Characterization of VRC01, a potent and broadly neutralizing anti-HIV mAb, produced in transiently and stably transformed tobacco

The proposed clinical trial in Africa of VRC01, a potent broadly neutralizing antibody (bNAb) capable of neutralizing 91% of known HIV-1 isolates, raises concerns about testing a treatment which will be too expensive to be accessible by the most important target population, the poor in under-developed regions such as sub-Saharan Africa. Here, we report the expression of VRC01 in plants as an economic alternative to conventional mammalian-cell-based production platforms. The heavy and light chain genes of VRC01 were cloned onto a single vector, pTRAk.2, which was transformed into Nicotiana benthamiana or Nicotiana tabacum using transient and stable expression production systems respectively. VRC01 has been successfully expressed transiently in plants with expression level of approximately 80 mg antibody/kg; stable transgenic lines expressing up to 100 mg antibody/kg were also obtained. Plant-produced VRC01 from both systems showed a largely homogeneous N-glycosylation profile with a single dominant glycoform. The binding kinetics to gp120 IIIB (approximately 1 nM), neutralization of HIV-1 BaL or a panel of 10 VRC01-sensitive HIV-1 Env pseudoviruses of VRC01 produced in transient and stable plants were also consistent with VRC01 from HEK cells

Muscle recruitment strategies can reduce joint loading during level walking

Joint inflammation, with consequent cartilage damage and pain, typically reduces functionality and affects activities of daily life in a variety of musculoskeletal diseases. Since mechanical loading is an important determinant of the disease process, a possible conservative treatment is the unloading of joints. In principle, a neuromuscular rehabilitation program aimed to promote alternative muscle recruitments could reduce the loads on the lower-limb joints during walking. The extent of joint load reduction one could expect from this approach remains unknown. Furthermore, assuming significant reductions of the load on the affected joint can be achieved, it is unclear whether, and to what extent, the other joints will be overloaded. Using subject-specific musculoskeletal models of four different participants, we computed the muscle recruitment strategies that minimised the hip, knee and ankle contact force, and predicted the contact forces such strategies induced at the other joints. Significant reductions of the peak force and impulse at the knee and hip were obtained, while only a minimal effect was found at the ankle joint. Adversely, the peak force and the impulse in non-targeted joints increased when aiming to minimize the load in an adjacent joint. These results confirm the potential of alternative muscle recruitment strategies to reduce the loading at the knee and the hip, but not at the ankle. Therefore, neuromuscular rehabilitation can be targeted to reduce the loading at affected joints but must be considered carefully in patients with multiple joints affected due to the potential adverse effects in non-targeted joints

Investigation of the dependence of joint contact forces on musculotendon parameters using a codified workflow for image-based modelling

The generation of subject-specific musculoskeletal models of the lower limb has become a feasible taskthanks to improvements in medical imaging technology and musculoskeletal modelling software.Nevertheless, clinical use of these models in paediatric applications is still limited for what concernsthe estimation of muscle and joint contact forces. Aiming to improve the current state of the art, amethodology to generate highly personalized subject-specific musculoskeletal models of the lower limbbased on magnetic resonance imaging (MRI) scans was codified as a step-by-step procedure and appliedto data from eight juvenile individuals. The generated musculoskeletal models were used to simulate 107gait trials using stereophotogrammetric and force platform data as input. To ensure completeness of themodelling procedure, muscles’ architecture needs to be estimated. Four methods to estimate muscles’maximum isometric force and two methods to estimate musculotendon parameters (optimal fiber lengthand tendon slack length) were assessed and compared, in order to quantify their influence on the models’output. Reported results represent the first comprehensive subject-specific model-based characterizationof juvenile gait biomechanics, including profiles of joint kinematics and kinetics, muscle forces and jointcontact forces. Our findings suggest that, when musculotendon parameters were linearly scaled from areference model and the muscle force-length-velocity relationship was accounted for in the simulations,realistic knee contact forces could be estimated and these forces were not sensitive the method used tocompute muscle maximum isometric force

Frequent use of emergency departments: an application to the paediatric context.

