Consensus paper on the management of acute isolated vertigo in the emergency department.

Acute vertigo is defined as the perception of movement of oneself or the surroundings in the absence of actual motion and it is a frequent cause for emergency department admissions. The utilization of medical resources and the duration of hospital stay for this kind of symptom is high. Furthermore, the efficiency of brain imaging in the acute phase is low, considering the limited sensitivity of both CT and MRI for diagnosing diseases that are the causes of central type of vertigo. Relying on imaging tests can provide false reassurance in the event of negative results or prolong the in-hospital work-up improperly. On the other hand, clinical examinations, notably the assessment of nystagmus' features, have proven to be highly accurate and efficient when performed by experts. Literature data point out that emergency physicians often do not employ these skills or use them incorrectly. Several clinical algorithms have been introduced in recent years with the aim of enhancing the diagnostic accuracy of emergency physicians when evaluating this specific pathology. Both the 'HINTS and 'STANDING' algorithms have undergone external validation in emergency physician hands, showing good diagnostic accuracy. The objective of this consensus document is to provide scientific evidence supporting the clinical decisions made by physicians assessing adult patients with acute vertigo in the emergency department, particularly in cases without clear associated neurological signs. The document aims to offer a straightforward and multidisciplinary approach. At the same time, it tries to delineate benchmarks for the formulation of local diagnostic and therapeutic pathways, as well as provide a base for the development of training and research initiatives.

GMP-Grade Manufacturing and Quality Control of a Non-Virally Engineered Advanced Therapy Medicinal Product for Personalized Treatment of Age-Related Macular Degeneration.

The introduction of new therapeutics requires validation of Good Manufacturing Practice (GMP)-grade manufacturing including suitable quality controls. This is challenging for Advanced Therapy Medicinal Products (ATMP) with personalized batches. We have developed a person-alized, cell-based gene therapy to treat age-related macular degeneration and established a vali-dation strategy of the GMP-grade manufacture for the ATMP; manufacturing and quality control were challenging due to a low cell number, batch-to-batch variability and short production duration. Instead of patient iris pigment epithelial cells, human donor tissue was used to produce the transfected cell product ("tIPE"). We implemented an extended validation of 104 tIPE productions. Procedure, operators and devices have been validated and qualified by determining cell number, viability, extracellular DNA, sterility, duration, temperature and volume. Transfected autologous cells were transplanted to rabbits verifying feasibility of the treatment. A container has been engineered to ensure a safe transport from the production to the surgery site. Criteria for successful validation and qualification were based on tIPE's Critical Quality Attributes and Process Parameters, its manufacture and release criteria. The validated process and qualified operators are essential to bring the ATMP into clinic and offer a general strategy for the transfer to other manufacture centers and personalized ATMPs.

In Vivo and Ex Vivo Gene Electrotransfer in Ophthalmological Disorders.

The aim of this document is to present an overview of gene electrotransfer in ophthalmological disorders. In order to ensure an adequate variety of the assessed studies, several electronic databases were considered and studies published between January 1998 and December 2021 were analysed. Three investigators carried out data extraction and analysis, focusing on both technical (i.e., electrical protocol, type of electrode, plasmid) and medical (i.e., type of study, threated disease) aspects and highlighting the main differences in terms of results obtained. Moreover, the IGEA experience in the project "Transposon-based, targeted ex vivo gene therapy to treat age-related macular degeneration" (TargetAMD) was reported in the results section. No clinical trial was found on international literature and on ClinicalTrials.gov. Twelve preclinical studies were found including in vivo and ex-vivo applications. The studied showed that electrotransfer could be very efficient for plasmid DNA transfection. Many attempts such as modification of the electric field, buffers and electrodes have been made and the optimization of electric field setting seems to be very important. Using this technique, gene replacement can be designed in cases of retinal inheritance or corneal disease and a wide range of human eye diseases could, in the future, benefitfrom these gene therapy technologies.

Impact of the number of electric pulses on cell electrochemotherapy in vitro: Limits of linearity and saturation.

In this study the evolution in the efficiency of electrochemotherapy (reversible electroporation) with pulse number was assessed in vitro. Experiments were performed using 100 µs pulses at different electric field intensities and the chemotherapeutic agent bleomycin. Additionally, electrical impedance spectroscopy measurements were used as a different method to study in real time the changes produced on cells with pulse number during trains of consecutive pulses. Our results show that the relation between pulse number and the observed outcome is complex and difficult to fully characterize. This relation can display a highly linear behaviour up to a certain number of pulses and/or field intensity applied. However, the relation between the number of pulses and the observed outcome always evolves to a saturation or at least a reduction in the electric field effects that is displayed when either electric field intensity or pulse number are increased. An exponential model was found to best describe this relation within the range of experimental conditions considered. Electrical impedance measurements confirmed the results and gave a more precise quantification of this dependence. The study highlights the importance that pulse number has in the electrochemotherapy protocols and establishes some limits in the use of this parameter.

Electrochemotherapy in the Treatment of Bone Metastases: A Phase II Trial.

