DNA base editing corrects common Hemophilia A mutations and restores factor VIII expression in vitro and ex-vivo models.

BACKGROUND: Replacement and non-replacement therapies effectively control bleedings in Hemophilia A (HA) but imply lifelong interventions. The authorized gene addition therapy could provide a cure but still poses questions on durability. F8 gene correction would definitively restore factor VIII (FVIII) production, as shown in animal models through nucleases mediating homologous recombination (HR). However, low efficiency and potential off-target double-strand break (DSB) still limit HR translatability. OBJECTIVES: To correct common model single point mutations leading to severe HA through the recently developed DSB/HR-independent base (BE) and prime (PE) editing approaches. METHODS: Screening for efficacy of BE/PE systems in HEK293T transiently expressing FVIII variants and validation at DNA (sequencing) and protein (ELISA; aPTT) level in stable clones. Evaluation of rescue in engineered blood outgrowth endothelial cells (BOEC) by lentiviral-mediated delivery of BE. RESULTS AND CONCLUSIONS: Transient assays identified the best-performing BE/PE systems for each variant, with the highest rescue of FVIII expression (up to 25% of rFVIIIwt) for the p.R2166* and p.R2228Q mutations. In stable clones we demonstrated that the mutation reversion on DNA (∼24%) was consistent with the rescue of FVIII secretion and activity 20-30%). The lentiviral-mediated delivery of the selected BE systems was attempted in engineered BOEC harboring the p.R2166* and p.R2228Q variants, which led to an appreciable and dose-dependent rescue of secreted functional FVIII. Overall data provide the first proof-of-concept for effective BE/PE-mediated correction of HA-causing mutations, which encourage studies in mouse models to develop a personalized cure for large cohorts of patients though a single intervention.

Efficacy of rehabilitation interventions on functioning in patients with hemophilic arthropathy: A systematic review with meta-analysis.

BACKGROUND: Hemophilic arthropathy is a detrimental condition that crucially affects functional outcomes in hemophilic patients. In recent years, due to the advances in systemic therapies, growing attention has been raised in the rehabilitation field in order to improve functional outcomes of hemophilic patients. However, the optimal rehabilitation modalities in these patients are far from being fully characterized. OBJECTIVE: The present study aimed to assess the effects of different rehabilitation interventions on physical functioning and health-related quality of life of hemophilic arthropathic patients. METHODS: The review followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement. Five databases were systematically searched for randomized controlled trials (RCTs) published until June 22nd, 2023. The selection criteria included adult patients with hemophilia A and B receiving rehabilitation interventions. The outcomes were muscle strength, physical function, pain intensity, physical performance, and health-related quality of life. RESULTS: Out of 1,743 identified records, 17 studies were included in the qualitative synthesis. Rehabilitation interventions were categorized into exercise intervention, fascial therapy, and multimodal intervention. The findings suggested positive outcomes in terms of muscle modifications, range of motion improvements, joint health enhancements, pain intensity reduction, and quality of life improvements. More in detail, meta-analyses showed significant improvements in pain intensity [ES: -1.10 cm (-1.37, -0.82), p< 0.00001], joint health [ES: -1.10 (-1.38, -0.82), p< 0.00001], In accordance, exercise interventions showed significant benefits in terms of joint health [ES: -2.54 (-3.25, -1.83), p< 0.00001)] and quality of life [ES: 1.17 (0.48, 1.86), p< 0.0000)]. CONCLUSION: Rehabilitation interventions have a positive impact on functional outcomes and health-related quality of life of hemophilic arthropathic patients. Further studies are needed to better elucidate the role of a comprehensive intervention combining different rehabilitation approaches to treat hemophilic arthropathy.

GP64-pseudotyped lentiviral vectors target liver endothelial cells and correct hemophilia A mice.

Lentiviral vectors (LV) are efficient vehicles for in vivo gene delivery to the liver. LV integration into the chromatin of target cells ensures their transmission upon proliferation, thus allowing potentially life-long gene therapy following a single administration, even to young individuals. The glycoprotein of the vesicular stomatitis virus (VSV.G) is widely used to pseudotype LV, as it confers broad tropism and high stability. The baculovirus-derived GP64 envelope protein has been proposed as an alternative for in vivo liver-directed gene therapy. Here, we perform a detailed comparison of VSV.G- and GP64-pseudotyped LV in vitro and in vivo. We report that VSV.G-LV transduced hepatocytes better than GP64-LV, however the latter showed improved transduction of liver sinusoidal endothelial cells (LSEC). Combining GP64-pseudotyping with the high surface content of the phagocytosis inhibitor CD47 further enhanced LSEC transduction. Coagulation factor VIII (FVIII), the gene mutated in hemophilia A, is naturally expressed by LSEC, thus we exploited GP64-LV to deliver a FVIII transgene under the control of the endogenous FVIII promoter and achieved therapeutic amounts of FVIII and correction of hemophilia A mice.

