Assessment of Nontechnical Skills During Resuscitation: Validation in the Italian Version of the TEAM.

SUMMARY STATEMENT: Nontechnical skills (hereinafter referred to as NTS), such as task management, leadership, situational awareness, communication, and decision making contribute to safe and efficient team performance. The importance during cardiopulmonary resuscitation is being increasingly emphasized. We carried out the intercultural adaptation of the TEAM score in Italian and to evaluate the reliability and validity of the resulting Italian version (i-TEAM). A forward-backward translation was made with the author called i-TEAM. Psychometric properties of the i-TEAM score were evaluated, including acceptability, construct validity, and interrater reliability. We divided the participants into 3 groups based on their experience, and we verified if there was a correlation between the final score NTS of i-TEAM and the groups. The Cronbach coefficient was 0.91 for the Total i-TEAM score. The descriptive statistics showed that there was no correlation between NTS score and experience (group). Our results show that i-TEAM has psychometric properties similar to the original score.

Epidermal Growth Factor Receptor Targeting in Colorectal Carcinoma: Antibodies and Patient-Derived Organoids as a Smart Model to Study Therapy Resistance.

Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide. Therefore, the need for new therapeutic strategies is still a challenge. Surgery and chemotherapy represent the first-line interventions; nevertheless, the prognosis for metastatic CRC (mCRC) patients remains unacceptable. An important step towards targeted therapy came from the inhibition of the epidermal growth factor receptor (EGFR) pathway, by the anti-EGFR antibody, Cetuximab, or by specific tyrosine kinase inhibitors (TKI). Cetuximab, a mouse-human chimeric monoclonal antibody (mAb), binds to the extracellular domain of EGFR thus impairing EGFR-mediated signaling and reducing cell proliferation. TKI can affect the EGFR biochemical pathway at different steps along the signaling cascade. Apart from Cetuximab, other anti-EGFR mAbs have been developed, such as Panitumumab. Both antibodies have been approved for the treatment of KRAS-NRAS wild type mCRC, alone or in combination with chemotherapy. These antibodies display strong differences in activating the host immune system against CRC, due to their different immunoglobulin isotypes. Although anti-EGFR antibodies are efficient, drug resistance occurs with high frequency. Resistant tumor cell populations can either already be present before therapy or develop later by biochemical adaptations or new genomic mutations in the EGFR pathway. Numerous efforts have been made to improve the efficacy of the anti-EGFR mAbs or to find new agents that are able to block downstream EGFR signaling cascade molecules. Indeed, we examined the importance of analyzing the anti-EGFR antibody-drug conjugates (ADC) developed to overcome resistance and/or stimulate the tumor host's immunity against CRC growth. Also, patient-derived CRC organoid cultures represent a useful and feasible in vitro model to study tumor behavior and therapy response. Organoids can reflect tumor genetic heterogeneity found in the tissue of origin, representing a unique tool for personalized medicine. Thus, CRC-derived organoid cultures are a smart model for studying the tumor microenvironment and for the preclinical assay of anti-EGFR drugs.

Antibody-Drug Conjugate Made of Zoledronic Acid and the Anti-CD30 Brentuximab-Vedotin Exert Anti-Lymphoma and Immunostimulating Effects.

Relevant advances have been made in the management of relapsed/refractory (r/r) Hodgkin Lymphomas (HL) with the use of the anti-CD30 antibody-drug conjugate (ADC) brentuximab-vedotin (Bre-Ved). Unfortunately, most patients eventually progress despite the excellent response rates and tolerability. In this report, we describe an ADC composed of the aminobisphosphonate zoledronic acid (ZA) conjugated to Bre-Ved by binding the free amino groups of this antibody with the phosphoric group of ZA. Liquid chromatography-mass spectrometry, inductively coupled plasma-mass spectrometry, and matrix-assisted laser desorption ionization-mass spectrometry analyses confirmed the covalent linkage between the antibody and ZA. The novel ADC has been tested for its reactivity with the HL/CD30+ lymphoblastoid cell lines (KMH2, L428, L540, HS445, and RPMI6666), showing a better titration than native Bre-Ved. Once the HL-cells are entered, the ADC co-localizes with the lysosomal LAMP1 in the intracellular vesicles. Also, this ADC exerted a stronger anti-proliferative and pro-apoptotic (about one log fold) effect on HL-cell proliferation compared to the native antibody Bre-Ved. Eventually, Bre-Ved-ZA ADC, in contrast with the native antibody, can trigger the proliferation and activation of cytolytic activity of effector-memory Vδ2 T-lymphocytes against HL-cell lines. These findings may support the potential use of this ADC in the management of r/r HL.

