, while the second represents a more general population.The discrimination ability of the models in the external validation CHS population was comparable to that of the models developed and trained on the clinical trial population, as well as similar to the performance of the individual risk models developed in previous research.27 The sensitivity analysis conducted to examine the impact of varying sets of physicians’ severity weights, showed that physicians with different views can affect the final recommendation result. Yet, for 50% of the patient population, there was consensus regarding the recommendation despite the variance in the severity weightings. For the rest of the patients, whose benefit-harm ratios were closer to one and less inclined to one direction, individual preferences (physician or patient) may be a more prominent factor in the final treatment decision. This illustrates that the tool offers support to decision-making rather than replacing human clinical judgment, through balancing the integration of empirically derived individualized evidence with built-in flexibility for shared decision making in clinical practice.When translating across populations and contexts, there are also considerable variations in perspectives of the importance of potential benefits and risks.17 Accordingly, the tool’s interface has been designed to integrate both graphical illustrations and numerical representation of the individualized baseline risks and estimated treatment effects, along with a final treatment recommendation and its strength . Two different measures of risk were included because they have been found to be understandable and interpretable for patients.28 Physicians can, therefore, determine whether specific outcomes or adverse event risks, or the final recommendation (using the suggested severity ranks or adapting them) are most informative for the treatment decision. Beyond just providing a risk score or a yes/no treatment recommendation, it is beneficial to include outputs such as the strength of the benefit-harm ratio, which is particularly important among patients for whom the treatment decision does not clearly incline to one direction .A valid methodology for reaching an individualized, evidence-based treatment decision, however, is not enough to assure implementation in clinical practice. The transformation of theory to practice depends on the usability and alignment of analytic outputs with clinicians’ perceptions and preferences.This study had notable limitations to consider. First, the timeline of the SPRINT trial allowed an evaluation of the absolute risks for a relatively short time period of three years. A longer follow-up period of five or ten years would be preferable for the iARR/iARI results, but likely would not have changed the final treatment recommendation . Second, the limited sample size of the SPRINT trial posed a challenge in stabilizing the coefficients of the prediction models thus needing to train the model using multiple bootstraps.29 As in other studies, the individualized estimates in the current study are limited to approximations of conditional average effects for patients similar to the individual patient. Related to data measurement limitations, in the translation of clinical trial data to individualized EHR-based risk for an external population, there can be discrepancies between the variable measurements used in the trials and the variable measurements found in an existing EHR database. The potential for these differences in measurements emphasizes the importance of comparing the performance of the developed models in the clinical trial and the external populations, which in the current study yielded relatively consistent performance. Furthermore, although we externally validated the prediction models, we were not able to validate the SPRINT results or the final treatment recommendation in the CHS population because the real-world context does not confer randomization to treatment. Nonetheless, since the risk models were externally validated, and their outputs translate directly to the treatment recommendation, the recommendation is likely to be valid in the CHS population. Finally, we have not yet evaluated the tool in a clinical setting, but we have demonstrated feasibility and the potential benefits.Importantly, truly individualized treatment effects are not ascertainable due to the lack of the ability to determine the counterfactual and to observe all relevant factors.Following an approach such as the one proposed in this study would allow for the translation of clinical trial data into an individualized clinical decision support tool that could potentially be replicated for many types of individualized dichotomous clinical decisions . EHR systems provide a suitable setting for the implementation of this kind of tool, presenting physicians with an instant summary of the relevant clinical trials tailored to the characteristics of their patients. The tool can be automatically presented in the EHR only for patients who meet the inclusion criteria of the relevant trial. Rather than being an added burden to the patient visit, such a tool has the potential to facilitate the physician’s treatment decision-making and patient engagement by computing what is missing in practice: estimated individualized benefits and harms based on clinical trial evidence, that can be integrated with subjective perception of severity and combined into a single recommendation.30 randomized clinical trial, whose methods and results have been previously described.32 This trial compared intensive management of systolic blood pressure targeting <120 mmHg with standard management targeting < 140 mmHg in patients with hypertension and high cardiovascular risk and no diabetes. The trial was stopped early because the average rate of cardiovascular events and all-cause mortality were significantly lower with intensive treatment.In order to demonstrate the