International prognostic score for asymptomatic early-stage chronic lymphocytic leukemia.

Most patients with chronic lymphocytic leukemia (CLL) are diagnosed with early-stage disease and managed with active surveillance. The individual course of patients with early-stage CLL is heterogeneous, and their probability of needing treatment is hardly anticipated at diagnosis. We aimed at developing an international prognostic score to predict time to first treatment (TTFT) in patients with CLL with early, asymptomatic disease (International Prognostic Score for Early-stage CLL [IPS-E]). Individual patient data from 11 international cohorts of patients with early-stage CLL (n = 4933) were analyzed to build and validate the prognostic score. Three covariates were consistently and independently correlated with TTFT: unmutated immunoglobulin heavy variable gene (IGHV), absolute lymphocyte count higher than 15 × 109/L, and presence of palpable lymph nodes. The IPS-E was the sum of the covariates (1 point each), and separated low-risk (score 0), intermediate-risk (score 1), and high-risk (score 2-3) patients showing a distinct TTFT. The score accuracy was validated in 9 cohorts staged by the Binet system and 1 cohort staged by the Rai system. The C-index was 0.74 in the training series and 0.70 in the aggregate of validation series. By meta-analysis of the training and validation cohorts, the 5-year cumulative risk for treatment start was 8.4%, 28.4%, and 61.2% among low-risk, intermediate-risk, and high-risk patients, respectively. The IPS-E is a simple and robust prognostic model that predicts the likelihood of treatment requirement in patients with early-stage CLL. The IPS-E can be useful in clinical management and in the design of early intervention clinical trials.

Daratumumab for relapsed or refractory AL amyloidosis with high plasma cell burden.

Daratumumab, an anti-CD38 antibody, is effective in AL amyloidosis with low tumor burden. Data of daratumumab treatment in patients with AL amyloidosis but high tumor burden (≥10% bone marrow plasma cells) are limited. We report retrospective data of 10 consecutive patients with high tumor burden treated with daratumumab for relapsed/refractory AL amyloidosis. The median age at diagnosis was 62.3 years; all patients had cardiac involvement, and six (60%) patients had renal involvement. Median bone marrow plasma cell infiltration was 15% (range 10%-40%), and the median difference between involved and noninvolved free light-chains (dFLC) was 446 mg/L (range 102-1392 mg/L). Patients had a median of three prior lines of therapy, including bortezomib in all patients and lenalidomide in seven (70%) patients. The median time to first hematological response was 14 days (range 7-28 days), and the median time to best hematological response was 64 days (range 7-301 days). The hematological overall response was 90%, with high-quality response (≥ very good partial remission [VGPR]) in 70% of the patients. Fifty percent of the patients had a cardiac response after a median of 3.8 months (range 0.7-9.1). Infusion-related adverse events ≤ grade 2 occurred in seven (70%) patients and grade 3 adverse events in one patient. After a median follow-up time of 10 months, eight (80%) patients continued to receive daratumumab. We conclude that daratumumab is a very effective and safe treatment option in AL patients with relapsed/refractory disease and high disease burden at diagnosis. Daratumumab leads to rapid disease control and improvement of organ function.

Safety and efficacy of reduced dosage ketoprofen with or without tramadol for long-term treatment of osteoarthritis in dogs: a randomized clinical trial.

BACKGROUND: This study aimed to evaluate the safety and efficacy of reduced-dosage ketoprofen with or without tramadol in dogs. Five healthy dogs receiving standard-dosage ketoprofen (2 mg/kg SC, then 1 mg/kg PO daily) comprised Group A. Twenty dogs with osteoarthritis were randomized to receive reduced-dosage ketoprofen (0.5 mg/kg SC once; 0.25 mg/kg PO daily) alone (Group B) or in combination with tramadol (5 mg/kg/day PO) (Group C). Treatments were administered for 28 days. Platelet aggregation time (PAT), gastrointestinal (GI) endoscopy and glomerular filtration rate (GFR) were performed up to 60 days after treatment initiation. Pain was scored using a validated clinical metrology instrument up to D120. Data were analyzed with general linear mixed model for repeated measures (α = 0.05). RESULTS: PAT was not different between groups but was increased with time for all groups. GI lesion scores were higher in Group A than Groups B and C (day 28; P = 0.005) and were increased with time for Group A (P = 0.005). GFR was lower in Group A than Groups B and C (day 28; P < 0.01) and were decreased with time for group A (P < 0.001). Standard-dosage ketoprofen administration resulted in clinically relevant adverse effects. Pain score decreased in both treated groups (B and C) from D0 to D28. Need of rescue analgesia from D29 to D120 was higher in Group B than in Group C (P = 0.039). CONCLUSIONS: The long-term safety profile of reduced-dosage ketoprofen is similar whether the drug is administered alone or in combination with tramadol to dogs with osteoarthritis. Analgesic efficacy of the combination looks attractive.