INTRODUCTION: Bone metastatic disease is a major cause of pain and decreased quality of life in patients with cancer. In addition to systemic therapy and pain control with narcotic analgesics, standard local treatments include palliation with radiation therapy and surgery. However, 20-30 % of patients do not respond to conventional treatments, increasing the interest in alternative therapies. We present the results of a new minimally invasive technique in the treatment of bone metastases. METHODS: Twenty-nine patients affected by painful bone metastases were treated with electrochemotherapy (ECT) from July 2009 to July 2011; the mean age was 60 years (range 37-87); 21 patients received a previous ineffective local treatment; the appendicular skeleton was affected in 15 patients while in 14 patients other sites were involved. ECT was performed using the Cliniporator Vitae under fluoroscopy or CT guidance depending on the site of the lesion. Clinical response was assessed using VAS scale and objective tumour response was evaluated according to the MD Anderson criteria for bone metastases. RESULTS: All patients well tolerated the procedure and no intraoperative or postoperative complications were observed. At a mean follow-up of 7 months, 24 patients were available for evaluation. 84 % of the patients (20 out of 24) referred improvement of pain ≥50 % with reduction of narcotics consumption. Radiographic evaluation after 3 months in 20 evaluable patients, showed "partial response" in 1 patient, "stable disease" in 17 and "progression" in two cases. DISCUSSION: Results reported in this study demonstrated ECT to be safe and feasible in the treatment of painful bone metastases even when other previous treatments were ineffective. Pain and disease progression control was achieved in the majority of the patients with consequent improvement of quality of life. CONCLUSION: ECT should be considered a new feasible tool in the treatment of bone metastases in place or in combination with standard treatments; further developments are required to extend the use of this technique to spine metastases.

Feasibility of Electroporation in Bone and in the Surrounding Clinically Relevant Structures: A Preclinical Investigation.

Skeletal metastases are a common cause of severe morbidity, reduction in quality of life and often early mortality. Consequently, improvements in therapies are necessary. Electroporation uses electric energy to alter cancer cell membrane permeability and enhance the local uptake of chemotherapeutics, thus leading to local tumor control. The aim of this study was to investigate the feasibility and safety of delivering electric field protocols causing electroporation of healthy bone and structures of clinical relevance using small and large animal models. Reversible electroporation was used in the rabbit sciatic nerve by applying 2 series of 8 pulses 100ms long at 1000 V/cm. Irreversible electroporation was used in rabbit distal femur condyles and in sheep vertebral body by applying 120 pulses 100ms long at 1750 V/cm. Any effect on surrounding sensitive structures was investigated. Reversible electroporation of sciatic nerve was associated with transient foot functional deficit that completely recovered at 30 days. Irreversible electroporation removed cells from trabeculae in the femurs of rabbits and in the vertebral body of sheep. After irreversible protocol, histology and microtomography demonstrated that the trabecular structure was maintained, the presence of new bone marrow cells, osteoblasts, and mineral apposition characterized by new trabeculae thinner than controls (P = .005) and a significant reduction in the ablated areas (-225%, P = .0219). Spinal cord, vertebral pedicles and spinal nerves showed transient edema in the absence of functional or structural alterations. Collectively, these results show that electroporation can be safely applied to bone even in the proximity of neuronal structures.

Modeling the positioning of single needle electrodes for the treatment of breast cancer in a clinical case.

BACKGROUND: Breast cancer is the most common cancer in women worldwide and is the second most common cause of cancer death in women. Electrochemotherapy (ECT) used in early-phase clinical trials for the treatment of primary breast cancer resulted in a not complete tumor necrosis in most cases. The present study was undertaken to analyze the feasibility to use ECT to treat patients with histologically proven unifocal ductal breast cancer. In particular, results of ECT treatment in a clinical case are compared with the ones of a simplified 3D dosimetric model. METHODS: This clinical study was conducted with the pulse generator Cliniporator Vitae (IGEA, Carpi, Italy). ECT procedures were performed according to ESOPE standard operating procedures. Five single needle electrodes were used with one positioned in the center of the tumor, and the other four distributed around the nodule. Histological images of the resected tumor are compared with the maps of the electric field obtained with a simplified 3D model in Comsol Multiphysics v 4.3. RESULTS: The results of the clinical case demonstrated a reduced efficacy of the ECT treatment described. The proposed simple numerical model of the breast tumor located in a low conductive tissue suggests that this is due to the reduced electric field induced inside the tumor with such 5 electrodes placement. However, where the electric field is predicted higher than the reversible electroporation threshold (E>400 V/cm), also the histological images confirm the necrosis of the target with a good agreement between the modeled and clinical results. CONCLUSIONS: The results suggest the dependence of the effectiveness of the treatment on the careful placement of the electrodes. A detailed planned procedure for the tumor analysis after the treatment is also needed in order to better correlate the single electrode positions and the histological images. Simulation models could be used to identify better electrodes configuration in planning the experimental protocol for ECT treatment of breast tumors.

