Preclinical risk assessment strategy to mitigate the T-cell dependent immunogenicity of protein biotherapeutics: State of the art, challenges and future perspectives.

Protein therapeutics hold a prominent role and have brought significant diversity in efficacious medicinal products. Not just monoclonal antibodies and different antibody formats (pegylated antigen-binding fragments, bispecifics, antibody-drug conjugates, single chain variable fragments, nanobodies, dia-, tria- and tetrabodies), but also purified blood products, growth factors, recombinant cytokines, enzyme replacement factors, fusion proteins are all good instances of therapeutic proteins that have been developed in the past decades and approved for their value in oncology, immune-oncology, and autoimmune diseases discovery programs. Although there was an ingrained belief that fully humanized proteins were expected to have limited immunogenicity, adverse effects associated with immune responses to biological therapies raised some concern in biotech companies. Consequently, drug developers are designing strategies to assess potential immune responses to protein therapeutics during both the preclinical and clinical phases of development. Despite the many factors that can contribute to protein immunogenicity, T cell- (thymus-) dependent (Td) immunogenicity seems to play a crucial role in the development of anti-drug antibodies (ADAs) to biologics. A broad range of methodologies to predict and rationally assess Td immune responses to protein drugs has been developed. This review aims to briefly summarize the preclinical immunogenicity risk assessment strategy to mitigate the risk of potential immunogenic candidates coming towards clinical phases, discussing the advantages and limitations of these technologies, and suggesting a rational approach for assessing and mitigating Td immunogenicity.

Salvianolic Acid B Strikes Back: New Evidence in the Modulation of Expression and Activity of Matrix Metalloproteinase 9 in MDA-MB-231 Human Breast Cancer Cells.

Salvianolic acid B (SalB) is a bioactive compound from Salviae miltiorrhizae, one of the most important traditional herbal medicines widely used in several countries for the treatment of cardiovascular diseases. The aim of this study was to evaluate the in vitro effect of SalB on the expression and the activity of matrix metalloproteinase 9 (MMP-9), a zinc-dependent proteolytic enzyme, in human MDA-MB-231 breast cancer cells. This cellular model is characterized by a marked invasive phenotype, supported by a high constitutive expression of MMPs, especially gelatinases. SalB was first of all evaluated by in silico approaches primarily aimed at predicting the main pharmacokinetic parameters. The most favorable interaction between the natural compound and MMP-9 was instead tested by molecular docking analysis that was subsequently verified by an enzymatic inhibition assay. MDA-MB-231 cells were treated with SalB 5 µM and 50 µM for 24 h and 48 h. The conditioned media obtained from treated cells were then analyzed by gelatin zymography and reverse zymography to, respectively, evaluate the MMP-9 activity and the presence of TIMP-1. The expression of the enzyme was then evaluated by Western blot on conditioned media and by analysis of transcripts through reverse transcriptase-polymerase chain reaction (RT-PCR). The in silico approach showed the ability of SalB to interact with the catalytic zinc ion of the enzyme, with a plausible competitive mode of action. The analysis of conditioned culture media showed a reduction in MMP-9 activity and the concomitant decrease in the enzyme concentration, partially confirmed by analysis of transcripts. SalB showed the ability to modulate the function of MMP-9 in MDA-MB-231 cells. To our knowledge, this is the first time in which the role of SalB on MMP-9 in a highly invasive cellular model is investigated. The obtained results impose further and more specific evaluations in order to obtain a better understanding of the biochemical mechanisms that regulate the interaction between this natural compound and the MMP-9.

Brucella ceti and Brucella pinnipedialis genome characterization unveils genetic features that highlight their zoonotic potential.