Fiber-Based SERS-Fluidic Polymeric Platforms for Improved Optical Analysis of Liquids.

Downsizing surface-enhanced Raman spectroscopy (SERS) within microfluidic devices has opened interesting perspectives for the development of low-cost and portable (bio)sensors for the optical analysis of liquid samples. Despite the research efforts, SERS-fluidic devices still rely either on the use of expensive bulky set-ups or on polymeric devices giving spurious background signals fabricated via expensive manufacturing processes. Here, polymeric platforms integrating fluidics and optics were fabricated with versatile designs allowing easy coupling with fiber-based Raman systems. For the first time, anti-fouling photocurable perfluoropolyether (PFPE) was explored for high-throughput SERS-integrating chip fabrication via replica molding of negative stamps obtained through standard and advanced fabrication processes. The PFPE devices comprised networks of channels for fluid handling and for optical fiber housing with multiple orientations. Embedded microfeatures were used to control the relative positioning of the fibers, thus guaranteeing the highest signal delivering and collection. The feasibility of PFPE devices as fiber-based SERS fluidic platforms was demonstrated through the straightforward acquisition of Raman-SERS spectra of a mixture of gold nanoparticles as SERS substrates with rhodamine 6G (Rh6G) at decreasing concentrations. In the presence of high-performing gold nanostars, the Rh6G signal was detectable at dilutions down to the nanomolar level even without tight focusing and working at low laser power-a key aspect for analyte detection in real-world biomedical and environmental applications.

Acquired haemophilia A in southern Switzerland from 2013 to 2019: a case series.

AIMS OF THE STUDY: Acquired haemophilia A is a rare disease with an annual incidence of 1.48 per million. Based on clinical observations, we suspect a higher incidence in southern Switzerland, and aimed at providing local epidemiological data, and clinical information regarding diagnosis, treatment and outcome in our region. METHODS: All adult patients with acquired haemophilia A treated between 2013 and 2019 in our facility were included in the present retrospective analysis. RESULTS: We treated 11 patients with acquired haemophilia A between 2013 and 2019, resulting in an annual incidence of 4.5 per million (95% confidence interval [CI] 0-9.0). Median delay from first symptoms to diagnosis was 4.5 days, and the median age at diagnosis was 79 years (range 23-87). Possible causative conditions were: pregnancy (n = 1), polyarteritis nodosa (n = 1), myelodysplastic syndrome (n = 1), chronic human immunodeficiency virus (HIV) (n = 1), and HIV postexposure prophylaxis (n = 1). In five patients no underlying or associated condition was identified. Median activated partial thromboplastin time (aPTT)) at baseline was 79 seconds (65-117; ref. value <38 sec), and FVIII:C 2.15% (<1-3.75%). A FVIII:C <1% was present in 4/10 patients. Median FVIII-inhibitor titre was 10.3 BU/ml (2.4-75.0 BU/ml). All patients had bleeding symptoms, 5/10 patients had major bleedings, and 7/10 patients were treated with bypassing agents. All patients received corticosteroids; 7/10 patients received immunosuppressive combination therapy. FVIII levels of ≥50% were achieved after a median of 40 days (8-62). One patient had a severe immunosuppressive therapy-related infection. An 87-years-old woman died for reasons not related to acquired haemophilia A or immunosuppressive therapy. CONCLUSIONS: Acquired haemophilia A is a rare disease, but manageable despite the advanced patient age and comorbidities. Its incidence in Southern Switzerland is higher than previously suspected.

