Reduced sulfatide content in deferoxamine-induced senescent HepG2 cells

Iron chelators, such as deferoxamine, exert an anticancer effect by altering the activity of biomolecules critical for regulation of the cell cycle, cell metabolism, and apoptotic processes. Thus, iron chelators are sometimes used in combination with radio- and/or chemotherapy in the treatment of cancer. The possibility that deferoxamine could induce a program of senescence similar to radio- and/or chemotherapy, fostering adaptation in the treatment of cancer cells, is not fully understood. Using established biochemical techniques, biomarkers linked to lipid composition, and coherent anti-Stokes Raman scattering microscopy, we demonstrated that hepatocellular carcinoma-derived HepG2 cells survive after deferoxamine treatment, acquiring phenotypic traits and representative hallmarks of senescent cells. The results support the view that deferoxamine acts in HepG2 cells to produce oxidative stress-induced senescence by triggering sequential mitochondrial and lysosomal dysfunction accompanied by autophagy blockade. We also focused on the lipidome of senescent cells after deferoxamine treatment. Using mass spectrometry, we found that the deferoxamine-induced senescent cells presented marked remodeling of the phosphoinositol, sulfatide, and cardiolipin profiles, which all play a central role in cell signaling cascades, intracellular membrane trafficking, and mitochondria functions. Detection of alterations in glycosphingolipid sulfate species suggested modifications in ceramide generation, and turnover is frequently described in cancer cell survival and resistance to chemotherapy. Blockade of ceramide generation may explain autophagic default, resistance to apoptosis, and the onset of senescence

Deep ensemble learning and transfer learning methods for classification of senescent cells from nonlinear optical microscopy images

The success of chemotherapy and radiotherapy anti-cancer treatments can result in tumor suppression or senescence induction. Senescence was previously considered a favorable therapeutic outcome, until recent advancements in oncology research evidenced senescence as one of the culprits of cancer recurrence. Its detection requires multiple assays, and nonlinear optical (NLO) microscopy provides a solution for fast, non-invasive, and label-free detection of therapy-induced senescent cells. Here, we develop several deep learning architectures to perform binary classification between senescent and proliferating human cancer cells using NLO microscopy images and we compare their performances. As a result of our work, we demonstrate that the most performing approach is the one based on an ensemble classifier, that uses seven different pre-trained classification networks, taken from literature, with the addition of fully connected layers on top of their architectures. This approach achieves a classification accuracy of over 90%, showing the possibility of building an automatic, unbiased senescent cells image classifier starting from multimodal NLO microscopy data. Our results open the way to a deeper investigation of senescence classification via deep learning techniques with a potential application in clinical diagnosis

Noninvasive morpho-molecular imaging reveals early therapy-induced senescence in human cancer cells

Anticancer therapy screening in vitro identifies additional treatments and improves clinical outcomes. Systematically, although most tested cells respond to cues with apoptosis, an appreciable portion enters a senescent state, a critical condition potentially driving tumor resistance and relapse. Conventional screening protocols would strongly benefit from prompt identification and monitoring of therapy-induced senescent (TIS) cells in their native form. We combined complementary all-optical, label-free, and quantitative microscopy techniques, based on coherent Raman scattering, multiphoton absorption, and interferometry, to explore the early onset and progression of this phenotype, which has been understudied in unperturbed conditions. We identified TIS manifestations as early as 24 hours following treatment, consisting of substantial mitochondrial rearrangement and increase of volume and dry mass, followed by accumulation of lipid vesicles starting at 72 hours. This work holds the potential to affect anticancer treatment research, by offering a label-free, rapid, and accurate method to identify initial TIS in tumor cells

Full-Spectrum CARS Microscopy of Cells and Tissues with Ultrashort White-Light Continuum Pulses