The Gram-negative bacteria Brucella ceti and Brucella pinnipedialis circulate in marine environments primarily infecting marine mammals, where they cause an often-fatal disease named brucellosis. The increase of brucellosis among several species of cetaceans and pinnipeds, together with the report of sporadic human infections, raises concerns about the zoonotic potential of these pathogens on a large scale and may pose a threat to coastal communities worldwide. Therefore, the characterization of the B. ceti and B. pinnipedialis genetic features is a priority to better understand the pathological factors that may impact global health. Moreover, an in-depth functional analysis of the B. ceti and B. pinnipedialis genome in the context of virulence and pathogenesis was not undertaken so far. Within this picture, here we present the comparative whole-genome characterization of all B. ceti and B. pinnipedialis genomes available in public resources, uncovering a collection of genetic tools possessed by these aquatic bacterial species compared to their zoonotic terrestrial relatives. We show that B. ceti and B. pinnipedialis genomes display a wide host-range infection capability and a polyphyletic phylogeny within the genus, showing a genomic structure that fits the canonical definition of closeness. Functional genome annotation led to identifying genes related to several pathways involved in mechanisms of infection, others conferring pan-susceptibility to antimicrobials and a set of virulence genes that highlight the similarity of B. ceti and B. pinnipedialis genotypes to those of Brucella spp. displaying human-infecting phenotypes.

Oncology and Palliative Care Nurses' Knowledge and Attitudes Toward Artificial Nutrition and Hydration for Patients at End of Life in Italy: A Cross-sectional Survey.

BACKGROUND: Nutrition is a basic patients' need and an important component of nursing. End-of-life cancer patients can experience difficulties in eating and drinking, sometimes requiring artificial nutrition and hydration (ANH). Nurses' knowledge and attitudes greatly influence nursing care. Nurses may lack knowledge of ANH. To date, no study has explored the knowledge and attitudes of oncology and palliative care nurses toward end-of-life ANH. OBJECTIVE: To describe oncology and palliative care nurses' knowledge of and attitudes toward ANH. METHODS: A multicenter cross-sectional study was conducted in 7 regions of Italy using the Italian version of the Questionnaire on Knowledge and Attitudes in Providing ANH for Terminal Cancer Patients. Attitudes were divided into benefits and burdens of providing ANH. Data were analyzed by descriptive statistics. RESULTS: High percentages of oncology and palliative care nurses showed reasonable knowledge about ANH. More disagreement was evidenced about benefits and burdens of ANH, and lower importance was given to them. CONCLUSIONS: Some misconceptions and a low level of awareness exist among some nurses about ANH. More nursing studies should be conducted and extended to different care settings. Moreover, guidelines should be reinforced with a nursing perspective to better define nurses' role in the decision-making process on ANH. IMPLICATIONS FOR PRACTICE: This exploration of nurses' knowledge of and attitudes toward ANH in end-of-life cancer patients can contribute to an understanding of how nurses can be involved in better meeting cancer patients' nutritional needs at the end of life.

Physical, Nutritional, and Sensory Properties of Cheese Obtained from Goats Fed a Dietary Supplementation with Olive Leaves.

The aim of this study is to evaluate the physical, nutritional, and sensory properties of cheese obtained from goats fed a dietary supplementation with olive leaves (OL). Thirty Saanen goats were randomly allocated into two groups of 15 goats each, (1) a control group fed with a standard diet (CG), and (2) an experimental group (EG) fed an OL-enriched diet. The trial lasted for 30 days. The milk of each group was then collected and used to produce Caciotta cheese, which was analyzed at the beginning and at the end of the ripening period (60 days). The results showed a positive effect of dietary OL supplementation in improving the fatty acid profiles due to the significant increase of unsaturated fatty acids, mostly α-linolenic acid (C18:3 n-3), with the consequent reduction of the ω-6/ω-3 ratio, a condition commonly associated with an increased health functionality of food products. Moreover, improved oxidative stability was observed in cheese during ripening, a presumable consequence of the transfer into the milk of dietary bioactive compounds, mainly polyphenols of high biological value, and credited as a marked antioxidant potential. Furthermore, reduced lipolytic action was observed in 60-day ripened cheese, even if no significant changes in sensory properties were evidenced.

Nutrigenomic Effects of Long-Term Grape Pomace Supplementation in Dairy Cows.