The impact of microfluidics in high-throughput drug-screening applications.

Drug discovery is an expensive and lengthy process. Among the different phases, drug discovery and preclinical trials play an important role as only 5-10 of all drugs that begin preclinical tests proceed to clinical trials. Indeed, current high-throughput screening technologies are very expensive, as they are unable to dispense small liquid volumes in an accurate and quick way. Moreover, despite being simple and fast, drug screening assays are usually performed under static conditions, thus failing to recapitulate tissue-specific architecture and biomechanical cues present in vivo even in the case of 3D models. On the contrary, microfluidics might offer a more rapid and cost-effective alternative. Although considered incompatible with high-throughput systems for years, technological advancements have demonstrated how this gap is rapidly reducing. In this Review, we want to further outline the role of microfluidics in high-throughput drug screening applications by looking at the multiple strategies for cell seeding, compartmentalization, continuous flow, stimuli administration (e.g., drug gradients or shear stresses), and single-cell analyses.

Pitfalls of Early Systemic Corticosteroids Home Therapy in Older Patients with COVID-19 Pneumonia.

Corticosteroids have been widely used for acute respiratory distress syndrome (ARDS), but their role in the early phase of SARS-CoV-2 infection is controversial. Our study aimed to determine the effectiveness of early corticosteroid therapy (ECT) in preventing the progression of disease, reducing the escalation of care and improving clinical outcome in older patients hospitalized for COVID-19 pneumonia. A total of 90 subjects (47.7% women; mean age = 82.3 ± 6.7 years) were enrolled. ECT was administered to 33 out of 90 patients before the hospitalization. At admission, no difference was detected in median SOFA score (2, IQR:2 vs. 2, IQR: 2). We found a significant difference in mean PaO2/FiO2 ratio during the first week of hospitalization between ECT patients and controls (F = 5.49, p = 0.002) and in mean PaO2/FiO2 ratio over time (F = 6.94, p < 0.0001). We detected no-significant differences in terms of in-hospital mortality and transfer to ICU between ECT patients and controls (27.1% vs. 22.8%, respectively, p = 0.63). ECT was associated with worse clinical outcomes, showing no benefit in attenuating the progression of the disease or reducing the escalation of care. These findings are crucial given the current pandemic, and further studies are needed to provide additional data on the optimal timing of initiating corticosteroid treatment.

Computational fluid dynamic models as tools to predict aerosol distribution in tracheobronchial airways.

Aerosol and pollutants, in form of particulates 5-8 µm in main size face every day our respiratory system as natural suspension in air or forced to be inhaled as a coadjutant in a medical therapy for respiratory diseases. This inhalation happens in children to elderly, women and men, healthy or sick and disable people. In this paper we analyzed the inhalation of aerosol in conditions assimilable to the thermal therapy. We use a computational fluid dynamic 3D model to compute and visualize the trajectories of aerosol (3-7-10-25 µm) down to the sixth generation of bronchi, in a steady and dynamic condition (7 µm) set as breath cycle at rest. Results, compared to a set of milestone experimental studies published in literature, allow the comprehension of particles behavior during the inhalation from mouth to bronchi sixth generation, the visualization of jet at larynx constriction and vortices, in an averaged characteristic rigorous geometrical model including tracheal rings. Results on trajectories and deposition show the importance of the including transient physiological breath cycle on aerosol deposition analyses. Numerical and graphical results, may enable the design of medical devices and protocols to make the inhalations more effective in all the users' population.

Bioprinting of Matrigel Scaffolds for Cancer Research.