Locally enhanced chemotherapy by electroporation: clinical experiences and perspective of use of electrochemotherapy.

Electroporation is used to enhance drug diffusion and gene delivery into the cytosol. The combination of electroporation and cytotoxic drugs, electrochemotherapy (ECT), is used to treat metastatic tumor nodules located at the skin and subcutaneous tissue. The objective response rate following a single session of treatment exceeds 80%, with minimal toxicity for the patients. The efficacy of ECT in the bone and visceral metastasis is currently investigated, and Phase II studies have been completed. ECT has been used to treat skin primary tumors, except melanoma, and is under investigation for locally advanced pancreatic cancer. Early evidence suggests that treatment of tumor nodules with ECT recruits components of the immune system and eliciting a systemic immune response against cancer is a challenging clinical perspective. Considering the proven safety in several different clinical applications electroporation should be viewed as a clinical platform technology with wide perspectives for use in ECT, gene therapy and DNA vaccination.

Targeting autophagy potentiates tyrosine kinase inhibitor-induced cell death in Philadelphia chromosome-positive cells, including primary CML stem cells.

Imatinib mesylate (IM), a potent inhibitor of the BCR/ABL tyrosine kinase, has become standard first-line therapy for patients with chronic myeloid leukemia (CML), but the frequency of resistance increases in advancing stages of disease. Elimination of BCR/ABL-dependent intracellular signals triggers apoptosis, but it is unclear whether this activates additional cell survival and/or death pathways. We have shown here that IM induces autophagy in CML blast crisis cell lines, CML primary cells, and p210BCR/ABL-expressing myeloid precursor cells. IM-induced autophagy did not involve c-Abl or Bcl-2 activity but was associated with ER stress and was suppressed by depletion of intracellular Ca2+, suggesting it is mechanistically nonoverlapping with IM-induced apoptosis. We further demonstrated that suppression of autophagy using either pharmacological inhibitors or RNA interference of essential autophagy genes enhanced cell death induced by IM in cell lines and primary CML cells. Critically, the combination of a tyrosine kinase inhibitor (TKI), i.e., IM, nilotinib, or dasatinib, with inhibitors of autophagy resulted in near complete elimination of phenotypically and functionally defined CML stem cells. Together, these findings suggest that autophagy inhibitors may enhance the therapeutic effects of TKIs in the treatment of CML.

Design of an irreversible electroporation system for clinical use.

Irreversible electroporation is an ablation modality in which microseconds, high-voltage electrical pulses are applied to induce cell necrosis in a target tissue. To perform irreversible electroporation it is necessary to use a medical device specifically designed for this use. The design of an irreversible electroporation system is a complex task in which the effective delivery of high energy pulses and the safety of the patient and operator are equally important. Pulses of up to 3000 V of amplitude and 50 A of current need to be generated to irreversibly electroporate a target volume of approximately 50 to 70 cm3 with as many as six separate electrodes; therefore, a traditional approach based on high voltage amplifiers becomes hard to implement. In this paper, we present the process that led to the first irreversible electroporator capable of such performances approved for clinical use. The main design choices and its architecture are outlined. Safety issues are also explained along with the solutions adopted.

High levels of the BCR/ABL oncoprotein are required for the MAPK-hnRNP-E2 dependent suppression of C/EBPalpha-driven myeloid differentiation.

The inability of myeloid chronic myelogenous leukemia blast crisis (CML-BC) progenitors to undergo neutrophil differentiation depends on suppression of C/EBPalpha expression through the translation inhibitory activity of the RNA-binding protein hnRNP-E2. Here we show that "oncogene dosage" is a determinant factor for suppression of differentiation in CML-BC. In fact, high levels of p210-BCR/ABL are required for enhanced hnRNP-E2 expression, which depends on phosphorylation of hnRNP-E2 serines 173, 189, and 272 and threonine 213 by the BCR/ABL-activated MAPK(ERK1/2). Serine/threonine to alanine substitution abolishes hnRNP-E2 phosphorylation and markedly decreases its stability in BCR/ABL-expressing myeloid precursors. Similarly, pharmacologic inhibition of MAPK(ERK1/2) activity decreases hnRNP-E2 binding to the 5'UTR of C/EBPalpha mRNA by impairing hnRNP-E2 phosphorylation and stability. This, in turn, restores in vitro and/or in vivo C/EBPalpha expression and G-CSF-driven neutrophilic maturation of differentiation-arrested BCR/ABL(+) cell lines, primary CML-BC(CD34+) patient cells and lineage-negative mouse bone marrow cells expressing high levels of p210-BCR/ABL. Thus, increased BCR/ABL oncogenic tyrosine kinase activity is essential for suppression of myeloid differentiation of CML-BC progenitors as it is required for sustained activation of the MAPK(ERK1/2)-hnRNP-E2-C/EBPalpha differentiation-inhibitory pathway. Furthermore, these findings suggest the inclusion of clinically relevant MAPK inhibitors in the therapy of CML-BC.