Search

Flavia Ferrantelli,1,* Claudia Arenaccio,1,* Francesco Manfredi,1 Eleonora Olivetta,1 Chiara Chiozzini,1 Patrizia Leone,1 Zulema Percario,2 Alessandro Ascione,1 Michela Flego,1 Paola Di Bonito,3 Luisa Accardi,3 Maurizio Federico1 1National Center for Global Health, Istituto Superiore Di Sanità (ISS), Rome, Italy; 2Department of Science, Roma Tre University, Rome, Italy; 3Department of Infectious Diseases, Istituto Superiore Di Sanità (ISS), Rome, Italy*These authors contributed equally to this workCorrespondence: Maurizio FedericoNational Center for Global Health, Istituto Superiore Di Sanità, Viale Regina Elena, 299, Rome 00161, ItalyTel +39-06-4990-3605Fax +39-06-49903210Email maurizio.federico@iss.itPurpose: Single-chain variable fragments (scFvs) are one of the smallest antigen-binding units having the invaluable advantage to be expressed by a unique short open reading frame (ORF). Despite their reduced size, spontaneous cell entry of scFvs remains inefficient, hence precluding the possibility to target intracellular antigens. Here, we describe an original strategy to deliver scFvs inside target cells through engineered extracellular vesicles (EVs). This approach relies on the properties of a Human Immunodeficiency Virus (HIV)-1 Nef mutant protein referred to as Nefmut. It is a previously characterized Nef allele lacking basically all functions of wt Nef, yet strongly accumulating in the EV lumen also when fused at its C-terminus with a foreign protein. To gain the proof-of-principle for the efficacy of the proposed strategy, the tumor-promoting Human Papilloma Virus (HPV)16-E7 protein was considered as a scFv-specific intracellular target. The oncogenic effect of HPV16-E7 relies on its binding to the tumor suppressor pRb protein leading to a dysregulated cell duplication. Interfering with this interaction means impairing the HPV16-E7-induced cell proliferation.Methods: The Nefmut gene was fused in frame at its 3ʹ-terminus with the ORF coding for a previously characterized anti-HPV16-E7 scFv. Interaction between the Nefmut-fused anti-HPV16-E7 scFv and the HPV16-E7 protein was tested by both confocal microscope and co-immunoprecipitation analyses on co-transfected cells. The in cis anti-proliferative effect of the Nefmut/anti-HPV16-E7 scFv was assayed by transfecting HPV16-infected cells. The anti-proliferative effect of EVs engineered with Nefmut/anti-HPV16-E7 scFv on HPV16-E7-expressing cells was evaluated in two ways: i) through challenge with purified EVs by a Real-Time Cell Analysis system and ii) in transwell co-cultures by an MTS-based assay.Results: The Nefmut/anti-HPV16-E7 scFv chimeric product is efficiently uploaded in EVs, binds HPV16-E7, and inhibits the proliferation of HPV16-E7-expressing cells. Most important, challenge with cell-free EVs incorporating the Nefmut/anti-HPV16-E7 scFv led to the inhibition of proliferation of HPV16-E7-expressing cells. The proliferation of these cells was hindered also when they were co-cultured in transwells with cells producing EVs uploading Nefmut/anti-HPV16-E7 scFv.Conclusion: Our data represent the proof-of-concept for the possibility to target intracellular antigens through EV-mediated delivery of scFvs. This finding could be relevant to design novel methods of intracellular therapeutic interventions.Keywords: single-chain variable fragments, Human Papilloma Virus E7 protein, exosomes, human immunodeficiency virus Nef protein, scFv intracellular delivery