Frequent users of Emergency Departments (EDs) represent a particularly interesting category of users since they account to a small percentage of patients but they affect considerably accesses, overcrowding and the overall costs of ED activities. The literature on the topic is vast and it allows to delineate a profile of frequent users identifying socio-demographic (age, gender, nationality) and clinical (chronic diseases, drugs and alcohol abuse; psychic illnesses) risk factors. However, most of the studies on the topic limit the analysis to one-year period or to a single study site and there is no consensus on the definition of frequent use. Frequent users are generally defined as those patients reporting a number of accesses per year beyond a certain threshold. The selection of the threshold is often based on previous literature or on percentiles but the definitions vary considerably and the choice tends to be subjective. In this study, the focus will be placed on paediatric patients, with reference to which the contributions in the literature are still very limited. The objective is to identify the most important drivers of ED frequent use in the 19 EDs of Liguria region (Italy) during a three-year period (2013-2015). The dataset contains 287,242 accesses referred to 144,895 under 14 patients and it includes information on patients\u2019 characteristics and on their clinical pathway. To overcome the limitations connected to previous definitions of frequent use, we exploit the availability of data on three years; this allows to define, not only frequent use, but also its duration (i.e. One-shot / Multiple shot frequent use) and intensity (Normal, High, Very high). By the use of logit and multinomial logit regressions we identify a set of risk factors associated to frequent use and to the different forms of frequent use. Results show that even if frequent users represent a small share of patients (9%) they contribute to roughly 25% of accesses. Chronic conditions are the most relevant determinants of frequent use (particularly mental disorders, diseases of the respiratory system) but also foreign nationality turns out to be an important predictor. Differences emerge in the impact of regressors on the different forms of frequent use defined according to its duration and intensity. The study represents an important tool to support policy-making and to discriminate between the potentially preventable frequent use (i.e. inapFrequent users of Emergency Departments (EDs) represent a particularly interesting category of users since they account to a small percentage of patients but they affect considerably accesses, overcrowding and the overall costs of ED activities. The literature on the topic is vast and it allows to delineate a profile of frequent users identifying socio-demographic (age, gender, nationality) and clinical (chronic diseases, drugs and alcohol abuse; psychic illnesses) risk factors. However, most of the studies on the topic limit the analysis to one-year period or to a single study site and there is no consensus on the definition of frequent use. Frequent users are generally defined as those patients reporting a number of accesses per year beyond a certain threshold. The selection of the threshold is often based on previous literature or on percentiles but the definitions vary considerably and the choice tends to be subjective. In this study, the focus will be placed on paediatric patients, with reference to which the contributions in the literature are still very limited. The objective is to identify the most important drivers of ED frequent use in the 19 EDs of Liguria region (Italy) during a three-year period (2013-2015). The dataset contains 287,242 accesses referred to 144,895 under 14 patients and it includes information on patients\u2019 characteristics and on their clinical pathway. To overcome the limitations connected to previous definitions of frequent use, we exploit the availability of data on three years; this allows to define, not only frequent use, but also its duration (i.e. One-shot / Multiple shot frequent use) and intensity (Normal, High, Very high). By the use of logit and multinomial logit regressions we identify a set of risk factors associated to frequent use and to the different forms of frequent use. Results show that even if frequent users represent a small share of patients (9%) they contribute to roughly 25% of accesses. Chronic conditions are the most relevant determinants of frequent use (particularly mental disorders, diseases of the respiratory system) but also foreign nationality turns out to be an important predictor. Differences emerge in the impact of regressors on the different forms of frequent use defined according to its duration and intensity. 150 05 March 2018, IISES Annual Conference, Sevilla ISBN ISBN 978-80-87927-45-8, IISES The study represents an important tool to support policy-making and to discriminate between the potentially preventable frequent use (i.e. inapFrequent users of Emergency Departments (EDs) represent a particularly interesting category of users since they account to a small percentage of patients but they affect considerably accesses, overcrowding and the overall costs of ED activities. The literature on the topic is vast and it allows to delineate a profile of frequent users identifying socio-demographic (age, gender, nationality) and clinical (chronic diseases, drugs and alcohol abuse; psychic illnesses) risk factors. However, most of the studies on the topic limit the analysis to one-year period or to a single study site and there is no consensus on the definition of frequent use. Frequent users are generally defined as those patients reporting a number of accesses per year beyond a certain threshold. The selection of the threshold is often based on previous literature or on percentiles but the definitions vary considerably and the choice tends to be subjective. In this study, the focus will be placed on paediatric patients, with reference to which the contributions in the literature are still very limited. The objective is to identify the most important drivers of ED frequent use in the 19 EDs of Liguria region (Italy) during a three-year period (2013-2015). The dataset contains 287,242 accesses referred to 144,895 under 14 patients and it includes information on patients\u2019 characteristics and on their clinical pathway. To overcome the limitations connected to previous definitions of frequent use, we exploit the availability of data on three years; this allows to define, not only frequent use, but also its duration (i.e. One-shot / Multiple shot frequent use) and intensity (Normal, High, Very high). By the use of logit and multinomial logit regressions we identify a set of risk factors associated to frequent use and to the different forms of frequent use. Results show that even if frequent users represent a small share of patients (9%) they contribute to roughly 25% of accesses. Chronic conditions are the most relevant determinants of frequent use (particularly mental disorders, diseases of the respiratory system) but also foreign nationality turns out to be an important predictor. Differences emerge in the impact of regressors on the different forms of frequent use defined according to its duration and intensity. 150 05 March 2018, IISES Annual Conference, Sevilla ISBN ISBN 978-80-87927-45-8, IISES The study represents an important tool to support policy-making and to discriminate between the potentially preventable frequent use (i.e. inapappropriate use) and that associated to complex medical conditions, such as chronic conditions