The increasing demand for more animal products put pressure on improving livestock production efficiency and sustainability. In this context, advanced animal nutrition studies appear indispensable. Here, the effect of grape pomace (GP), the polyphenol-rich agricultural by-product, was evaluated on Holstein-Friesian cows' whole-blood transcriptome, milk production and composition. Two experimental groups were set up. The first one received a basal diet and served as a control, while the second one received a 7.5% GP-supplemented diet for a total of 60 days. Milk production and composition were not different between the group; however, the transcriptome analysis revealed a total of 40 genes significantly affected by GP supplementation. Among the most interesting down-regulated genes, we found the DnaJ heat-shock protein family member A1 (DNAJA1), the mitochondrial fission factor (MFF), and the impact RWD domain protein (IMPACT) genes. The gene set enrichment analysis evidenced the positive enrichment of 'interferon alpha (IFN-α) and IFN-γ response', 'IL6-JAK-STAT3 signaling' and 'complement' genes. Moreover, the functional analysis denoted positive enrichment of the 'response to protozoan' and 'negative regulation of viral genome replication' biological processes. Our data provide an overall view of the blood transcriptomic signature after a 60-day GP supplementation in dairy cows which mainly reflects a GP-induced immunomodulatory effect.

Dietary Grape Pomace Supplementation in Dairy Cows: Effect on Nutritional Quality of Milk and Its Derived Dairy Products.

Grape pomace (GP) is the main solid by-product of winemaking and represents a rich source of potent bioactive compounds which could display a wide range of beneficial effects in human health for their association with reduced risk of several chronic diseases. Several studies have proposed the use of GP as a macro-ingredient to obtain economically worthwhile animal feedstuffs naturally enriched by polyphenols and dietary fibers. Moreover, the research carried out in this field in the last two decades evidences the ability of GP to induce beneficial effects in cow milk and its derived dairy products. First of all, a general increase in concentration of polyunsaturated fatty acids (PUFA) was observed, and this could be considered the reflection of the high content of these compounds in the by-product. Furthermore, an improvement in the oxidative stability of dairy products was observed, presumably as a direct consequence of the high content of bioactive compounds in GP that are credited with high and well-characterized antioxidant functions. Last but not least, particularly in ripened cheeses, volatile compounds (VOCs) were identified, arising both from lipolytic and proteolytic processes and commonly associated with pleasant aromatic notes. In conclusion, the GP introduction in the diet of lactating cows made it possible to obtain dairy products characterized by improved nutritional properties and high health functionality. Furthermore, the presumable improvement of organoleptic properties seems to be effective in contributing to an increase in the consumer acceptability of the novel products. This review aims to evaluate the effect of the dietary GP supplementation on the quality of milk and dairy products deriving from lactating dairy cows.

Effect of dietary grape marc on fresh and refrigerated boar semen.

In several studies there has been evaluation of the dietary addition of antioxidants to improve the quality of fresh and stored semen in domestic animals. Grape marc (GM), as the residue of vinification, contains large amounts of polyphenols with antioxidant, anti-inflammatory, antimicrobial, and antiaging effects. In this study, two regimens of dietary GM supplementation (2% and 4%) were tested regarding effects on the characteristics and lipid peroxidation of fresh and stored semen. The dietary supplementation of GM improved sperm characteristics in fresh semen, especially at 4% of GM. There were greater values for all kinetic variables and membrane integrity, and lesser values for sperm abnormalities and lipid peroxidation with 2% and 4% GM supplementation. Lipid peroxidation of the pellet was less with both 2% and 4% GM supplementation. During sperm storage, the dietary supplementation of GM improved the quality of sperm, with greater values for kinetic variables and membrane integrity at day 15 of storage. In the present study, supplementation of GM in boar diets improved fresh semen characteristics and reduced the lipid peroxidation of ejaculated spermatozoa, possibly due to the effect of polyphenols present in the GM. Consequently, this likely resulted in improved sperm quality during storage.

Chemical-nutritional quality and oxidative stability of milk and dairy products obtained from Friesian cows fed with a dietary supplementation of dried grape pomace.

BACKGROUND: The aim of the study was to evaluate the nutritional properties of milk and cheese obtained from Friesian cows fed with a diet supplemented with dried grape pomace, a by-product of the oenological industry, which is very rich in polyphenols. This approach is inspired by the increasing interest in foods containing functional ingredients that may have beneficial effects on human health. During the testing period, analyses of the chemical and nutritional properties of milk and dairy products derived from it were performed; particular attention was given to the effect of cheese ripening on the oxidative stability and fermentation process, evaluating respectively the presence of malondialdehyde and γ-aminobutyric acid. RESULTS: Dietary enrichment with grape pomace did not affect the milk composition but induced modifications in the fatty acid profiles in both milk and cheese with an increase in concentration of linoleic acid, trans-vaccenic acid, rumenic acid and total n-6 fatty acids. Moreover, after 30 days of cheese ripening, an increased oxidative stability and an increased concentration of γ-aminobutyric acid were found. CONCLUSIONS: Our results indicated a general improvement in nutritional parameters of milk and related cheese obtained from Friesian cows that received the feeding enrichment with dried grape pomace. Further analysis should be performed to improve knowledge of the chemical and microbiological mechanisms at the source of these findings. © 2019 Society of Chemical Industry.