To evaluate the relationship between HDL-Cholesterol (HDL-C), hypertension, and left ventricular hypertrophy (LVH) in a large sample of Caucasian youths with overweight/obesity (OW/OB). A cross-sectional multicenter study was performed in 1469 youths (age 6–16 years) with OW/OB observed in the period 2016–2020. An additional independent sample of 244 youths with an echocardiographic evaluation, observed in a single center was analyzed. The sample was divided in six quantiles (Q) of HDL-C: Q1: >56, Q2: ≤56 > 51, Q3: ≤51 > 45, Q4: ≤45 > 41, Q5: ≤41 > 39, Q6: <39 mg/dL. The nadir of the relationship was identified in youths in the first quantile. Among HDL-Cholesterol quantiles the distribution of hypertension was non-linear with a percentage of 25.0%, 40.1%, 33.6%, 31.3%, 35.2% and 39.7% in the six quantiles, respectively. The percentage of LVH was 21.8%, 43.6%, 48.8%, 35.5%, 38.5% and 52.0% in the six quantiles, respectively. The highest odds [95%Cl] of hypertension were 2.05 (1.33–3.16) (P < 0.01) in Q2, 1.67 (1.10–2.55) (P < 0.05) in Q3 and 1.59 (1.05–2.41) (P < 0.05) in Q6 vs Q1. The odds of LVH were 3.86 (1.15–10.24) (P < 0.05) in Q2, 4.16 (1.58–10.91) (P < 0.05) in Q3 and 3.60 (1.44–9.02) (P < 0.05) in Q6 vs Q1, independently by centers, age, sex, prepubertal stage, and body mass index. Contrary to the common belief, the present study shows that high levels of HDL-C may be not considered a negative predictor of hypertension and LVH, two risk factors for future CV disease. • Association between HDL-Cholesterol and cardiovascular risk is non-linear in adults. • Whether a similar slope is present in youths with obesity is unexplored. • 1469 youths with obesity were stratified across six quantiles of HDL-Cholesterol. • A U-shaped curve between HDL-Cholesterol quantiles and hypertension was found. • Targeting only low HDL-Cholesterol as a risk factor may be misleading. [ABSTRACT FROM AUTHOR]

Paola Di Bonito,1 Chiara Chiozzini,2 Claudia Arenaccio,2 Simona Anticoli,2 Francesco Manfredi,2 Eleonora Olivetta,2 Flavia Ferrantelli,2 Emiliana Falcone,3 Anna Ruggieri,3 Maurizio Federico2 1Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Rome, Italy; 2National AIDS Center, Istituto Superiore di Sanità, Rome, Italy; 3Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Rome, Italy Abstract: We recently proved that exosomes engineered in vitro to deliver high amounts of HPV E7 upon fusion with the Nefmut exosome-anchoring protein elicit an efficient anti-E7 cytotoxic T lymphocyte immune response. However, in view of a potential clinic application of this finding, our exosome-based immunization strategy was faced with possible technical difficulties including industrial manufacturing, cost of production, and storage. To overcome these hurdles, we designed an as yet unproven exosome-based immunization strategy relying on delivery by intramuscular inoculation of a DNA vector expressing Nefmut fused with HPV E7. In this way, we predicted that the expression of the Nefmut/E7 vector in muscle cells would result in a continuous source of endogenous (ie, produced by the inoculated host) engineered exosomes able to induce an E7-specific immune response. To assess this hypothesis, we first demonstrated that the injection of a Nefmut/green fluorescent protein-expressing vector led to the release of fluorescent exosomes, as detected in plasma of inoculated mice. Then, we observed that mice inoculated intramuscularly with a vector expressing Nefmut/E7 developed a CD8+ T-cell immune response against both Nef and E7. Conversely, no CD8+ T-cell responses were detected upon injection of vectors expressing either the wild-type Nef isoform of E7 alone, most likely a consequence of their inefficient exosome incorporation. The production of immunogenic exosomes in the DNA-injected mice was formally demonstrated by the E7-specific CD8+ T-cell immune response we detected in mice inoculated with exosomes isolated from plasma of mice inoculated with the Nefmut/E7 vector. Finally, we provide evidence that the injection of Nefmut/E7 DNA led to the generation of effective antigen-specific cytotoxic T lymphocytes whose activity was likely part of the potent, therapeutic antitumor effect we observed in mice implanted with TC-1 tumor cells. In summary, we established a novel method to generate immunogenic exosomes in vivo by the intramuscular inoculation of DNA vectors expressing the exosome-anchoring protein Nefmut and its derivatives. Keywords: nanovesicles, cytotoxic T lymphocytes, HIV-1 Nef, DNA vectors