Hemostasis and endothelial functionality: the double face of coagulation factors.

Hemostasis is a sophisticated sequence of events aimed to repair vessel injury. This process occurs in combination with angiogenesis, which leads to new blood vessel formation helping in the wound repair and facilitating tissue healing. The fine mechanisms that regulate hemostasis and angiogenesis are well described, but for long time, coagulation factors (CFs) have been considered merely players in the coagulation cascade. However, several experimental evidences highlight the crucial functions of these CFs in regulating endothelial functionality, especially in the angiogenic process. Some of these CFs (e.g. thrombin and tissue factor) have been widely investigated and have been described to trigger intracellular signaling related to endothelial cell (EC) functionality. For others (e.g. factor VIII and thrombomodulin), potential receptors and molecular mechanisms have not been fully elucidated but some data show their potential to induce EC response. This review focuses on the emerging roles of selected CFs in regulating EC functions, especially highlighting their ability to activate signaling pathways involved in the angiogenesis, migration, proliferation and endothelial barrier stability.

Immune tolerance promotion by LSEC-specific lentiviral vector-mediated expression of the transgene regulated by the stabilin-2 promoter.

Liver sinusoidal endothelial cells (LSECs) are specialized endocytic cells that clear the body from blood-borne pathogens and waste macromolecules through scavenger receptors (SRs). Among the various SRs expressed by LSECs is stabilin-2 (STAB2), a class H SR that binds to several ligands, among which endogenous coagulation products. Given the well-established tolerogenic function of LSECs, we asked whether the STAB2 promoter (STAB2p) would enable us to achieve LSEC-specific lentiviral vector (LV)-mediated transgene expression, and whether the expression of this transgene would be maintained over the long term due to tolerance induction. Here, we show that STAB2p ensures LSEC-specific green fluorescent protein (GFP) expression by LV in the absence of a specific cytotoxic CD8+ T cell immune response, even in the presence of GFP-specific CD8+ T cells, confirming the robust tolerogenic function of LSECs. Finally, we show that our delivery system can partially and permanently restore FVIII activity in a mouse model of severe hemophilia A without the formation of anti-FVIII antibodies. Overall, our findings establish the suitability of STAB2p for long-term LSEC-restricted expression of therapeutic proteins, such as FVIII, or to achieve antigen-specific immune tolerance in auto-immune diseases.

Hydrothermal Transformation of Eggshell Calcium Carbonate into Apatite Micro-Nanoparticles: Cytocompatibility and Osteoinductive Properties.

The eggshell is a biomineral consisting of CaCO3 in the form of calcite phase and a pervading organic matrix (1-3.5 wt.%). Transforming eggshell calcite particles into calcium phosphate (apatite) micro-nanoparticles opens the door to repurposing the eggshell waste as materials with potential biomedical applications, fulfilling the principles of the circular economy. Previous methods to obtain these particles consisted mainly of two steps, the first one involving the calcination of the eggshell. In this research, direct transformation by a one-pot hydrothermal method ranging from 100-200 °C was studied, using suspensions with a stoichiometric P/CaCO3 ratio, K2HPO4 as P reagent, and eggshells particles (Ø < 50 µm) both untreated and treated with NaClO to remove surface organic matter. In the untreated group, the complete conversion was achieved at 160 °C, and most particles displayed a hexagonal plate morphology, eventually with a central hole. In the treated group, this replacement occurred at 180 °C, yielding granular (spherulitic) apatite nanoparticles. The eggshell particles and apatite micro-nanoparticles were cytocompatible when incubated with MG-63 human osteosarcoma cells and m17.ASC murine mesenchymal stem cells and promoted the osteogenic differentiation of m17.ASC cells. The study results are useful for designing and fabricating biocompatible microstructured materials with osteoinductive properties for applications in bone tissue engineering and dentistry.

A sustainable one-pot method to transform seashell waste calcium carbonate to osteoinductive hydroxyapatite micro-nanoparticles.

