USE OF GENETICALLY MODIFIED MICE TO STUDY THE ANTI-INFLAMMATORY AND IMMUNOMODULATORY ROLE OF HIGH-DENSITY LIPOPROTEINS DURING ATHEROSCLEROSIS DEVELOPMENT

Introduction: High-density lipoproteins (HDLs) have several anti-atherosclerotic/anti-inflammatory properties and the apolipoprotein A-I (apoA-I), the main protein component of HDL, plays a major role. The aim of the present project was to investigate the impact of apoA-I on atherosclerosis development, phenotype and inflammation, through the use of genetically modified mice. Methods: This study was performed in C57Bl/6 wild-type mice, resistant to atherosclerosis development, and in three athero-prone mouse lines: apoEKO, apoEKO with the additional deletion of murine apoA-I (dKO) and dKO overexpressing human apoA-I (hA-I). These animals were fed a chow diet for 22 weeks. Cholesterolemia was quantified by FPLC analysis; atherosclerosis development was evaluated at the whole aorta by en-face analysis and at the aortic sinus and common coronary arteries by histology. Skin biopsies were processed for both light (LM) and transmission electron microscopy (TEM); moreover, lipids were extracted from skin and the lipid content were quantified. Finally, skin draining lymph nodes and spleens were harvested for histology and leukocyte populations were investigated by flow cytometry. Results: As expected, in wild-type mice all the cholesterol was found into the HDL fractions and in apoEKO mice the majority of cholesterol was present into the VLDL/LDL fractions. In dKO animals HDL-cholesterol was instead almost absent and VLDL/LDL-cholesterol was about 30% lower compared to apoEKO mice. hA-I mice were characterized by a large HDL-cholesterol peak and by a marked presence of VLDL/LDL particles although less prominent that in apoEKO mice. En-face analysis showed that dKO and apoEKO mice had a similar extent of atherosclerotic plaques at the aortic arch (6.9\ub15.6%, 7.2\ub15.5%, respectively). No atherosclerosis was instead observed in wild-type as well as in hA-I mice. At the aortic sinus, dKO mice showed a significant increase in lesion development compared to both apoEKO and hA-I mice (6.2\ub10.5x105\u3bcm2 vs. 3.2\ub10.7x105\u3bcm2 and 0.4\ub10.3x105\u3bcm2, p<0.001). As expected, no atherosclerotic plaques were found at this district in wild-type mice. In addition, preliminary data suggest that dKO, but not apoEKO mice develop significant atherosclerotic plaques at the common coronary arteries (76,25\ub18,62% and 0\ub10%, respectively). LM analysis displayed that the skin of EKO and h-A-I mice was comparable with C57Bl/6 mice, whereas dKO animals showed an increase in dermal thickness and the presence of foam cells and lymphocytes in reticular dermis. TEM analysis revealed the accumulation of cholesterol cleft in the papillary dermis and of cholesterol crystals within foam cells. Quantification of the skin lipid content showed a strong accumulation of both unesterified and esterified cholesterol in dKO mouse skin. The weights of skin lymph nodes in dKO mice were higher compared to those of the other mouse lines. Histological analysis showed the presence of foamy macrophages, granulomatous reactions surrounding cholesterol crystals and dilated sinuses. Conversely, spleen weight and histology were unaffected by genotypes. In peripheral blood, skin draining lymph nodes and spleen, dKO mice showed a significantly higher percentage of CD4+ T effector memory lymphocytes and a reduced percentage of T na\uefve lymphocytes compared to other experimental group. The percentage of monocytoid or plasmacytoid dendritic cells, B lymphocytes and monocyte subsets in secondary lymphoid organs were not influenced by the genotype. Conclusion: Taken together our results demonstrate that murine apoA-I deletion into an apoEKO background markedly exacerbates atherosclerosis development both at the aortic sinus and common coronary arteries. ApoA-I deficiency is associated with an impaired cholesterol homeostasis in the skin. This model reproduces for the first time the cutaneous phenotype of human apoA-I deficiency, characterized by xanthomatous deposition in the absence of a hyperlipidemic status. In addition, our data show that apoA-I deletion is related to an enlargement of skin draining lymph nodes which are characterized by foamy macrophages accumulation and cholesterol deposition, predisposing to granulomatous reactions. Finally, the new murine model displays activation and increase in CD4+ T effector memory lymphocytes in peripheral blood and lymphoid organs