Paola Di Bonito,1 Linda Petrone,1 Gabriele Casini,2 Iolanda Francolini,2 Maria Grazia Ammendolia,3 Luisa Accardi,1 Antonella Piozzi,2 Lucio D’Ilario,2 Andrea Martinelli2 1Department of Infectious, Parasitic and Immune-mediated Diseases, Italian National Institute of Health, 2Department of Chemistry, Sapienza University of Rome, Rome, Italy; 3Department of Technology and Health, Italian National Institute of Health, Rome, Italy Background: Poly(L-lactide) (PLLA) is a biodegradable polymer currently used in many biomedical applications, including the production of resorbable surgical devices, porous scaffolds for tissue engineering, nanoparticles and microparticles for the controlled release of drugs or antigens. The surfaces of lamellar PLLA single crystals (PLLAsc) were provided with amino groups by reaction with a multifunctional amine and used to adsorb an Escherichia coli-produced human papillomavirus (HPV)16-E7 protein to evaluate its possible use in antigen delivery for vaccine development.Methods: PLLA single crystals were made to react with tetraethylenepentamine to obtain amino-functionalized PLLA single crystals (APLLAsc). Pristine and amino-functionalized PLLAsc showed a two-dimensional microsized and one-dimensional nanosized lamellar morphology, with a lateral dimension of about 15–20 µm, a thickness of about 12 nm, and a surface specific area of about 130 m2/g. Both particles were characterized and loaded with HPV16-E7 before being administered to C57BL/6 mice for immunogenicity studies. The E7-specific humoral-mediated and cell-mediated immune response as well as tumor protective immunity were analyzed in mice challenged with TC-1 cancer cells.Results: Pristine and amino-functionalized PLLAsc adsorbed similar amounts of E7 protein, but in protein-release experiments E7-PLLAsc released a higher amount of protein than E7-APLLAsc. When the complexes were dried for observation by scanning electron microscopy, both samples showed a compact layer, but E7-APLLAsc showed greater roughness than E7-PLLAsc. Immunization experiments in mice showed that E7-APLLAsc induced a stronger E7-specific immune response when compared with E7-PLLAsc. Immunoglobulin G isotyping and interferon gamma analysis suggested a mixed Th1/Th2 immune response in both E7-PLLAsc-immunized and E7-APLLAsc-immunized mice. However, only the mice receiving E7-APLLAsc were fully protected from TC-1 tumor growth after three doses of vaccine.Conclusion: Our results show that APLLA single crystals improve the immunogenicity of HPV16-E7 and indicate that E7-APLLAsc could be used for development of an HPV16 therapeutic vaccine against HPV16-related tumors. Keywords: poly(L-lactide), lamellar crystals, human papillomavirus, HPV16-E7, therapeutic vaccine

Mindfulness-based stress reduction, a complementary and alternative therapy, is able to decrease cancer-related fatigue, and stress and to improve the quality of life in cancer patients. Some studies evaluated if mindfulness-based stress reduction could improve some cardiometabolic and cancer risk factors, including systemic chemokines, growth factors, and pro-inflammatory biomarkers (e.g., C-reactive protein, Interleukin-1). In this narrative review, we highlight the pleiotropic beneficial effects of mindfulness-based stress reduction and its clinical impact on cardiovascular and cancer risk factors among patients with cancer in different stages. Moreover, improvements in the overall quality of life, sleep quality, and immune functions [changes in plasma levels of interleukin-4 (IL-4), interferon-γ (INF-γ), and interleukin-10 (IL-10)] will also be discussed. Albeit few clinical studies available in the literature, evidenced the beneficial effects of mindfulness-based stress reduction on the immune and cardiometabolic profile in cancer patients, providing important insights into the closest collaboration between psycho-oncologists, oncologists, and cardiologists. [ABSTRACT FROM AUTHOR]