Cancer is one of the most life-threatening diseases worldwide. Despite the huge efforts, the failure rate of therapies remains high due to cells heterogeneity, so physiologically relevant models are strictly necessary. Bioprinting is a technology able to form highly complex 3D tissue models and enables the creation of large-scale constructs. In cancer research, Matrigel® is the most widely used matrix, but it is hardly bioprinted pure, without the use of any other bioink as reinforcement. Its complex rheological behavior makes the control with a standard bioprinting process nearly impossible. In this work, we present a customized bioprinting strategy to produce pure Matrigel® scaffolds with good shape fidelity. To this aim, we realized a custom-made volumetric dispensing system and performed printability evaluations. To determine optimal printing parameters, we analyzed fibers spreading ratio on simple serpentines. After identifying an optimal flow rate of 86.68 ± 5.77 µL/min and a printing speed of 10 mm/min, we moved forward to evaluate printing accuracy, structural integrity and other key parameters on single and multi-layer grids. Results demonstrated that Matrigel® was able to maintain its structure in both simple and complex designs, as well as in single and multilayer structures, even if it does not possess high mechanical strength. In conclusion, the use of volumetric dispensing allowed printing pure Matrigel® constructs with a certain degree of shape fidelity on both single and multiple layers.

Improving cell seeding efficiency through modification of fiber geometry in 3D printed scaffolds.

Cell seeding on 3D scaffolds is a very delicate step in tissue engineering applications, influencing the outcome of the subsequent culture phase, and determining the results of the entire experiment. Thus, it is crucial to maximize its efficiency. To this purpose, a detailed study of the influence of the geometry of the scaffold fibers on dynamic seeding efficiency is presented. 3D printing technology was used to realize polylactic acid porous scaffolds, formed by fibers with a non-circular cross-sectional geometry, named multilobed to highlight the presence of niches and ridges. An oscillating perfusion bioreactor was used to perform bidirectional dynamic seeding of MG63 cells. The fiber shape influences the fluid dynamic parameters of the flow, affecting values of fluid velocity and wall shear stress. The path followed by cells through the scaffold fibers is also affected and results in a larger number of adhered cells in multilobed scaffolds compared to scaffolds with standard pseudo cylindrical fibers. Geometrical and fluid dynamic features can also have an influence on the morphology of adhered cells. The obtained results suggest that the reciprocal influence of geometrical and fluid dynamic features and their combined effect on cell trajectories should be considered to improve the dynamic seeding efficiency when designing scaffold architecture.

Shear-Induced Encapsulation into Red Blood Cells: A New Microfluidic Approach to Drug Delivery.

Encapsulating molecules into red blood cells (RBCs) is a challenging topic for drug delivery in clinical practice, allowing to prolong the residence time in the body and to avoid toxic residuals. Fluidic shear stress is able to temporary open the membrane pores of RBCs, thus allowing for the diffusion of a drug in solution with the cells. In this paper, both a computational and an experimental approach were used to investigate the mechanism of shear-induced encapsulation in a microchannel. By means of a computational fluid dynamic model of a cell suspension, it was possible to calculate an encapsulation index that accounts for the effective shear acting on the cells, their distribution in the cross section of the microchannel and their velocity. The computational model was then validated with micro-PIV measurements on a RBCs suspension. Finally, experimental tests with a microfluidic channel showed that, by choosing the proper concentration and fluid flow rate, it is possible to successfully encapsulate a test molecule (FITC-Dextran, 40 kDa) into human RBCs. Cytofluorimetric analysis and confocal microscopy were used to assess the RBCs physiological shape preservation and confirm the presence of fluorescent molecules inside the cells.

Molecular screening test in familial forms of cerebral cavernous malformation: the impact of the Multiplex Ligation-dependent Probe Amplification approach.

Object The purpose of this study was to underline the effectiveness of molecular analysis in cerebral cavernous angioma, with special attention to the familial forms. Methods Multiplex Ligation-dependent Probe Amplification analysis integrates the consecutive sequence analysis of the 3 genes (Krit1/CCM1, MGC4607/CCM2, and PDCD10/CCM3) known to be responsible for cerebral cavernous malformation lesions. Results The Multiplex Ligation-dependent Probe Amplification analysis revealed a new mutation, a heterozygous exon 9/10 deletion of Krit1, in the proband and in all affected family members. Conclusions The identification of the molecular defect allows physicians to screen family members at risk and to identify affected individuals before the onset of clinical symptoms caused by the presence of lesions.

Signal transduction pathways of mantle cell lymphoma: a phosphoproteome-based study.