Preliminary validity of the Draw a Shape Test for upper extremity assessment in multiple sclerosis

Multiple sclerosisEsclerosi múltipleEsclerosis múltipleObjective To validate the smartphone sensor-based Draw a Shape Test – a part of the Floodlight Proof-of-Concept app for remotely assessing multiple sclerosis-related upper extremity impairment by tracing six different shapes. Methods People with multiple sclerosis, classified functionally normal/abnormal via their Nine-Hole Peg Test time, and healthy controls participated in a 24-week, nonrandomized study. Spatial (trace accuracy), temporal (mean and variability in linear, angular, and radial drawing velocities, and dwell time ratio), and spatiotemporal features (trace celerity) were cross-sectionally analyzed for correlation with standard clinical and brain magnetic resonance imaging (normalized brain volume and total lesion volume) disease burden measures, and for capacity to differentiate people with multiple sclerosis from healthy controls. Results Data from 69 people with multiple sclerosis and 18 healthy controls were analyzed. Trace accuracy (all shapes), linear velocity variability (circle, figure-of-8, spiral shapes), and radial velocity variability (spiral shape) had a mostly fair/moderate-to-good correlation (|r| = 0.14–0.66) with all disease burden measures. Trace celerity also had mostly fair/moderate-to-good correlation (|r| = 0.18–0.41) with Nine-Hole Peg Test performance, cerebellar functional system score, and brain magnetic resonance imaging. Furthermore, partial correlation analysis related these results to motor impairment. People with multiple sclerosis showed greater drawing velocity variability, though slower mean velocity, than healthy controls. Linear velocity (spiral shape) and angular velocity (circle shape) potentially differentiate functionally normal people with multiple sclerosis from healthy controls. Interpretation The Draw a Shape Test objectively assesses upper extremity impairment and correlates with all disease burden measures, thus aiding multiple sclerosis-related upper extremity impairment characterization.This research was funded by F. Hoffmann-La Roche Ltd, Basel, Switzerland

Tablet splitting in elderly patients with dementia: The case of quetiapine

Quetiapine is an atypical antipsychotic approved for treating schizophrenia, bipolar depression, and mania but is frequently used in an off-label manner to control the behavioral and psychological symptoms of dementia in elderly patients with dementia. Due to the need to personalize doses for elderly patients with dementia, quetiapine tablet manipulation is widespread in hospital settings, long-term care facilities, and patient homes. The aim of this study was to assess the impact of the different splitting techniques on quetiapine fumarate tablets by analysing the obtained sub-divided tablets and to discuss compliance with the European Pharmacopoeia limits on whole and split tablets. Quetiapine fumarate tablets of two dose strengths were taken at random (in a number able to assure a power of 0.8 during statistical comparison) and were split with a kitchen knife or tablet cutter. The weight and the drug content were determined for each half tablet. The obtained data were compared to the European Pharmacopoeia limits. The differences between the different splitting techniques were statistically tested. Data showed that split tablets, independently of the dose strength and the technique employed, were not compliant with the European Pharmacopoeia specifications for both entire and subdivided tablets in terms of weight and content uniformity. Thus, such a common practice could have potential effects on treatment efficacy and toxicity, especially when also considering the fragility of the elderly target population in which polypharmacotherapy is very common. These results indicate a compelling need for flexible quetiapine formulations that can assure more accurate dose personalization

Osteopontin: A New Facilitating Factor in Alopecia Areata Pathogenesis?