EGFR-Targeted Antibody-Drug Conjugate to Different Aminobisphosphonates: Direct and Indirect Antitumor Effects on Colorectal Carcinoma Cells.

Antibody--drug conjugates (ADCs) are a promising delivery system that involves linking a monoclonal antibody (mAb) to a specific drug, such as a cytotoxic agent, to target tumor cells. This new class of antitumor therapy acts as a "biological missile" that can destroy tumor cells while increasing the therapeutic index and decreasing toxicity. One of the most critical factors in ADC design is selecting a target antigen that is highly expressed on the surface of cancer cells. In this study, we conjugated Cetuximab (Cet), a monoclonal antibody that targets the epidermal growth factor receptor (EGFR), to aminobisphosphonates (N-BPs) such as ibandronate (IBA) or risedronate (RIS) or zoledronate (ZA). Cetuximab is administered to patients with metastatic colorectal carcinoma (mCRC) with a wild-type (WT) EGFR transduction pathway. Also, it is well established that N-BPs can trigger the antitumor activity of Vδ2 T cells in both in vitro and in vivo experimental models. The resulting ADCs were added in co-culture to assess the effect on CRC cell line proliferation and sensitivity to Vδ2 T antitumor lymphocytes in comparison with the native antibody. These assays have been performed both in conventional and 3D spheroid cultures. We found that all three ADCs can increase the inhibitory effect on cell proliferation of the WT-EGFR cell line Caco-2 while only Cet-RIS and Cet-ZA can increase the cytotoxicity mediated by Vδ2 T cells against both WT and EGFR-mutated CRC cell lines (Caco-2, DLD-1, and HCT-116). Also, the ADCs can trigger the cell proliferation of Vδ2 T cells present in peripheral blood and tumor specimens. Our findings indicate that anti-EGFR antibodies bound to N-BPs can improve the antitumor effects of the native antibody possibly increasing the therapeutic effect.

Factors Associated with SARS-CoV-2 Infection Evaluated by Antibody Response in a Sample of Workers from the Emilia-Romagna Region, Northern Italy.

Factors associated with SARS-CoV-2 infection risk are still debated. This case-control study aims to investigate the possible relationship between SARS-CoV-2 infection, evaluated through antibody response, and the main sociodemographic, occupational, clinical-anamnestic, and biochemical factors in a population of Modena province (Northern Italy), mainly workers. Both workers who voluntarily joined the screening campaign proposed by companies and self-referred individuals who underwent serological testing were enrolled. Subjects with antibody positivity were recruited as cases (n = 166) and subjects tested negative (n = 239) as controls. A questionnaire on sociodemographic, occupational, and clinical data was administered through telephone interviews. Serum zinc/iron/copper/chromium/nickel, vitamins D/B12, folates, triglycerides, and LDL/HDL/total cholesterol were measured. Cases lived more often in urban areas (61.8% vs. 57%). Cases and controls did not differ significantly by working macrocategories, but the percentage of workers in the ceramic sector was higher among cases. Low adherence to preventive measures in the workplace was more frequent among seropositives. Folate concentration was significantly lower among cases. Therefore, adequate folate levels, living in rural areas, and good adherence to preventive strategies seem protective against infection. Workers in the ceramic sector seem to be at greater risk; specific factors involved are not defined, but preventive interventions are needed.

Alarm Fatigue in Nursing Students Undertaking Clinical Training in Intensive Care Units: A Multicenter Study.

BACKGROUND: The frequency of alarms from monitors and other electro-medical devices is of great utility but can increase the professional's workload and expose nurses in the intensive care unit to Alarm Fatigue. A recent study suggested that students in training can also experience the problem during their first clinical experiences in intensive care. Unfortunately, no data are available about the Italian panorama. To explore Alarm Fatigue among Bachelor of Science in Nursing students at the end of their internship experience in intensive care settings. METHODS: Multicenter cross-sectional design. A convenience sample of nurses from 3 Italian university hospitals was recruited. The students completed the revised version of the "Alarm Fatigue questionnaire-ita" at the end of the clinical internship in intensive care settings. RESULTS: 130 nursing students were enrolled (response rate 59.36%). The overall level of Alarm Fatigue was Me= 24.5 IQR [17.5, 30.5]. In addition, 9.23% of the sample reported errors or near misses related to Alarm Fatigue during the internship experience. The alarm fatigue level was higher in students who committed "errors/almost errors" (p=0.038) and in "student workers" (p=0.005). DISCUSSION: The extent of alarm fatigue experienced by nursing students requires developing a preventive strategy.