We have developed a straightforward, one-pot, low-temperature hydrothermal method to transform oyster shell waste particles (bCCP) from the species Crassostrea gigas (Mg-calcite, 5 wt% Mg) into hydroxyapatite (HA) micro/nanoparticles. The influence of the P reagents (H3PO4, KH2PO4, and K2HPO4), P/bCCP molar ratios (0.24, 0.6, and 0.96), digestion temperatures (25-200 °C), and digestion times (1 week-2 months) on the transformation process was thoroughly investigated. At 1 week, the minimum temperature to yield the full transformation significantly reduced from 160 °C to 120 °C when using K2HPO4 instead of KH2PO4 at a P/bCCP ratio of 0.6, and even to 80 °C at a P/bCCP ratio of 0.96. The transformation took place via a dissolution-reprecipitation mechanism driven by the favorable balance between HA precipitation and bCCP dissolution, due to the lower solubility product of HA than that of calcite at any of the tested temperatures. Both the bCCP and the derived HA particles were cytocompatible for MG-63 human osteosarcoma cells and m17.ASC murine mesenchymal stem cells, and additionally, they promoted the osteogenic differentiation of m17.ASC, especially the HA particles. Because of their physicochemical features and biological compatibility, both particles could be useful osteoinductive platforms for translational applications in bone tissue engineering.

Macrophage Reprogramming via the Modulation of Unfolded Protein Response with siRNA-Loaded Magnetic Nanoparticles in a TAM-like Experimental Model.

New therapeutic strategies are required in cancer therapy. Considering the prominent role of tumor-associated macrophages (TAMs) in the development and progression of cancer, the re-education of TAMs in the tumor microenvironment (TME) could represent a potential approach for cancer immunotherapy. TAMs display an irregular unfolded protein response (UPR) in their endoplasmic reticulum (ER) to endure environmental stress and ensure anti-cancer immunity. Therefore, nanotechnology could be an attractive tool to modulate the UPR in TAMs, providing an alternative strategy for TAM-targeted repolarization therapy. Herein, we developed and tested polydopamine-coupled magnetite nanoparticles (PDA-MNPs) functionalized with small interfering RNAs (siRNA) to downregulate the protein kinase R (PKR)-like ER kinase (PERK) expression in TAM-like macrophages derived from murine peritoneal exudate (PEMs). After the evaluation of the cytocompatibility, the cellular uptake, and the gene silencing efficiency of PDA-MNPs/siPERK in PEMs, we analyzed their ability to re-polarize in vitro these macrophages from M2 to the M1 inflammatory anti-tumor phenotype. Our results indicate that PDA-MNPs, with their magnetic and immunomodulator features, are cytocompatible and able to re-educate TAMs toward the M1 phenotype by PERK inhibition, a UPR effector contributing to TAM metabolic adaptation. These findings can provide a novel strategy for the development of new tumor immunotherapies in vivo.

Boosting intracellular sodium selectively kills hepatocarcinoma cells and induces hepatocellular carcinoma tumor shrinkage in mice.

Pharmacological treatments for advanced hepatocellular carcinoma (HCC) have a partial efficacy. Augmented Na+ content and water retention are observed in human cancers and offer unexplored targets for anticancer therapies. Na+ levels are evaluated upon treatments with the antibiotic cation ionophore Monensin by fluorimetry, ICP-MS, 23Na-MRI, NMR relaxometry, confocal or time-lapse analysis related to energy production, water fluxes and cell death, employing both murine and human HCC cell lines, primary murine hepatocytes, or HCC allografts in NSG mice. Na+ levels of HCC cells and tissue are 8-10 times higher than that of healthy hepatocytes and livers. Monensin further increases Na+ levels in HCC cells and in HCC allografts but not in primary hepatocytes and in normal hepatic and extrahepatic tissue. The Na+ increase is associated with energy depletion, mitochondrial Na+ load and inhibition of O2 consumption. The Na+ increase causes an enhancement of the intracellular water lifetime and death of HCC cells, and a regression and necrosis of allograft tumors, without affecting the proliferating activity of either HCCs or healthy tissues. These observations indicate that HCC cells are, unlike healthy cells, energetically incapable of compensating and surviving a pharmacologically induced Na+ load, highlighting Na+ homeostasis as druggable target for HCC therapy.

A tryptophan metabolite prevents depletion of circulating endothelial progenitor cells in systemic low-grade inflammation.