Osteopontin (OPN) is a multifunctional glycophosphoprotein secreted by many cell types, including osteoblasts, lymphocites, macrophages, epithelial cells, and vascular smooth muscle cells. It has been implicated in many physiological and pathological processes, such as cell-mediated immunity, inflammation, cell survival, and tumor invasion and metastasis. Osteopontin has multiple emerging roles in cutaneous biology and pathology and OPN involvement has been emphasized in Th1-mediated diseases such as psoriasis. Alopecia areata (AA) is a form of non-scarring hair loss affecting anagen stage hair follicles with a multifactorial autoimmune pathogenesis characterized by a prevalent Th1 cytokine profile. Given the role of osteopontin in Th1-mediated inflammation, we have postulated that OPN may be involved in AA pathogenesis. The aim of our study was to investigate plasma OPN level in alopecia areata before and after DPCP treatment. Our results showed that OPN plasma levels in patients with alopecia areata were higher than in healthy controls, but patients achieving complete recovery after DPCP treatment did not show a statistically significant reduction of OPN plasma levels.</p

Oral drug therapy in elderly with dysphagia: between a rock and a hard place!

Demographic indicators forecast that by 2050, the elderly will account for about one-third of the global population. Geriatric patients require a large number of medicines, and in most cases, these products are administered as solid oral solid dosage forms, as they are by far the most common formulations on the market. However, this population tends to suffer difficulties with swallowing. Caregivers in hospital geriatric units routinely compound in solid oral dosage forms for dysphagic patients by crushing the tablets or opening the capsules to facilitate administration. The manipulation of a tablet or a capsule, if not clearly indicated in the product labeling, is an off-label use of the medicine, and must be supported by documented scientific evidence and requires the patient's informed consent. Compounding of marketed products has been recognized as being responsible for an increased number of adverse events and medical errors. Since extemporaneous compounding is the rule and not the exception in geriatrics departments, the seriousness and scope of issues caused by this daily practice are probably underestimated. In this article, the potential problems associated with the manipulation of authorized solid oral dosage forms are discussed

The Economic Burden of Multiple Myeloma. Definition of a Model for Forecasting Patients’ Costs

Background: The aim of this study was to evaluate healthcare costs in a single-centre population of patients with multiple myeloma (MM), in an attempt to develop a model for forecasting costs. Methods: A cohort of 387 MM patients, diagnosed at Policlinico San Matteo (Pavia, Italy), between 2002 and 2014, was analysed grouping patients into those eligible (n=223) or not eligible (n=164) for transplantation. After descriptive statistics, the benchmark model - Ordinary Least Squares - and different variations of the Generalized Linear Model were adopted. Results: The average total cost per patient was around €28,500 for patients not eligible for transplantation and around €87,000 for the eligible ones. The difference in marginal costs for transplant-eligible patients was probably due to higher costs for hospitalisation and the costs of the transplant procedure itself. The analysis highlighted four determinants useful for building a model to forecast expenditure: age, bortezomib use, lenalidomide use, and number of lines of therapies. The two most important determinants of expenditure were use of the novel agents and the total number of lines of therapy, which reflects a higher number of doses and a greater need for accesses to hospital. Conclusion: In conclusion, using a Generalized Linear Model, we identified four determinants in our cohort which were useful for building a model to predict expenditure for MM patients. Although the analysis was performed in a particular setting in a single hospital, the model could be applied to any scenario of patients

Magnetic Resonance Imaging Visualization of Vulnerable Atherosclerotic Plaques at the Brachiocephalic Artery of Apolipoprotein E Knockout Mice by the Blood-pool Contrast Agent B22956/1

The aim of this study was to identify, by magnetic resonance imaging (MRI), the ability of the blood-pool contrast agent B22956/1 to detect atherosclerotic plaques developing at the brachiocephalic artery of apolipoprotein E knockout (apoE-KO) mice and to possibly identify vulnerable atherosclerotic lesions. After high-fat feeding for 8 or 12 weeks, MRIs of brachiocephalic arteries were acquired before and after B22956/1 administration; then vessels were removed and analyzed by histology. B22956/1 injection caused a rapid increase in plaque signal enhancement and plaque to muscle contrast values, which remained stable up to 70 minutes. A linear correlation between signal enhancement and macrophage content was found 10 minutes after B22956/1 injection ( p < .01). Signal enhancement and plaque to muscle contrast values correlated with macrophage content 40 minutes after contrast agent administration ( p < .01). Finally, 70 minutes after B22956/1 infusion, plaque to muscle contrast significantly correlated with the percentage of stenosis ( p < .005). B22956/1 administration to high fat-fed apoE-KO mice resulted in a rapid enhancement of atherosclerotic plaques and in a great ability to rapidly visualize vulnerable plaques, characterized by a high macrophage content. These results suggest that B22956/1 could represent an interesting tool for the identification of atherosclerotic plaques potentially leading to acute cardiovascular events