Tbx20 Is Required in Mid-Gestation Cardiomyocytes and Plays a Central Role in Atrial Development.

RationaleMutations in the transcription factor TBX20 (T-box 20) are associated with congenital heart disease. Germline ablation of Tbx20 results in abnormal heart development and embryonic lethality by embryonic day 9.5. Because Tbx20 is expressed in multiple cell lineages required for myocardial development, including pharyngeal endoderm, cardiogenic mesoderm, endocardium, and myocardium, the cell type-specific requirement for TBX20 in early myocardial development remains to be explored.ObjectiveHere, we investigated roles of TBX20 in midgestation cardiomyocytes for heart development.Methods and resultsAblation of Tbx20 from developing cardiomyocytes using a doxycycline inducible cTnTCre transgene led to embryonic lethality. The circumference of developing ventricular and atrial chambers, and in particular that of prospective left atrium, was significantly reduced in Tbx20 conditional knockout mutants. Cell cycle analysis demonstrated reduced proliferation of Tbx20 mutant cardiomyocytes and their arrest at the G1-S phase transition. Genome-wide transcriptome analysis of mutant cardiomyocytes revealed differential expression of multiple genes critical for cell cycle regulation. Moreover, atrial and ventricular gene programs seemed to be aberrantly regulated. Putative direct TBX20 targets were identified using TBX20 ChIP-Seq (chromatin immunoprecipitation with high throughput sequencing) from embryonic heart and included key cell cycle genes and atrial and ventricular specific genes. Notably, TBX20 bound a conserved enhancer for a gene key to atrial development and identity, COUP-TFII/Nr2f2 (chicken ovalbumin upstream promoter transcription factor 2/nuclear receptor subfamily 2, group F, member 2). This enhancer interacted with the NR2F2 promoter in human cardiomyocytes and conferred atrial specific gene expression in a transgenic mouse in a TBX20-dependent manner.ConclusionsMyocardial TBX20 directly regulates a subset of genes required for fetal cardiomyocyte proliferation, including those required for the G1-S transition. TBX20 also directly downregulates progenitor-specific genes and, in addition to regulating genes that specify chamber versus nonchamber myocardium, directly activates genes required for establishment or maintenance of atrial and ventricular identity. TBX20 plays a previously unappreciated key role in atrial development through direct regulation of an evolutionarily conserved COUPT-FII enhancer