SB202190 Predicts BRAF-Activating Mutations in Primary Colorectal Cancer Organoids via Erk1-2 Modulation.

The p38 inhibitor SB202190 is a necessary component of the medium used for normal colorectal mucosa cultures. Sato et al. suggested that the primary activity of SB202190 may be EGFR signaling stabilization, causing an increased phosphorylation of Erk1-2 sustaining organoid proliferation. However, the growth of some colorectal cancer (CRC)-derived organoid cultures is inhibited by this molecule via an unknown mechanism. We biochemically investigated SB202190 activity on a collection of 25 primary human CRC organoids, evaluating EGFR, Akt and Erk1-2 activation using Western blot. We found that Erk1-2 phosphorylation was induced by SB202190 in 20 organoid cultures and inhibited in 5 organoid cultures. A next-generation sequencing (NGS) analysis revealed that the inhibition of p-Erk1-2 signaling corresponded to the cultures with BRAF mutations (with four different hits, one being undescribed), while p-Erk1-2 induction was apparently unrelated to other mutations involving the EGFR pathway (Her2, KRAS and NRAS). We found that SB202190 mirrored the biochemical activity of the BRAF inhibitor Dabrafenib, known to induce the paradoxical activation of p-Erk1-2 signaling in BRAF wild-type cells. SB202190 was a more effective inhibitor of BRAF-mutated organoid growth in the long term than the specific BRAF inhibitors Dabrafenib and PLX8394. Overall, SB202190 can predict BRAF-activating mutations in patient-derived organoids, as well as allowing for the identification of new BRAF variants, preceding and enforcing NGS data.

Priming of Colorectal Tumor-Associated Fibroblasts with Zoledronic Acid Conjugated to the Anti-Epidermal Growth Factor Receptor Antibody Cetuximab Elicits Anti-Tumor Vδ2 T Lymphocytes.

Tumor-associated fibroblasts (TAF) exert immunosuppressive effects in colorectal carcinoma (CRC), impairing the recognition of tumor cells by effector lymphocytes, including Vδ2 T cells. Herein, we show that CRC-derived TAF can be turned by zoledronic acid (ZA), in soluble form or as antibody-drug conjugate (ADC), into efficient stimulators of Vδ2 T cells. CRC-TAF, obtained from patients, express the epidermal growth factor receptor (EGFR) and the butyrophilin family members BTN3A1/BTN2A1. These butyrophilins mediate the presentation of the phosphoantigens, accumulated in the cells due to ZA effect, to Vδ2 T cells. CRC-TAF exposed to soluble ZA acquired the ability to trigger the proliferation of Vδ2 T cells, in part represented by effector memory cells lacking CD45RA and CD27. In turn, expanded Vδ2 T cells exerted relevant cytotoxic activity towards CRC cells and CRC-TAF when primed with soluble ZA. Of note, also the ADC made of the anti-EGFR cetuximab (Cet) and ZA (Cet-ZA), that we recently described, induced the proliferation of anti-tumor Vδ2 T lymphocytes and their activation against CRC-TAF. These findings indicate that ZA can educate TAF to stimulate effector memory Vδ2 T cells; the Cet-ZA ADC formulation can lead to the precise delivery of ZA to EGFR+ cells, with a double targeting of TAF and tumor cells.

microRNAs in Liquid Biopsy: The Way to a Simple and Rapid Test for Early Colorectal Cancer Diagnosis?

About 15% of colorectal cancers (CRCs) are diagnosed as advanced, metastatic stage IV, a patient condition with an average survival of 2 [...].

Immune Checkpoint Receptor/Ligand Expression and Chemotherapy in Colorectal Cancer.

Immune checkpoint (IC) molecules act as receptors, expressed on immune effector cells, that are able to recognize specific ligands in normal or tumor cells [...].

Evolution of 3D Cultures: Toward Tailored Preclinical Models.

The identification and validation of simple, reliable and reproducible three dimensional (3D) in vitro culture systems represent a major challenge in the field of anticancer drug development [...].

The In Vitro Adaptation of Patient-Derived Organoids Suggests Alternative Strategies against CMS1 Colorectal Cancer: When the Microenvironment Does Make the Difference.

Colorectal cancer (CRC) is a relatively slow-growing tumor that can be treated successfully when identified in the early stages [...].

10th Anniversary of Cells: Advances in Cellular Immunology-Regulation of Autoimmune Response and Antitumor Reactivity: Are They Two Side of the Same Coin?