Background: Chronic systemic inflammation reduces the bioavailability of circulating endothelial progenitor cells (EPCs). Indoleamine 2,3-dioxygenase 1 (IDO1), a key enzyme of immune tolerance catalyzing the initial step of tryptophan degradation along the so-called l-kynurenine (l-kyn) pathway, that is induced by inflammatory stimuli and exerts anti-inflammatory effects. A specific relationship between IDO1 activity and circulating EPC numbers has not yet been investigated. Methods: In this study, circulating EPCs were examined in mice treated with low doses of lipopolysaccharide (LPS) to mimic low-grade inflammation. Moreover, the association between IDO1 activity and circulating EPCs was studied in a cohort of 277 patients with variable systemic low-grade inflammation. Results: Repeated low doses of LPS caused a decrease in circulating EPCs and l-kyn supplementation, mimicking IDO1 activation, significantly increased EPC numbers under homeostatic conditions preventing EPC decline in low-grade endotoxemia. Accordingly, in patients with variable systemic low-grade inflammation, there was a significant interaction between IDO1 activity and high-sensitivity C-reactive protein (hs-CRP) in predicting circulating EPCs, with high hs-CRP associated with significantly lower EPCs at low IDO1 activity but not at high IDO1 activity. Interpretation: Overall, these findings demonstrate that systemic low-grade inflammation reduces circulating EPCs. However, high IDO1 activity and l-kyn supplementation limit circulating EPC loss in low-grade inflammation.

Directed self-assembly of a xenogeneic vascularized endocrine pancreas for type 1 diabetes.

Intrahepatic islet transplantation is the standard cell therapy for ß cell replacement. However, the shortage of organ donors and an unsatisfactory engraftment limit its application to a selected patients with type 1 diabetes. There is an urgent need to identify alternative strategies based on an unlimited source of insulin producing cells and innovative scaffolds to foster cell interaction and integration to orchestrate physiological endocrine function. We previously proposed the use of decellularized lung as a scaffold for ß cell replacement with the final goal of engineering a vascularized endocrine organ. Here, we prototyped this technology with the integration of neonatal porcine islet and healthy subject-derived blood outgrowth endothelial cells to engineer a xenogeneic vascularized endocrine pancreas. We validated ex vivo cell integration and function, its engraftment and performance in a preclinical model of diabetes. Results showed that this technology not only is able to foster neonatal pig islet maturation in vitro, but also to perform in vivo immediately upon transplantation and for over 18 weeks, compared to normal performance within 8 weeks in various state of the art preclinical models. Given the recent progress in donor pig genetic engineering, this technology may enable the assembly of immune-protected functional endocrine organs.

Cardiac Differentiation Promotes Focal Adhesions Assembly through Vinculin Recruitment.

Cells of the cardiovascular system are physiologically exposed to a variety of mechanical forces fundamental for both cardiac development and functions. In this context, forces generated by actomyosin networks and those transmitted through focal adhesion (FA) complexes represent the key regulators of cellular behaviors in terms of cytoskeleton dynamism, cell adhesion, migration, differentiation, and tissue organization. In this study, we investigated the involvement of FAs on cardiomyocyte differentiation. In particular, vinculin and focal adhesion kinase (FAK) family, which are known to be involved in cardiac differentiation, were studied. Results revealed that differentiation conditions induce an upregulation of both FAK-Tyr397 and vinculin, resulting also in the translocation to the cell membrane. Moreover, the role of mechanical stress in contractile phenotype expression was investigated by applying a uniaxial mechanical stretching (5% substrate deformation, 1 Hz frequency). Morphological evaluation revealed that the cell shape showed a spindle shape and reoriented following the stretching direction. Substrate deformation resulted also in modification of the length and the number of vinculin-positive FAs. We can, therefore, suggest that mechanotransductive pathways, activated through FAs, are highly involved in cardiomyocyte differentiation, thus confirming their role during cytoskeleton rearrangement and cardiac myofilament maturation.

Therapeutic potential of fetal liver cell transplantation in hemophilia A mice.