Oxaprozin: A new hope in the modulation of matrix metalloproteinase 9 activity.

Oxaprozin (4,5-diphenyl-2-oxazolepropionic acid) is a non-steroidal, analgesic and antipyretic propionic acid derivative, whose activity in treating inflammatory disorders is well known. The aim of this study was to investigate the ability of oxaprozin to modulate the activity of matrix metalloproteinase 9 (MMP-9), a zinc-dependent endopeptidase involved in a wide range of physiological and pathological events associated with extracellular matrix (ECM) remodelling. The interaction between oxaprozin and MMP-9 was firstly investigated in silico by molecular docking and analysis with LIGPLOT software. Subsequently, the potential inhibitory activity of oxaprozin against MMP-9 and the possible mechanism of the ligand-enzyme interaction were investigated in vitro. Taking into account the in silico findings, MMP-9 can be considered a potential target of oxaprozin, which seems to be able to chelate the catalytic zinc ion through the nitrogen of the oxazole ring and the carboxylate moiety. Moreover, one of the phenyl rings interact with the S1' inhibitor-binding pocket through hydrophobic interaction. Gelatin zymography and enzymatic inhibition assay confirmed the potential role of oxaprozin as a competitive inhibitor of MMP-9. These observations sound particularly interesting if we consider the pathological role of MMP-9, especially evident in inflammatory conditions and cancer. This work may represent a starting point to improve the understanding of the role of oxaprozin, as well as its structural analogues, in modulating the MMP-9 function.

Could Music Minimize Discomfort and Pain During Office-Based ENT Surgery?

BACKGROUND: Video-assisted endoscopic radiofrequency inferior turbinate volume reduction (RFVTR) is one of the most common surgical therapies for inferior turbinate hypertrophy (ITH). Despite all the technical and surgical advancement, it is advisable to reduce as low as possible the intraoperative discomfort. The aim of this study is to evaluate the role of music in reducing patient discomfort during RFVTR. MATERIALS AND METHODS: Twenty-three patients with chronic nasal obstruction due to ITH and candidate to RFVTR are included. Before the procedure each patient filled in a completed Italian version of the state anxiety questionnaire (State-Trait Anxiety Inventory), SNOT 22 questionnaire, VAS, and chose their favourite music to be played during RFVTR. All patients evaluate the intraoperative discomfort with a visual analog scale (VAS) and for each patient, vital parameters such as blood pressure and heart rate were recorded 15 minutes before the procedure, during and after RFVTR. RESULTS: The intraoperative VAS scores during listening to music (5.7 ± 2.42 vs 6.7 ± 1.97; p< 0.05) were significantly lower, such as systolic BP (133.5 ±17.2 vs 136.78 ±16.8; p< 0.05) and heat rate (80.3 ±14.9 vs 81.7 ±15.5; p NS). During our survey, most of the patients preferred listening to classical music and none preferred rock music. No correlation was found between STAI 1-2 and intraoperative surgical discomfort evaluated both with VAS and cardiac parameters (systolic BP and HR). CONCLUSIONS: Music can be useful as a complementary method to control anxiety and reduce perception of pain in an office-based procedure, such as the RFVTR. The patient is more relaxed and experiences less discomfort; thus the surgeon and nurse can work with more confidence.

RNA Sequencing-Based Whole-Transcriptome Analysis of Friesian Cattle Fed with Grape Pomace-Supplemented Diet.