The innate and adaptive arms of the immune system are involved in maintaining organism homeostasis [...].

Targeting of colorectal cancer organoids with zoledronic acid conjugated to the anti-EGFR antibody cetuximab.

BACKGROUND: Antibody-drug conjugates (ADC) are essential therapeutic options to treat solid and hematological cancers. The anti-epidermal growth factor-receptor (EGFR) antibody cetuximab (Cet) is used for the therapy of colorectal carcinoma (CRC). Anti-CRC Vδ2 cytolytic T lymphocytes can be elicited by the priming of tumor cells with the aminobisphosphonate zoledronic acid (ZA) and consequent presentation of isopentenyl pyrophosphates through butyrophilin (BTN) family members such as BTN3A1 and BTN2A1. A major drawback that impairs the targeting of ZA to CRC is the bone tropism of aminobisphosphonates. METHODS: The phosphoric group of ZA was linked to free amino groups of Cet in the presence of imidazole following the labeling of phosphoric groups of DNA to amino groups of proteins. The generation of Cet-ZA ADC was confirmed by matrix assisted laser desorption ionization mass spectrometry and inductively coupled plasma-mass spectrometry analysis. Thirteen CRC organoids were obtained with a chemically defined serum-free medium in Geltrex domes. Proliferation and activation of cytolytic activity against CRC organoids by Vδ2 T cells was detected with flow cytometry, crystal violet and cytotoxic probe assays and image analysis. Immunohistochemistry and quantification of BTN3A1 or BTN2A1 expression and the number of tumor infiltrating Vδ2 T cells in CRC were performed by automatic immunostaining, whole slide scanning and computerized analysis of digital pathology imaging. RESULTS: The novel ADC Cet-ZA was generated with a drug antibody ratio of 4.3 and displayed a reactivity similar to the unconjugated antibody. More importantly, patient-derived CRC organoids, or CRC tumor cell suspensions, could trigger the expansion of Vδ2 T cells from peripheral blood and tumor infiltrating lymphocytes when primed with Cet-ZA. Furthermore, Cet-ZA triggered Vδ2 T cell-mediated killing of CRC organoids. The expression of BTN3A1 and BTN2A1 was detected not only in CRC organoids but also in CRC specimens, together with a considerable amount of tumor infiltrating Vδ2 T cells. CONCLUSIONS: These findings are proof of concept that the Cet-ZA ADC can be used to target specifically CRC organoids and may suggest a new experimental approach to deliver aminobisphosphonates to EGFR+ solid tumors.

Mesenchymal Stem/Stromal Cells as a Therapeutic Tool in Cell-Based Therapy and Regenerative Medicine: An Introduction Expertise to the Topical Collection.

We are pleased to present this opening editorial, introducing our topical collection, "The New Era of Mesenchymal Stromal/Stem Cell Functional Application: State of the Art, Therapeutic Challenges and Future Directions" [...].

A multi-organ-on-chip to recapitulate the infiltration and the cytotoxic activity of circulating NK cells in 3D matrix-based tumor model.

The success of immunotherapeutic approaches strictly depends on the immune cells interaction with cancer cells. While conventional in vitro cell cultures under-represent the complexity and dynamic crosstalk of the tumor microenvironment, animal models do not allow deciphering the anti-tumor activity of the human immune system. Therefore, the development of reliable and predictive preclinical models has become crucial for the screening of immune-therapeutic approaches. We here present an organ-on-chip organ on chips (OOC)-based approach for recapitulating the immune cell Natural Killer (NK) migration under physiological fluid flow, infiltration within a 3D tumor matrix, and activation against neuroblastoma cancer cells in a humanized, fluid-dynamic environment. Circulating NK cells actively initiate a spontaneous "extravasation" process toward the physically separated tumor niche, retaining their ability to interact with matrix-embedded tumor cells, and to display a cytotoxic effect (tumor cell apoptosis). Since NK cells infiltration and phenotype is correlated with prognosis and response to immunotherapy, their phenotype is also investigated: most importantly, a clear decrease in CD16-positive NK cells within the migrated and infiltrated population is observed. The proposed immune-tumor OOC-based model represents a promising approach for faithfully recapitulating the human pathology and efficiently employing the immunotherapies testing, eventually in a personalized perspective. An immune-organ on chip to recapitulate the tumor-mediated infiltration of circulating immune cells within 3D tumor model.

Seroprevalence Survey of Anti-SARS-CoV-2 Antibodies in a Population of Emilia-Romagna Region, Northern Italy.