suggested process of translating clinical trial results into individualized treatment recommendations, we used data that was made available from the Systolic Blood Pressure Intervention Trial (SPRINT)Electronic health record (EHR) data from Clalit Health Services (CHS) members were used for external validation of analytic outputs which were developed using the SPRINT population as part of this study. CHS is an integrated payer/provider health care organization that provides primary, specialty, and inpatient healthcare to over half of the population in Israel (close to 4.5 million people), where there is universal healthcare coverage. All relevant patient data were extracted from the central data warehouse collating this information from inpatient, outpatient, laboratory, pharmacy, and other clinical settings in CHS.This study was conducted, in part, using SPRINT-POP Research Materials obtained from the National Heart Lung and Blood Institute (NHLBI) Biologic Specimen and Data Repository Information Coordinating Centre, and does not necessarily reflect the opinions or views of the SPRINT-POP or the NHLBI. The study received approval from the Institutional Review Board Committee (IRBC) of Meir Hospital of CHS. Participants from the SPRINT gave written informed consent as part of the clinical trial. For the retrospective analysis using CHS data, the IRBC approval included an exemption regarding obtaining written informed consent.The study was divided into three phases: (1) develop models of individualized risk estimations for multiple positive and negative outcomes; (2) translate these predictions into a single treatment recommendation; and (3) integrate them into a clinically-relevant tool concept to support practicing physicians' decision making. In the first phase, eight prediction models of benefits and harms related to intensive blood pressure lowering were developed based on SPRINT data, which was collected between 2010 and 2015 (median follow-up period of 3.26 years) with baseline characteristics defined at the study entry date for each participant. These models were externally validated for their ability to predict outcomes in a 3-year period (between 2013 and 2015) in the CHS population, with baseline characteristics as of the index date, defined as January 1, 2013. The risk estimates from the eight models were weighted and consolidated into an intensive vs. non-intensive treatment recommendation, which was tested against the SPRINT randomization. Finally, a clinical decision support tool was designed to incorporate the analytic outputs while also accounting for the practicing physician’s needs during a patient visit.32 For the prediction models’ external validation, CHS members who met the same inclusion and exclusion criteria as in the SPRINT study were included. The CHS cohort of patients had a diagnosis of hypertension and two elevated blood pressure measurements as of the index date. The SPRINT's inclusion and exclusion criteria that could be assessed from the EHR data were applied  that are commonly considered to be associated with high blood pressure: myocardial infarction, stroke, acute decompensated heart failure, and cardiovascular death. Additionally, four prediction models were developed for adverse events (treatment's harms) that were reported by the SPRINT study to occur more with intensive blood pressure treatment: hypotension, syncope, electrolyte abnormality, and acute kidney injury. For both outcomes and adverse events, only non-composite outcomes were selected.6 Missing predictor values were imputed using multiple imputation functions by Van Buuren et al.33 to create 5 imputed data sets.34 Further details regarding aspects of the multiple imputation and variable selection methods are described in the supplement  on the entire SPRINT population, drawn randomly with replacement.38 and second using a weighted Cox by the Fine-Gray method for competing risks in order to receive a valid estimation of the baseline risk (considering death from any cause as the competing event for each outcome).39 In addition, to assure lack of overfitting, we calculated a set of out-of-sample models for each SPRINT participant (for both the cause-specific and competing risk models), using the average coefficients of all bootstraps in which he or she did not participate (about one third of the bootstraps).40Each model training in each bootstrap was evaluated twice: first using a simple Cox regression (cause-specific) in order to obtain coefficients that represent the true nature of the treatment effect on each outcome;26 The AUCs and their variances were calculated using 1,250 bootstraps (with 250 from each imputed data set). To create complete independence between stages, these were newly created bootstraps, not those used for the model fitting stage. Calibration was assessed by comparing average predicted risks with observed percentages of events, stratified by deciles of risk.26 This analysis was done separately in each imputed data set and then averaged.The models' performance in predicting the baseline risk within the SPRINT population was evaluated for discrimination by examining area under the receiver-operator-characteristic curves (AUC) and for calibration. The performance was evaluated using the competing risk coefficients of the final models and out-of-sample models.41 of seven of the prediction models in predicting the baseline risk were conducted in the CHS population . Details on variable definitions from the CHS EHR data are described in Supplementary Table External validation for the performance4 This process was repeated for each adverse event by subtracting the off-treatment risk from the on-treatment risk, thus creating four individual-absolute-risk-increase (iARI) measures.The translation of the risk estimates to a treatment recommendation was intended to mimic the clinical decision-making process undertaken by physicians, considering both the potential