Coherent anti-StokesRaman scattering (CARS) microscopyis an emergingnonlinear vibrational imaging technique that delivers label-free chemicalmaps of cells and tissues. In narrowband CARS, two spatiotemporallysuperimposed picosecond pulses, pump and Stokes, illuminate the sampleto interrogate a single vibrational mode. Broadband CARS (BCARS) combinesnarrowband pump pulses with broadband Stokes pulses to record broadvibrational spectra. Despite recent technological advancements, BCARSmicroscopes still struggle to image biological samples over the entireRaman-active region (400-3100 cm(-1)). Here,we demonstrate a robust BCARS platform that answers this need. Oursystem is based on a femtosecond ytterbium laser at a 1035 nm wavelengthand a 2 MHz repetition rate, which delivers high-energy pulses usedto produce broadband Stokes pulses by white-light continuum generationin a bulk YAG crystal. Combining such pulses, pre-compressed to sub-20fs duration, with narrowband pump pulses, we generate a CARS signalwith a high (<9 cm(-1)) spectral resolution inthe whole Raman-active window, exploiting both the two-color and three-colorexcitation mechanisms. Aided by an innovative post-processing pipeline,our microscope allows us to perform high-speed (approximate to 1 ms pixeldwell time) imaging over a large field of view, identifying the mainchemical compounds in cancer cells and discriminating tumorous fromhealthy regions in liver slices of mouse models, paving the way forapplications in histopathological settings

Refractory mucocutaneous leishmaniasis resolved with combination treatment based on intravenous pentamidine, oral azole, aerosolized liposomal amphotericin B, and intralesional meglumine antimoniate

Introduction: Mucocutaneous leishmaniasis (MCL) is a complication of tegumentary leishmaniasis, causing potentially life-threatening lesions in the ear, nose, and throat (ENT) region, and most commonly due to Leishmania (Viannia) braziliensis. We report a case of relapsing MCL in an Italian traveler returning from Argentina. Case description: A 65-year-old Italian male patient with chronic kidney disease, arterial hypertension, prostatic hypertrophy, and type-2 diabetes mellitus was referred for severe relapsing MCL acquired in Argentina. ENT examination showed severe diffuse pharyngolaryngeal edema and erythema, partially obstructing the airways. A nasopharyngeal biopsy revealed a lymphoplasmacytic inflammation and presence of Leishmania amastigotes, subsequently identified as L. (V.) braziliensis by hsp70 PCR-RFLP analysis and sequencing. Despite receiving four courses of liposomal amphotericine B (L-AmB) and two courses of miltefosine over a 2-year period, the patient presented recurrence of symptoms a few months after the end of each course.After the patient was referred to us, a combined treatment was started with intravenous pentamidine 4 mg/kg on alternate days for 10 doses, followed by one dose per week for an additional seven doses, intralesional meglumine antimoniate on the nasal lesion once per week for six doses, oral azoles for three months, and aerosolized L-AmB on alternate days for three months.The treatment led to regression of mucosal lesions and respiratory symptoms. Renal function temporarily worsened, and the addition of insulin was required to maintain glycemic compensation after pentamidine discontinuation. Conclusions: This case highlights the difficulties in managing a life-threatening refractory case of MCL in an Italian traveler with multiple comorbidities. Even though parenteral antimonial derivatives are traditionally considered the treatment of choice for MCL, they are relatively contraindicated in cases of chronic kidney disease.The required dose adjustment in cases of impaired renal function is unknown, therefore the use of alternative drugs is recommended. This case was resolved with combination treatment, including aerosolized L-AmB, which had never been used before for MCL

Mortality rate of patients with cystic fibrosis on the waiting list and within one year after lung transplantation: a survey of Italian CF centers