Mantle cell lymphoma (MCL) is an incurable hematologic malignancy whose pathogenesis is only partly understood. The aim of the present study was to define a "core phosphoproteome" in MCL cell lines that is representative of primary MCL in order to improve knowledge of the signal transduction pathways involved in its tumorigenesis. We have analyzed phosphorylated proteins in several MCL cell lines by immobilized metal affinity chromatography and separation by 2-D PAGE, followed by RP-HPLC coupled with MS/MS identification. These data were correlated with information on copy number gains obtained by SNP-chip analysis. Several of the proteins identified could be linked to a specific signal transduction pathway, and have been recently recognized as important players in MCL pathogenesis, such as nuclear factor-kappaB (NF-kappaB) and phosphoinositide-3 kinase-mammalian target of rapamycin (PI3K-mTOR). However, our data also implicate a number of novel proteins and pathways in the pathobiology of MCL, one of which is mitochondrial signaling. A second-level analysis identified MAPK1, CK2, CK1, PKCzeta, and PKCepsilon as candidate upstream molecules. Our study provides new insights in MCL pathogenesis and helps to form the basis for testing new target-specific therapeutics.

Clinical usefulness of FDG-PET/CT scan imaging in the management of posttransplant lymphoproliferative disease.

BACKGROUND: Posttransplant lymphoproliferative disease (PTLD) is, aside skin cancer, the most common malignancy occurring after solid organ transplant in adults. Fluorodeoxyglucose (FDG) positron emission tomography (PET) has proved useful in the management of lymphomas. METHODS: We report our experience with the use of FDG-PET inline with computed tomography (CT) scanning in the management of four transplant recipients with histologically confirmed PTLD, including three monomorphic PTLDs and one polymorphic PTLD. RESULTS: FDG-PET/CT scan at diagnosis showed increased FDG uptake in all examined PTLD lesions, and the disease was upstaged on the basis of FDG-PET/CT scan results over conventional CT scanning in one patient. At the end of treatment, PET/CT scans no longer demonstrated FDG uptake in the original PTLD lesions in all patients. Complete remission of disease persisted for at least 1 year after diagnosis in all. CONCLUSIONS: Our results strongly support that FDG-PET scanning is highly specific for diagnosis and follow-up of PTLD. The clinical relevance of including FDG-PET/CT scanning in the management of PTLD should be evaluated in a larger prospective cohort study.

Glutamate-cysteine ligase polymorphism, hypertension, and male sex are associated with cardiovascular events. Biochemical and genetic characterization of Italian subpopulation.

BACKGROUND: Glutathione (GSH) is an important intravascular scavenger that protects endothelial cells from atherosclerosis. However, it is still unknown whether cardiovascular (CV) events are associated with metabolic and genetic factors, linked to GSH synthesis in an Italian subpopulation, and if a glutamate-cysteine ligase polymorphism within the catalytic subunit (GCLC) could affect blood and plasma GSH concentrations. METHODS: One hundred subjects, with or without CV risk factors, were enrolled to evaluate plasma and erythrocyte redox status (GSH, homocysteine, cysteine, cysteinylglycine), antioxidant vitamins (alpha-tocopherol and ascorbate), malondialdehyde, a lipid peroxidation product, and the presence of the GCLC-129 C/T polymorphism; an experimental hyperhomocysteinemia after methionine-induced stimulation of transsulfuration pathway was performed in 91% of enrolled subjects. Clinical, biochemical, and genetic variables were correlated with the presence of CV events (myocardial infarction, transient ischemic attacks, and stroke). RESULTS: By multiple logistic regression analysis, male sex (P = .027), hypertension (P = .001), and GCLC C/T genotype (P = .009) were the only variables associated with events. Plasma alpha-tocopherol content decreased postmethionine in the T allele subjects compared with wild type (P for time x group interaction = .001). Plasma-reduced GSH level was higher in C/T than in C/C genotype subjects at both time points (P for group = .03), whereas intracellular GSH concentration did not differ between the 2 genotype groups either at baseline or postmethionine. CONCLUSIONS: GCLC T allele, together with hypertension and male sex, is associated with CV events in our study population. Moreover, after stimulation of transsulfuration, intracellular GSH content is preserved in T allele subjects, probably by increases in GSH turnover and export, and consumption of alpha-tocopherol.