magnitude of the estimated benefits and harms, as well as their relative weight in terms of severity. The magnitude of predicted benefits of intensive blood pressure lowering was assessed by entering each patient’s individual characteristics into the cause-specific cardiovascular outcome models and calculating three-year individual absolute-risk-reduction (iARR) measures. On-treatment risk (intensive treatment variable set to yes) was subtracted from off-treatment risk for each specified outcome (intensive treatment variable set to no).31 The severity weights by each physician were averaged to create a final severity weight (SW) for each outcome.The severity of each cardiovascular outcome and serious adverse event was considered on a scale ranging from 0–10, through a blinded independent evaluation by 12 physicians with various relevant clinical backgrounds: general physicians, family physicians, internal medicine experts, cardiologists, nephrologists, and public health experts (two of each). The physicians were asked to assign a severity weight to each of the eight model outcomes and read the SPRINT definition of serious adverse events prior to their evaluation.j∙iARRj)/(∑SWk∙iARIk). A ratio >1 indicated that the benefits outweighed the harms, thus favoring intensive treatment.To determine the treatment recommendation for the individual patient, a benefit-harm ratio was calculated by dividing the sum of weighted benefits (positive effects' iARR) by the sum of the weighted harms (adverse events' iARI): . We evaluated the treatment’s effect on the annual incidence of all-cause mortality, the SPRINT's primary composite cardiovascular outcome, and a composite of the four serious adverse events. These outcomes were chosen to allow comparison to the original SPRINT results. To overcome the limitation of testing the recommendation in the same population in which the models were developed, this evaluation was performed using predictions of both the final and out-of-sample models. The analysis was carried out only on the first imputed data set . The treatment recommendation could not be externally validated, since the CHS population was not randomized to the two treatment groups.A clinical decision support tool was designed aiming to achieve practical utility and transparency of the analytic process that led to the final recommendation. Particular attention was given to the visual presentation and the type of measurements presented. Given different physicians’ preferences for measures of risk estimation, iARRs and iARIs were incorporated, as well as, individual-number-needed-to-treat (iNNT) for each cardiovascular outcome and individual-number-needed-to-harm (iNNH) for each adverse event. The tool’s interface was developed in iterations through receiving feedback from practicing clinicians of different specialties until a consensus was reached on willingness to implement the tool in their practice.P-values < 0.05 were considered significant (two-sided tests were used). Specific analyses for each step of the process have been outlined in the respective sections above.All analyses were conducted using R, CRAN version 3.5.2. Further information on research design is available in the SUPPLEMENTARY INFORMATIONReporting Summary checklist


The simple yet efficient and versatile fabrication of colloidal crystals was investigated based on the solidification-induced colloidal crystallization process with particle/water suspension as precursor. The resulting colloidal crystals were constituted by crystal grains with sizes ranging from several tens of micrometers to a few millimeters. Each of the grains had a close-hexagonal array of colloids, which endowed the bulk colloidal crystal powders with some specific optical properties. The freezing of water was shown as the major driving force to form colloidal crystal grains, which supersaturated the solution with nanoparticles and thus induced the formation and growth of colloidal crystal seeds. This process is intrinsically different from those conventional methods based on shearing force, surface tension, columbic interaction or magnetic interaction, revealing a new strategy to fabricate colloidal crystals in a convenient and efficient way. Photonic crystals (PCs) are periodic structures with a refractive index variation on the scale of the light wavelength. Since the pioneering work of Yablonovitch and John ,2,3, mucAn essential step towards the application of colloidal crystals is the convenient and efficient fabrication. Over the past years, various methods have been investigated for the preparation of colloidal crystals, which are mostly based on the self-assembly of colloidal nanoparticles. Among the most commonly-used methods, gravitational sedimentation is the earliest, which utilizes gravitational force to sink the particles to the substrate at a very slow rate, forming colloidal crystals ,22,23,24In this work, we developed a convenient and efficient method for preparing colloidal crystals through monodispersed-nanoparticle crystallization induced by the freezing of surrounding water. The products consisted of colloidal crystal grains with sizes ranging from several tens of micrometers to a few millimeters, and each of the grains had a close-hexagonal array of spheres. The fabrication process is very convenient and high-throughput with no need of careful control in manipulation. Besides, the strategy proposed here is intrinsically different from those conventional methods based on shearing force, surface tension, columbic interaction or magnetic interaction.