Recently, the European Society of Cardiology task force released a Consensus document (ESC-CD) on pediatric hypertension (HTN) supporting the use of normative tables (age range 6–16 years) for the diagnosis of HTN, while the Hypertension Canada Guidelines (HTN-CGs) proposed static cutoffs. We aimed to assess the prevalence of HTN by ESC-CD or HTN-CGs and their association with glomerular function and left ventricular (LV) geometry in youths with overweight/obesity (OW/OB). Data of 3446 youths were analyzed. HTN by was defined using normative tables (ESC-CD) or static cutoffs of BP ≥ 120/80 in children (age <12 years) and ≥130/85 mmHg in adolescents (age ≥12 years) (HTN-CGs). Mildly reduced glomerular filtration rate was defined by GFR <90 ≥ 60 mL/min/1.73 m2. Concentric LV hypertrophy (cLVH) was assessed in 500 youths and defined by LVH and high relative wall thickness as proposed by ESC-CD. Prevalence of HTN was 27.9% by ESC-CD and 22.7% by HTN-CGs. The association with mildly reduced glomerular filtration rate was significant only in hypertensive adolescents classified by HTN-CGs [Odds Ratio (OR), 95%Cl] 2.16 (1.44–3.24), whereas the association with cLVH was significant using both criteria: children OR 2.18 (1.29–3.67) by ESC-CD and 2.27 (1.32–3.89) by HTN-CGs; adolescents OR 2.62 (1.17–5.84) by ESC-CD and 2.83 (1.14–7.02) by HTN-CGs. Although static cutoffs may represent a simplification for HTN identification, tables by ESC-CD detect a higher number of hypertensive youths before a clear appearance of glomerular impairment, which offers advantages in terms of primary cardiovascular prevention. • Tables or static cutoffs of blood pressure are proposed for hypertension screening. • Tables provide higher hypertension prevalence than static cutoffs in obese youths. • Both criteria showed an association between hearth geometry and hypertension. • Static cutoffs for hypertension are associated with renal impairment in adolescents. • Tables provide advantages in terms of primary cardiovascular prevention. [ABSTRACT FROM AUTHOR]

Aim: To assess a new formula to improve the screening of isolated impaired glucose tolerance (IGT) in youth with overweight/obesity (OW/OB).Methods and Results: A cross-sectional study was performed in 1189 Caucasian youths with OW/OB aged 5-17 years, in whom impaired fasting glucose and high glycosylated hemoglobin were excluded. The sample was divided into training set (TS) (n = 883) and validation set (VS) (n = 306). Fasting (FG) and post-load plasma glucose, alanine aminotransferase (ALT), lipids and familial history for type 2 diabetes (FD) were available in all individuals. In the TS youths with IGT (n = 58, 7.0%) showed higher prevalence of female sex (FS), FD, and higher levels of FG, post-load glucose, ALT and lower levels of HDL-cholesterol vs individuals without IGT. The linear formula was obtained by logistic regression analysis in the TS: 0.05∗ALT + 0.07∗FG + 0.87∗FD + (0.06∗HDL∗ - 1) + 1∗FS. The best cut-off was 5.84. The performance of the formula vs IGT was: sensitivity: 0.74 and specificity: 0.71. Similar results were obtained in the VS.Conclusions: Using metabolic and anamnestic data we obtained a simple formula with a good performance for screening isolated IGT. This formula may support pediatricians to identify youths with OW/OB in whom the OGTT may be useful for detecting IGT. [ABSTRACT FROM AUTHOR]