Hemophilia A (HA) cell therapy approaches in pediatric individuals require suitable factor (F)VIII-producing cells for stable engraftment. Liver sinusoidal endothelial cells (LSEC) and hematopoietic stem cells (HSC) have been demonstrated to be suitable for the treatment of adult HA mice. However, after transplantation in busulfan (BU)-conditioned newborn mice, adult LSEC/HSC cannot efficiently engraft, while murine fetal liver (FL) hemato/vascular cells from embryonic day 11-13 of gestation (E11-E13), strongly engraft the hematopoietic and endothelial compartments while also secreting FVIII. Our aim was to investigate the engraftment of FL cells in newborn HA mice to obtain a suitable "proof of concept" for the development of a new HA treatment in neonates. Hence, we transplanted FL E11 or E13 cells and adult bone marrow (BM) cells into newborn HA mice with or without BU preconditioning. Engraftment levels and FVIII activity were assessed starting from 6 weeks after transplantation. FL E11-E13+ BU transplanted newborns reached up to 95% engraftment with stable FVIII activity levels observed for 16 months. FL E13 cells showed engraftment ability even in the absence of BU preconditioning, while FL E11 cells did not. BM BU transplanted newborn HA mice showed high levels of engraftment; nevertheless, in contrast to FL cells, BM cells cannot engraft HA newborns in BU non-conditioning regimen. Finally, none of the transplanted mice developed anti-FVIII antibodies. Overall, this study sheds some light on the therapeutic potential of healthy FL cells in the cure of HA neonatal/pediatric patients.

CD14+/CD31+ monocytes expanded by UM171 correct hemophilia A in zebrafish upon lentiviral gene transfer of factor VIII.

Emerging gene therapy clinical trials test the correction of hemophilia A (HA) by replacing factor VIII (FVIII) in autologous hematopoietic stem cells (HSCs). Although it is known that platelets, monocyte/macrophages, and mesenchymal stromal cells can secrete transgenic FVIII, a systematic examination of blood lineages as extrahepatic sources of FVIII, to our knowledge, has not yet been performed. In this study, we sought to provide a comprehensive map of native and lentivirus-based transgenic FVIII production from HSC stage to mature blood cells, through a flow cytometry analysis. In addition, we generated a model of transient HA in zebrafish based on antisense RNA, to assess the corrective potential of the FVIII-transduced HSCs. We discovered that FVIII production begins at the CD34+ progenitor stage after cytokine stimulation in culture. Among all mature white blood cells, monocytes are the largest producers of native FVIII and can maintain protein overexpression during differentiation from HSCs when transduced by a FVIII lentiviral vector. Moreover, the addition of the HSC self-renewal agonist UM171 to CD34+ cells during transduction expanded a subpopulation of CD14+/CD31+ monocytes with excellent ability to carry the FVIII transgene, allowing the correction of HA phenotype in zebrafish. Finally, the HA zebrafish model showed that f8 RNA is predominantly localized in the hematopoietic system at the larval stage, which indicates a potential contributory role of FVIII in hematopoiesis that warrants further investigation. We believe that this study may be of broad interest to hematologists and researchers striving to advance knowledge and permanent treatments for patients with HA.

Gut Microbiota Association with Diverticular Disease Pathogenesis and Progression: A Systematic Review.

INTRODUCTION: Growing evidence supports the role of the intestinal microbiome in the development of different intestinal and extraintestinal diseases. Diverticular disease (DD) is one of the most common disorders in western countries. In the last years, different articles have suggested a possible role of the intestinal microbiome in DD pathogenesis and in the development of acute diverticulitis (AD). This systematic review aimed to clarify the current knowledge on the role of the intestinal microbiome in colonic diverticulitis in different stages according to the 2009 PRISMA guidelines. MATERIALS AND METHODS: Two independent reviewers searched the literature in a systematic manner through online databases, including Medline, Scopus, Embase, Cochrane Oral Health Group Specialized Register, ProQuest Dissertations and Theses Database, and Google Scholar. Patients with any stage of disease were included. The Newcastle-Ottawa scale for case-control and cohort studies was used for the quality assessment of the selected articles. RESULTS: Overall, nine studies were included in the review. Only one article was focused on patients with AD, while all other articles only considered patients with DD without acute inflammation signs. Enterobacteriaceae seems to be the microbiota most associated with the disease, followed by Bifidobacteria. CONCLUSIONS: All the included studies showed great heterogeneity in population characteristics and sampling methods. Therefore, given the high prevalence of colonic diverticulitis in the general population, further studies are needed to clarify the role of the intestinal microbiome, paving the way to new target therapies with important social implications.

Liver-directed lentiviral gene therapy corrects hemophilia A mice and achieves normal-range factor VIII activity in non-human primates.