−1) to remove ethanol by repeated centrifugation and ultrasonic dispersion cycles. SiO2 colloidal suspensions with different volume fractions of particles  were prepared by quantitatively dispersing the silica particles in ultrapure water through sonication. The monodispersed carboxyl polystyrene (COOH–PS) latex particles with certain amounts of carboxyl groups covering the particle surface were purchased from Duke Scientific Corp., Palo Alto, CA, USA. They were diluted with ultrapure water to certain volume fractions.The monodispersed silica nanoparticles with diameters about 240 nm were synthesized using the Stöber method. The product was first washed with ethanol to remove impurities and unreacted tetraethoxysilane (TEOS) and then washed with ultrapure water (18.2 MΩ·cm−1) and then were instantly transferred to the freeze-drying chamber . The solid water in the frozen suspensions was removed by freeze-drying, producing porous blocks comprising of colloidal-crystal grains. The products were characterized morphologically by an optical microscope  equipped with white light as illumination. The microstructures of the colloidal-crystal grains were examined using a scanning electron microscope , where a thin layer of Au was sputtered on the samples prior to imaging. The reflectance spectra of the products were collected with various incident angles using a variable-angle spectroscope . Since the products were constituted by many small pieces of colloidal crystals, the appearance is pale with deterioration in visibility caused by strong scattering, which makes the characteristic reflectance spectra of the crystal hard to be detected. To facilitate the measurements, thin films composed of colloidal crystal grains were prepared according to the procedures shown in The latex suspensions  were frozen using liquid nitrogen, where the glass petri dish containing the latex suspension was placed near the surface of the liquid nitrogen in a closed environment. The suspensions solidified in a couple of minutes . Clearly, the experimental dots were near the theoretical line, implying that the reflection peaks of the film stemmed from the diffraction of the hexagonally closely packed particles. It is shown that the optical properties of the film arose from the colloidal crystal grains, which acted as building blocks.To characterize the optical properties of the colloidal crystals, the reflectance spectra of the films fabricated according to the procedures shown in The results presented above indicate that the porous blocks or films fabricated with our method were constituted by colloidal crystal grains with sizes ranging from several tens of microns to a few millimeters. The particles comprising the grains were hexagonally closely arranged. Based on these results, the formation mechanism is illustrated in Based on the mechanism proposed above, the particles in the colloidal suspension successively experienced being concentrated, precipitating, forming colloidal crystal seeds, growing large, and finally forming colloidal crystal grains. Hexagonally close-packed arrangement was the thermodynamically favored array fashion obtained when the interparticle repulsive interactions were not too strong. Thus, factors including the initial volume fraction and the surface-charge magnitude of the particles should have no obvious effects on the array fashion of the particles and thus the peak positions of their reflectance spectra. Thus, the only necessary action is to make sure the particles are well dispersed in the precursor. To verify the inference, the influences of the volume fraction and the surface charge of the particles in precursor on the reflectance spectra of the colloidal crystal films were investigated.2 colloidal suspensions with five different volume fractions of particles were prepared and used to fabricate colloidal crystal films. The reflectance of the films was measured at a fixed incident angle of 15°. Based on the spectra shown in SiOThe influence of the particle surface charge was also investigated, where the magnitude of the charge was tuned by adjusting the number of carboxyl groups covering the particle surface. Three batches of COOH–PS particles/water suspensions with different surface-charge magnitude were used to fabricate colloidal crystal films. The reflectance spectra of the films are shown in In addition, factors such as initial particle concentration, surface charge of the particle and solidifying speed of the liquid water may influence the order degree or average size of the elemental colloidal crystal grains, which would consequently affect the peak value and bandgap width of the reflection spectrum of the colloidal crystal film. This will be investigated in detail in our further work. Since there are many tiny colloidal crystals with size of tens of micrometers or even a few micrometers, which would lead to strong light scattering, the produced powders d had a pColloidal crystal powders and films with small free-standing colloidal crystal grains as building blocks were prepared based on the crystallization of monodispersed nanoparticles in particle/water suspension. The crystallization was induced by the freezing of liquid water, which made the solution be supersaturated with nanoparticles. The particles were hexagonally closely packed in their final arrangement and the structure was fixed by removing the solid water through freeze-drying. The initial volume fraction and surface-charge magnitude of the particles in the colloidal suspension showed no influence on the hexagonally close-packed array fashion of the particles. The strategy proposed here is an extremely convenient and highly efficient way to fabricate colloidal crystals, which has no need of careful control over the manipulation. In addition, it is intrinsically different from those conventional methods based on shearing force, surface tension, Columbic interaction or magnetic interaction. Since the crystallization of particles is induced by the solidification of water, various other kinds of solvents or monomers that can be solidified by lowering temperature or initiating polymerization may also be applied to fabricate other kinds of colloidal crystals. Thus, besides convenience and high-efficiency, the new strategy under study to prepare colloidal crystals is also versatile.