Estimation of the physiological mechanical conditioning in vascular tissue engineering by a predictive fluid-structure interaction approach.

The in vitro replication of physiological mechanical conditioning through bioreactors plays a crucial role in the development of functional Small-Caliber Tissue-Engineered Blood Vessels. An in silico scaffold-specific model under pulsatile perfusion provided by a bioreactor was implemented using a fluid-structure interaction (FSI) approach for viscoelastic tubular scaffolds (e.g. decellularized swine arteries, DSA). Results of working pressures, circumferential deformations, and wall shear stress on DSA fell within the desired physiological range and indicated the ability of this model to correctly predict the mechanical conditioning acting on the cells-scaffold system. Consequently, the FSI model allowed us to a priori define the stimulation pattern, driving in vitro physiological maturation of scaffolds, especially with viscoelastic properties.

Herringbone-like hydrodynamic structures in microchannels: A CFD model to evaluate the enhancement of surface binding.

Selected adsorption efficiency of a molecule in solution in a microchannel is strongly influenced by the convective/diffusive mass transport phenomena that supply the target molecule to the adsorption surface. In a standard microchannel with a rectangular cross section, laminar flow regime limits the fluid mixing, thus suggesting that mass transport conditions can be improved by the introduction of herringbone-like structures. Tuning of these geometrical patterns increases the concentration gradient of the target molecule at the adsorption surface. A computational fluid dynamic (CFD) study was performed to evaluate the relation between the geometrical herringbone patterns and the concentration gradient improvement in a 14 mm long microchannel. The results show that the inhomogeneity of the concentration gradient can provide an improved and localized adsorption under specific geometrical features, which can be tuned in order to adapt the adsorption pattern to the specific assay requirements.

Microcirculation in the murine liver: a computational fluid dynamic model based on 3D reconstruction from in vivo microscopy.

The liver is organized in hexagonal functional units - termed lobules - characterized by a rather peculiar blood microcirculation, due to the presence of a tangled network of capillaries - termed sinusoids. A better understanding of the hemodynamics that governs liver microcirculation is relevant to clinical and biological studies aimed at improving our management of liver diseases and transplantation. Herein, we built a CFD model of a 3D sinusoidal network, based on in vivo images of a physiological mouse liver obtained with a 2-photon microscope. The CFD model was developed with Fluent 16.0 (ANSYS Inc., Canonsburg, PA), particular care was taken in imposing the correct boundary conditions representing a physiological state. To account for the remaining branches of the sinusoids, a lumped parameter model was used to prescribe the correct pressure at each outlet. The effect of an adhered cell on local hemodynamics is also investigated for different occlusion degrees. The model here proposed accurately reproduces the fluid dynamics in a portion of the sinusoidal network in mouse liver. Mean velocities and mass flow rates are in agreement with literature values from in vivo measurements. Our approach provides details on local phenomena, hardly described by other computational studies, either focused on the macroscopic hepatic vasculature or based on homogeneous porous medium model.

Label-free identification of activated T lymphocytes through tridimensional microsensors on chip.

Label-free approaches to assess cell properties ideally suit the requirements of cell-based therapeutics, since they permit to characterize cells with minimal perturbation and manipulation, at the benefit of sample recovery and re-employment for treatment. For this reason, label-free techniques would find sensible application in adoptive T cell-based immunotherapy. In this work, we describe the label-free and single-cell detection of in vitro activated T lymphocytes in flow through an electrical impedance-based setup. We describe a novel platform featuring 3D free-standing microelectrodes presenting passive upstream and downstream extensions and integrated into microfluidic channels. We employ such device to measure the impedance change associated with T cell activation at electrical frequencies maximizing the difference between non-activated and activated T cells. Finally, we harness the impedance signature of unstimulated T cells to set a boundary separating activated and non-activated clones, so to characterize the selectivity and specificity of the system. In conclusion, the strategy here proposed highlights the possible employment of impedance to assess T cell activation in label-free.