Italy was the first Western European country to be severely hit by the COVID-19 pandemic. Variations in seroprevalence rates were reported according to geographical and temporal differences of previous surveys, as well as depending on demographic and occupational factors. In this cross-sectional study, we evaluated the prevalence of anti-SARS-CoV-2 antibodies in a population of the Emilia-Romagna region in Northern Italy after the first wave in the period from 26 September 2020−26 March 2021. We included 5128 subjects who voluntarily underwent serological tests to determine anti-SARS-CoV-2 antibody positivity, including both self-referred individuals (24.2%) and workers adhering to company screening programs (76.8%). Overall, seroprevalence was 11.3%, higher in self-referred (13.8%) than employed-referred (10.5%) individuals. A slightly higher seroprevalence emerged in women compared to men (12.3% and 10.7%), as well as in the extreme age categories (18.6% for 60−69 years, 18.0% for ≥70 years, and 17.1% for <20 years compared to 7.6% for 20−39 years). Healthcare professionals showed the highest prevalence of seropositivity (22.9%), followed by workers in direct contact with customers, such as the communication, finance, and tourism sectors (15.7%). Overall subgroups seroprevalence increased compared to the first wave data but the trends agreed between the first and subsequent waves, except for an increase in the younger age group and in the sector in direct contact with customers. Among the occupational categories, our study confirms that healthcare workers and workers in the sports sector were at high risk of exposure to SARS-CoV-2.

Generation of Tumor Spheroids to Evaluate T Cell and NK Cell Cytotoxicity.

Tumor cell spheroids are one of the three-dimensional (3D) culture systems that can be used to study the features of tumor cells in a more physiologic condition. Indeed, this 3D culture system has been validated in preclinical studies to select anticancer drugs. Tumor cell spheroids can be obtained through employing different procedures, and here I describe in detail how to obtain spheroids from tumor cell lines of colorectal carcinoma (CRC). I analyze the procedures employed to evaluate the phenotype and growth of tumor cells in this 3D culture system. Also, interaction with immune cells is considered. Indeed, it is relevant to define whether the antitumor effects exerted by different cytotoxic lymphocyte subsets are similar when tumor cells are used either as cells adherent to a plastic substrate or floating spheroids. To obtain optimal results in this complex system, some parameters must be considered, such as those related to poorly defined experimental variables including biological and biophysical features of tumor cell lines and the quality (purity and time to use after generation) of antitumor effector lymphocyte subsets. Also, I describe in detail the methods to generate these lymphocyte subsets and to characterize their cytotoxic potential and effectiveness in killing tumor cells. Human natural killer cells and T lymphocytes expressing the γδ T cell receptor (in particular Vδ2 T cells) are considered among the cytotoxic lymphocyte populations. Eventually, it should be possible to obtain reliable and feasible results to study the molecular mechanisms involved in recognition and killing of CRC spheroids exerted by effector lymphocytes. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Generation of tumor cell spheroids Basic Protocol 2: Evaluation of ATP content of spheroids Basic Protocol 3: Evaluation of antigen expression by cell spheroids Basic Protocol 4: Generation of effector lymphocytes Basic Protocol 5: Coculture of spheroids and lymphocytes Basic Protocol 6: Evaluation of lymphocyte cytotoxicity using crystal violet staining.

Comparison of Automated Ribotyping, spa Typing, and MLST in 108 Clinical Isolates of Staphylococcus aureus from Orthopedic Infections.

108 isolates of Staphylococcus aureus, belonging to six large ribogroups according to the automated Ribo-Printer® system, were studied with two highly used molecular methods for epidemiological studies, namely multi-locus sequence typing (MLST) and spa typing, followed by BURP and eBURST v3 analysis for clustering spa types and sequence (ST) types. The aim was to evaluate whether automated ribotyping could be considered a useful screening tool for identifying S. aureus genetic lineages with respect to spa typing and MLST. Clarifying the relationship of riboprinting with these typing methods and establishing whether ribogroups fit single clonal complexes were two main objectives. Further information on the genetic profile of the isolates was obtained from agr typing and the search for the mecA, tst genes, and the IS256 insertion sequence. Automated ribotyping has been shown to predict spa clonal complexes and MLST clonal complexes. The high cost and lower discriminatory power of automated ribotyping compared to spa and MSLT typing could be an obstacle to fine genotyping analyzes, especially when high discriminatory power is required. On the other hand, numerous advantages such as automation, ease and speed of execution, stability, typeability and reproducibility make ribotyping a reliable method to be juxtaposed to gold standard methods.