Liver gene therapy with adeno-associated viral (AAV) vectors delivering clotting factor transgenes into hepatocytes has shown multiyear therapeutic benefit in adults with hemophilia. However, the mostly episomal nature of AAV vectors challenges their application to young pediatric patients. We developed lentiviral vectors, which integrate in the host cell genome, that achieve efficient liver gene transfer in mice, dogs and non-human primates, by intravenous delivery. Here we first compare engineered coagulation factor VIII transgenes and show that codon-usage optimization improved expression 10-20-fold in hemophilia A mice and that inclusion of an unstructured XTEN peptide, known to increase the half-life of the payload protein, provided an additional >10-fold increase in overall factor VIII output in mice and non-human primates. Stable nearly life-long normal and above-normal factor VIII activity was achieved in hemophilia A mouse models. Overall, we show long-term factor VIII activity and restoration of hemostasis, by lentiviral gene therapy to hemophilia A mice and normal-range factor VIII activity in non-human primate, paving the way for potential clinical application.

Liver gene therapy with intein-mediated F8 trans-splicing corrects mouse haemophilia A.

Liver gene therapy with adeno-associated viral (AAV) vectors is under clinical investigation for haemophilia A (HemA), the most common inherited X-linked bleeding disorder. Major limitations are the large size of the F8 transgene, which makes packaging in a single AAV vector a challenge, as well as the development of circulating anti-F8 antibodies which neutralise F8 activity. Taking advantage of split-intein-mediated protein trans-splicing, we divided the coding sequence of the large and highly secreted F8-N6 variant in two separate AAV-intein vectors whose co-administration to HemA mice results in the expression of therapeutic levels of F8 over time. This occurred without eliciting circulating anti-F8 antibodies unlike animals treated with the single oversized AAV-F8 vector under clinical development. Therefore, liver gene therapy with AAV-F8-N6 intein should be considered as a potential therapeutic strategy for HemA.

Therapeutic correction of hemophilia A by transplantation of hPSC-derived liver sinusoidal endothelial cell progenitors.

Liver sinusoidal endothelial cells (LSECs) form the predominant microvasculature in the liver where they carry out many functions including the secretion of coagulation factor VIII (FVIII). To investigate the early origins of this lineage, we develop an efficient and scalable protocol to produce human pluripotent stem cell (hPSC)-derived LSEC progenitors characterized as venous endothelial cells (VECs) from different mesoderm subpopulations. Using a sensitive and quantitative vascular competitive transplantation assay, we demonstrate that VECs generated from BMP4 and activin A-induced KDR+CD235a/b+ mesoderm are 50-fold more efficient at LSEC engraftment than venous cells from BMP4 and WNT-induced KDR+CD235a/b- mesoderm. When transplanted into immunocompromised hemophilia A mice (NSG-HA), these VECs engraft the liver, proliferate, and mature to functional LSECs that secrete bioactive FVIII capable of correcting the bleeding phenotype. Together, these findings highlight the importance of appropriate mesoderm induction for generating hPSC-derived LSECs capable of functioning in a preclinical model of hemophilia A.

Nursing students' clinical placement experiences during the Covid-19 pandemic: A phenomenological study.

AIM: This study explored the clinical placement experiences of nursing students during the Covid-19 pandemic. BACKGROUND: The health emergency caused by Covid-19 required a rapid reorganisation of care settings. This reorganisation entailed revisiting the clinical placements settings and learning programs of Italian nursing faculties. Some Italian universities wanted to seize the health emergency as a learning opportunity enabling the nursing student to acquire additional knowledge and skills. DESIGN: We conducted a descriptive qualitative study employing a phenomenological approach. The study population was second and third-year nursing students. The students did their clinical placement in 5 Northern Italy hospitals, mainly in infectious diseases wards, intensive care and sub-intensive care units, emergency department, short-stay surgical units and internal medicine wards. In these departments, the inpatient wards were entirely converted into Covid-19 units. Ethical approval was obtained from the local ethics committee. METHODS: Semi-structured, open-ended interviews were conducted in March-April 2021 and analysed following a phenomenological approach. RESULTS: Twenty-one nursing students in their 2nd and 3rd academic year participated. Their average age was 24 years. 81% were female and 19% were male. Three main themes were generated: (i) Learning which surpasses technicalities; (ii) Confronting dignity issues; (iii) Feeling treated as an equal in the workspace. Students had to learn how to lower their fear and self-manage the emotional burden to be a caring presence for the patients who were intensely suffering from the disease and isolation. Attending a clinical practice placement in Covid-19 wards led them to focus on human dignity issues: participants realised how dignity was questioned and how they could become patients' advocates. Students also described that they felt part of the team, with their student role almost fading. CONCLUSIONS: This study describes that the most unpredictable public health emergency, such as Covid-19, can provide learning opportunities in the practice environment for nursing students. Students described feeling useful and capitalising on new competencies. Designing educational activities for nursing students concerning pandemic emergencies may be strategic for dealing with similar situations in the future.