Grape pomace (GPO), the main by-product of the wine making process, is a rich source of polyphenols with potent antioxidant properties. Recently, GPO has emerged as a potential feed additive in livestock nutrition, with several reports describing its beneficial effects on animals' overall health status or production traits. However, little is known about it from a molecular biology standpoint. In the present study, we report the first RNA sequencing-based whole-transcriptome profiling of Friesian calves fed with a GPO-supplemented diet. We identified 367 differentially expressed genes (p < 0.05) in the GPO-supplemented calves (n = 5), when compared with unsupplemented control group (n = 5). The pathway analysis showed that 'cholesterol lipid biosynthesis' was the most negatively-enriched (p < 0.001) pathway in the GPO-supplemented animals. In specific terms, five important genes coding for cholesterol biosynthesis enzymes, namely the Farnesyl-diphosphate Farnesyltransferase 1 (FDFT-1), Squalene Epoxidase (SQLE), NAD(P)-dependent Steroid Dehydrogenase-like (NSDHL), Methylsterol Monooxygenase (MSMO)-1, and Sterol-C5-desaturase (SC5D), two major transcription factors (the Sterol Regulatory Element-binding Transcription Factor 1 and 2), as well as the Low-Density Lipoprotein Receptor (LDLR), were all downregulated following GPO supplementation. Such an effect was mirrored by a reduction of blood cholesterol levels (p = 0.07) and a lowered (p < 0.001) Malondialdehyde (lipid oxidation marker) level in carcasses. We provide evidence on the effects of GPO-supplemented diets on the whole-transcriptome signature in veal calves, which mainly reflects an antioxidant activity.

[Validation of the Italian version of the GHAA-9 m questionnaire on patient satisfaction in digestive Endoscopy]. / Validazione della versione italiana del questionario GHAA-9 m sulla soddisfazione del paziente in endoscopia digestiva.

BACKGROUND: There isn't a validated questionnaire in Italian language to evaluate the quality perceived by the patient in Digestive Endoscopy. OBJECTIVE: validation of the translation of a questionnaire from English to Italian language to measure the level of patient satisfaction. MATERIALS AND METHODS: we conducted a prospective study on validation in Italian of a short questionnaire adapted for Endoscopy by the American Society of Gastrointestinal Endoscopy, the GHAA-9m. It's been tested with the technique of the questionnaire/interview on 80 outpatients who underwent in the month of September 2014 to endoscopic examinations of the gastrointestinal tract. The patients were divided into 2 groups of 40 patients: group 1 was administered before the questionnaire and subsequently the interview was conducted, on the contrary on the group 2 was administered before the interview and subsequently the questionnaire. The results of the two groups were compared using the inter-rater agreement. It was also evaluated the internal consistency of the questions. RESULTS: The results show that the instrument is experienced as simple and quick to use for patients. Data analysis allowed us to conclude that the Italian translation is valid and consistent. In the phase of the interview there were some aspects that suggest, in a development of this tool, some changes that could increase the accuracy and informational content. CONCLUSIONS: The Italian version of the questionnaire GHAA-9m has good validity, reliability, and shows property valuation comparable to those of the American version and can therefore be used in daily practice Digestive Endoscopy.

Impact of the latest TNM classification for gastric cancer: retrospective analysis on 94 D2 gastrectomies.

The aim of this study was to determine whether the latest edition of tumor-node-metastasis (TNM) classification provides reliable prognostic information. The fifth edition of TNM Classification of Malignant Tumors has introduced for gastric cancer the numeric count of involved lymph nodes whereas their topographic location was considered in earlier editions. For our study, data from 94 patients who underwent D2-gastrectomy were reviewed. The N-factor was scored according to both the Japanese Research Society for Gastric Cancer (JRSGC) classification (n) and, retrospectively, the latest TNM classification (N). Actuarial survival was calculated for both groups. The two staging systems showed similar stratification of actuarial survival with relation to N-stage; in the JRSGC classification no statistical differences were observed between n1 and n2 patients (62.7% vs. 52.5%; p = NS), whereas the 5th TNM classification showed a significant difference between N1 and N2 patients (68.5% vs. 45.0%; p = 0.04), and between N1 and the new category of N3 patients (68.5% vs. 45.0%, p = 0.03). It appears, therefore, that the numeric count of involved nodes may represent a more reliable indicator for single-case prognosis. Reclassification of all node-positive patients in our series caused an overall stage modification in 32.9% (31/94); 22 of those patients were reclassified to a less favorable stage (23.4%). In addition, 11.7% of patients (6/51) who were previously designated n1 were reclassified as N2, shifting from an expected actuarial survival after 72 months of 62.7% to 33.3%.