Preliminary validity of the Draw a Shape Test for upper extremity assessment in multiple sclerosis

Objective To validate the smartphone sensor-based Draw a Shape Test - a part of the Floodlight Proof-of-Concept app for remotely assessing multiple sclerosis-related upper extremity impairment by tracing six different shapes. Methods People with multiple sclerosis, classified functionally normal/abnormal via their Nine-Hole Peg Test time, and healthy controls participated in a 24-week, nonrandomized study. Spatial (trace accuracy), temporal (mean and variability in linear, angular, and radial drawing velocities, and dwell time ratio), and spatiotemporal features (trace celerity) were cross-sectionally analyzed for correlation with standard clinical and brain magnetic resonance imaging (normalized brain volume and total lesion volume) disease burden measures, and for capacity to differentiate people with multiple sclerosis from healthy controls. Results Data from 69 people with multiple sclerosis and 18 healthy controls were analyzed. Trace accuracy (all shapes), linear velocity variability (circle, figure-of-8, spiral shapes), and radial velocity variability (spiral shape) had a mostly fair/moderate-to-good correlation (|r| = 0.14-0.66) with all disease burden measures. Trace celerity also had mostly fair/moderate-to-good correlation (|r| = 0.18-0.41) with Nine-Hole Peg Test performance, cerebellar functional system score, and brain magnetic resonance imaging. Furthermore, partial correlation analysis related these results to motor impairment. People with multiple sclerosis showed greater drawing velocity variability, though slower mean velocity, than healthy controls. Linear velocity (spiral shape) and angular velocity (circle shape) potentially differentiate functionally normal people with multiple sclerosis from healthy controls. Interpretation The Draw a Shape Test objectively assesses upper extremity impairment and correlates with all disease burden measures, thus aiding multiple sclerosis-related upper extremity impairment characterization

Skin morphology in double apoA-I/apoE knock-out mice: a structural and ultrastructural study

Apolipoprotein(apo)A-I, the main protein component of high density lipoproteins (HDLs), plays a major role in cholesterol removal from peripheral tissues and increasing evidence supports its function as an important regulator of the immune response (Annema et al., 2013). The aim of the study was to evaluate the effect of apoA-I deficiency in dyslipidemic mice, when fed a low-fat/low-cholesterol diet. Three lines of male mice were considered: wild-type mice as controls, apoE-KO mice as dyslipidemic model (Zhang et al.,1992) and apoA-I/apoE double KO mice (DKO mice). Whereas in wild-type mice cholesterol circulates almost exclusively in HDLs, apoE-KO mice are hypercholesterolemic and cholesterol mostly circulates in low-density lipoproteins. In DKO mice, cholesterol levels are comparable to wild-type mice, but HDLs are almost absent and cholesterol entirely accumulates in low-density lipoproteins. In the present study, all animals were maintained on a low-fat/low-cholesterol diet up to 30 weeks of age. At sacrifice, skin biopsies from two different anatomical areas (thoracic and abdominal regions) were harvested from each animal and processed for both light (LM) and transmission electron microscopy (TEM). Whereas the skin of apoE-KO mice was comparable to that of control mice, LM analysis in DKO mice revealed an increase in dermal thickness and a massive presence of foam cells and lymphocytes. TEM analysis showed the presence of cholesterol clefts in the papillary dermis and inside foam cells in the reticular dermis. In conclusion, our results demonstrate that in DKO mice fed a low-fat/low-cholesterol diet, the lack of apoA-I is responsible for an aberrant skin morphology, with an exacerbated inflammatory response, possibly caused by a local cholesterol accumulation