A case history of gradual weight loss, bloat, dyschezia with dry faeces and laboured gait, resulting in sudden death after drinking water and associated with ingestion of pupal cocoons of Gonometa spp., was reported by keepers at Mmaditau crush in Botswana in 2013. The crush was a shared holding in a communal area with 15 registered animal keepers. The objective of this study was to profile the history, clinical signs, post-mortem findings, morbidity and mortality from the outbreak using the descriptive study method. Altogether, 81 cattle out of a total of 507 died of impaction from August to December 2013. On autopsy, a loosely connected mass of ingesta, intertwined in ropy silky strands, was observed. It was concluded that there is no readily accessible and available form of treatment at crush level, leaving only evasive husbandry practices as the feasible option. To aid evasive husbandry management practices, temporal and spatial monitoring of population dynamics of Gonometa spp. is recommended, particularly during a drought spell when animals are prone to develop pica, as the basis for an early warning system to farmers.Mortality in cattle associated with ingestion of cocoons ( Gonometa spp., the pupal stage follows from the final instar larval stage (caterpillar) about 5 weeks after the egg hatches, and in the context of Botswana’s climatic conditions it appears in September or October shortly after the first summer rains. The second generation of moths from the September and October cocoons appears in January and February and their offspring later produce cocoons at the end of March and early April. Owing to the onset of winter, cocoons produced in March and April and those that did not hatch in January and February enter diapause. This is a dormant stage that ends into the following September and October with a rise in daily minimum temperature. The predominant woody host species utilised by Gonometa postica and Gonometa rufobrunnea are acacia and mopane trees, respectively (Hartland-Rowe phane (larvae of Gonimbrasia belina) (Hartland-Rowe Gonometa spp. cocoons in Botswana. The aim of this study is to document the history, clinical signs, post-mortem findings, morbidity and mortality, and further discuss potential management practices that can be adopted in the context of the country’s extensive communal rearing system to avoid or limit future cases.Rumen impaction results from accumulation of indigestible ingesta in the rumen with limited aboral movement thereof. Such material could be urban and suburban garbage like plastics (Abdullahi, Usman & Mshalia Cattle were reported to be dying in increased numbers at Mmaditau crush in Zone 8 in the Central District of Botswana during the later months of the year 2013. The first death was reported to have occurred in August 2013, climaxing in mid-December 2013 and tapering to a halt in mid-February 2014. Related deaths with similar clinical signs were also reported in contiguous zones 6 and 7 . An epidClinical examination was carried out on two heifers that were regarded as typical of the cases seen and reported in the area. The heifers were in poor body condition, walked with a laboured gait and abducted forelimbs. Asymmetric abdominal distension, most pronounced in the left paralumbar fossa and indicative of bloat, was evident in both heifers. Straining to defecate with intermittent release of scanty dry stools was seen in one heifer, indicating dyschezia. The skin tenting test in both showed moderate-to-severe dehydration with pale mucous membranes. On auscultation, laboured breathing with audible grunts and wheezes coupled with nasal flaring was evident. Reticuloruminal motility was subdued in both animals.A full autopsy was not conducted on the animals as the owners were unwilling to kill them for further investigation. However, fresh and old ingesta masses harvested from previous cases that died were availed for partial post-mortem investigation. On observation the masses consisted of ingested materials adhering to silky strands that formed irregular ropes of various dimensions . Some maGonometa spp., largely on mopane and acacia trees (Environmental assessment in the vicinity of Mmaditau crush showed a high density of occupied and empty pupal cocoons of ia trees . Occupieia trees .No approval was required as this case report describes observations made during a clinical case attendance by employees of the Department of Veterinary Services, who are mandated through relevant statutes to do it as part of their routine extension service to farmers in the country.G. postica and G. rufobrunnea has only previously been reported in South Africa (Edwards Gonometa spp. Laboratory incubation of harvested cocoons confirmed the causal agent to be G. rufobrunnea as the emerged moths exhibited typically red forewings and laid white eggs with a distinctive black micropyle (Hartland-Rowe Mortality in cattle associated with the ingestion of cocoons of both  Edwards , Namibia Edwards  and Zimb Edwards . The hisPathogenesis associated with ingestion of cocoons is insidious. After ingestion, cocoons disintegrate in the rumen owing to both the actions of rumen acids and the mechanical effect of reticuloruminal motility, resulting in adhering and entangling of ingesta in the loosening silk strands. With stretching of the fine silk strands from degummed cocoons, ingesta adherence progresses gradually and they clump into a large mass causing rumen impaction Zumpt . The impGonometa spp. cocoons has enabled an estimation of the number of cocoons needed to spin 1 kg of spun silk to be 2326–4762 (Kioko, Raina & Mueke The intake threshold of cocoons needed to cause clinical signs and eventual death is not known. However, industrial processing of Gonometa spp. is grave as cases end up dying, unless surgical intervention is initiated to remove the impacted ingesta mass (Edwards The prognosis for rumen impaction cases associated with the ingestion of pupal cocoons of  Edwards . However