Enhanced Avidity Maturation of Antibody to Human Immunodeficiency Virus Envelope: DNA Vaccination with gp120-C3d Fusion Proteins

DNA vaccination can elicit both humoral and cellular immune responses and can confer protection against several pathogens. However, DNA vaccines expressing the envelope (Env) protein of human immunodeficiency virus (HIV) have been relatively ineffective at generating high titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, we report that fusion of Env and the complement component, C3d, in a DNA vaccine, enhances the titers of antibody to Env. Plasmids were generated that expressed a secreted form of Env (sgp120) from three isolates of HIV and these same forms fused to three tandem copies of the murine homologue of C3d (sgp120-3C3d). Analyses of titers and avidity maturation of the raised antibody indicated that immunizations with each of the sgp120-3C3d-expressing DNAs accelerated both the onset and the avidity maturation of antibody to Env. Originally published AIDS Research and Human Retroviruses, Vol. 17, No. 9, June 200

International Technology Transfer of a GCLP-Compliant HIV-1 Neutralizing Antibody Assay for Human Clinical Trials

The Collaboration for AIDS Vaccine Discovery/Comprehensive Antibody – Vaccine Immune Monitoring Consortium (CAVD/CA-VIMC) assisted an international network of laboratories in transferring a validated assay used to judge HIV-1 vaccine immunogenicity in compliance with Good Clinical Laboratory Practice (GCLP) with the goal of adding quality to the conduct of endpoint assays for Human Immunodeficiency Virus I (HIV-1) vaccine human clinical trials. Eight Regional Laboratories in the international setting (Regional Laboratories), many located in regions where the HIV-1 epidemic is most prominent, were selected to implement the standardized, GCLP-compliant Neutralizing Antibody Assay for HIV-1 in TZM-bl Cells (TZM-bl NAb Assay). Each laboratory was required to undergo initial training and implementation of the immunologic assay on-site and then perform partial assay re-validation, competency testing, and undergo formal external audits for GCLP compliance. Furthermore, using a newly established external proficiency testing program for the TZM-bl NAb Assay has allowed the Regional Laboratories to assess the comparability of assay results at their site with the results of neutralizing antibody assays performed around the world. As a result, several of the CAVD/CA-VIMC Regional Laboratories are now in the process of conducting or planning to conduct the GCLP-compliant TZM-bl NAb Assay as an indicator of vaccine immunogenicity for ongoing human clinical trials

PIN92 Quality of Life Among Hiv Patients: Results from the Ianua Clinical Trial

OBJECTIVES To understand the relationship between socio-demographic variables, clinical factors, highly active anti-retroviral therapy (HAART) and health related quality of life (QoL) in HIV-infected individuals participants in the IANUA multicenter study. METHODS Data relating to patients with HIV infection admitted to 3 infectious disease units in Genoa (Italy) between 2012 and 2014 are collected and analyzed. Univariate and multivariate association of demographic and clinical factors with QoL (computed using EQ-5D-3L) are examined. QoL determinants are assessed using a tobit model, while a logistic model is implemented in order to investigate the relation between specific patients characteristics and the likelihood of having higher QoL. RESULTS Results of the empirical modeling suggest that being Italian and having a job are positively associate with QoL, whereas being a female, taking other drugs in addition to anti-retroviral drugs and being subsidisied are negatively related to QoL. Among clinical factors, CD4 cell count level cannot be considered as significant predictor of QoL, while higher QoL seem to be defined by single tablet regimens. CONCLUSION The study investigates the major determinants of QoL among HIV patients and the results provide some informative tools useful to improve strategies aiming at maximizing QoL. As monitoring of QoL is nowadays a priority for clinicians, further work will be based on \u201cdynamic\u201d analysis comparing QoL at the initial time and QoL at 6-months follow up