Abstract Background Cystic Fibrosis (CF) Centers are involved in the decisions regarding the eligibility of CF patients with end-stage lung disease and timing for inclusion on waiting lists (WL) for lung transplantation (LT). There are currently no data on the mortality rates of Italian CF patients on WL and during the first year after LT and we aimed to assess these outcomes by surveying the CF Centers. Methods A survey was sent to Italian CF Centers which were requested to report the age at which all CF subjects included on the WL between 2010 and 2014 were included on the list, admitted to either standard or urgent LT, or had died either while on the WL or within the first 3 and 12 months after LT. All outcomes were recorded by December 31, 2015. Results Two hundred fifty-nine CF subjects were included on the WL during the 5-year study period. The mortality rate during the WL was 19.3% and was not associated with sex, age at inclusion on the WL or standard or urgent access to LT. 159 (61.4%) subjects underwent LT, 46 (28.9%) with urgent procedure. Deaths within the first 3 and 12 months after LT were significantly more prevalent in individuals who underwent urgent LT compared to those with standard LT (p < 0.01). Conclusions The mortality of Italian CF patients, included in our survey, was about twice that reported by the National Transplant Center for all LT indications, including CF, during the same time period and despite the introduction of urgent LT. The latter was associated with an unfavorable early outcome compared to standard LT

Optical Diffraction Tomography and Raman Confocal Microscopy for the Investigation of Vacuoles Associated with Cancer Senescent Engulfing Cells

Wild-type p53 cancer therapy-induced senescent cells frequently engulf and degrade neighboring ones inside a massive vacuole in their cytoplasm. After clearance of the internalized cell, the vacuole persists, seemingly empty, for several hours. Despite large vacuoles being associated with cell death, this process is known to confer a survival advantage to cancer engulfing cells, leading to therapy resistance and tumor relapse. Previous attempts to resolve the vacuolar structure and visualize their content using dyes were unsatisfying for lack of known targets and ineffective dye penetration and/or retention. Here, we overcame this problem by applying optical diffraction tomography and Raman spectroscopy to MCF7 doxorubicin-induced engulfing cells. We demonstrated a real ability of cell tomography and Raman to phenotype complex microstructures, such as cell-in-cells and vacuoles, and detect chemical species in extremely low concentrations within live cells in a completely label-free fashion. We show that vacuoles had a density indistinguishable to the medium, but were not empty, instead contained diluted cell-derived macromolecules, and we could discern vacuoles from medium and cells using their Raman fingerprint. Our approach is useful for the noninvasive investigation of senescent engulfing (and other peculiar) cells in unperturbed conditions, crucial for a better understanding of complex biological processes

Clinical epidemiology of chronic viral hepatitis B: A Tuscany real-word large-scale cohort study

AIM To build a regional database of chronic patients to define the clinical epidemiology of hepatitis B virus (HBV)- infected patients in the Tuscan public health care system. METHODS This study used a cross-sectional cohort design. We evaluated chronic viral hepatitis patients with HBV referred to the outpatient services of 16 hospital units. Information in the case report forms included main demographic data, blood chemistry data, viral hepatitis markers, instrumental evaluations, and eligibility for treatment or ongoing therapy and liver transplantation. RESULTS Of 4015 chronic viral hepatitis patients, 1096 (27.3%) were HBV infected. The case report form was correctly completed for only 833 patients (64% males, 36% females; mean age 50.1 ± 15.4). Of these HBV-infected patients, 73% were Caucasian, 21% Asian, 4% Central African, 1% North African and 1% American. Stratifying patients by age and nationality, we found that 21.7% of HBV-infected patients were aged &lt; 34 years (only 2.8% were Italian). The most represented routes of transmission were nosocomial/dental procedures (23%), mother-to-child (17%) and sexual transmission (12%). The most represented HBV genotypes were D (72%) and A (14%). Of the patients, 24.7% of patients were HBeAg positive, and 75.3% were HBeAg negative. Of the HBV patients 7% were anti-HDV positive. In the whole cohort, 26.9% were cirrhotic (35.8% aged &lt; 45 years), and 47% were eligible for or currently undergoing treatment, of whom 41.9 % were cirrhotic. CONCLUSION Only 27.3% of chronic viral hepatitis patients were HBV infected. Our results provide evidence of HBV infection in people aged &lt; 34 years, especially in the foreign population not protected by vaccination. In our cohort of patients, liver cirrhosis was also found in young adults