Low-density lipoprotein (LDL) receptor/transferrin fusion protein: in vivo production and functional evaluation as a potential therapeutic tool for lowering plasma LDL cholesterol

A soluble form of human low-density lipoprotein receptor (LDL-R) fused in frame with rabbit transferrin (LDL-Rs(hu)/Tf(rab)) is assessed in vivo as a therapeutic tool for lowering plasma LDL cholesterol. The cDNA encoding LDL-Rs(hu)/Tf(rab) is expressed in mice, using a hydrodynamics-based gene transfer procedure. The transgene is transcribed in the liver of transduced animals and the corresponding protein is secreted into the bloodstream. Circulating LDL-Rs(hu)/Tf(rab) binds LDL specifically, thus indicating that it is correctly processed through the cellular compartments in vivo. More importantly, the expression of LDL-Rs(hu)/Tf(rab) allows the removal of injected human (125)I-labeled LDL ((123)I-LDL) from the bloodstream of transduced CD1 mice, which show faster LDL plasma clearance, anticipating by approximately 90 min the same clearance value observed in control animals. A similar effect is observed in transduced LDL-R(-/-) mice, in which the clearance of injected human LDL depends solely on the presence of circulating LDL-Rs(hu) /Tf(rab). In these animals the extent of plasma LDL clearance is directly related to the concentration of LDL-Rs(hu)/Tf(rab) in the blood. Finally, LDL-Rs(hu)/Tf(rab) does not alter the pattern of LDL organ distribution: in transduced animals, as well as in control animals, liver and bladder are the predominantly labeled organs after (123)I-LDL injection. However, LDL-Rs(hu)/Tf(rab) has a quantitative effect on LDL tissue deposition: in treated animals LDL-Rs(hu)/Tf(rab) determines an increase in radioactivity in the liver at early times after (123)I-LDL injection and a progressive labeling of the bladder, starting 20 min after injection.     

Defibrotide for Sinusoidal Obstruction Syndrome/Veno-Occlusive Disease Prophylaxis in High-Risk Adult Patients: A Single-Center Experience Study

Sinusoidal obstruction syndrome (SOS), also known as hepatic veno-occlusive disease (VOD), is a serious complication after hematopoietic stem cell transplantation (HSCT). SOS/VOD usually occurs within 3 weeks of HSCT, but the 2016 European Society for Blood and Marrow Transplantation diagnosis criteria have been revised to include late forms. Prophylactic use of defibrotide is recommended in the pediatric setting, but its value remains uncertain in the adult population. We report here a single-center series of 63 adult patients considered at high risk for SOS/VOD who received defibrotide prophylaxis in combination with ursodeoxycholic acid between May 2012 and August 2016. The median duration of defibrotide therapy was 23 days. Bleeding occurred in 14 patients (21.5%). Defibrotide prophylaxis was discontinued in 7 patients (10.8%): 4 cases (6.3%) due to bleeding and 3 cases (4.6%) because of the need for antithrombotic therapy. Overall, SOS/VOD occurred in 4 cases (6.3%) within 21 days after HSCT (days 13 and 14) in 2 cases and late-onset SOS/VOD (days 57 and 58) in the other 2 cases. SOS/VOD was moderate in 1 case, very severe in 3 cases, with 2 deaths related to SOS/VOD. Cumulative incidence of grades II to IV acute graft-versus-host disease and transplant-associated thrombotic microangiopathy were 22.2% and 3.2%, respectively. With a median follow-up of 31 months (range, 10.7 to 60.3), the rates of 2-year overall survival, progression-free survival, incidence of relapse, and nonrelapse mortality were 56.5%, 49%, 28.7%, and 22.3%, respectively. In our experience defibrotide prophylaxis is associated with a low incidence of SOS/VOD after allogeneic HSCT in a high-risk adult population with an acceptable safety profile.     

Validation of the Italian version of the SBMA Functional Rating Scale as outcome measure

The Spinal and Bulbar Muscular Atrophy Functional Rating Scale (SBMAFRS) is an established rating instrument used to assess the functional status of patients with Spinal and Bulbar Muscular Atrophy (SBMA). Our aim was to validate an Italian version of the scale. We administered the SBMAFRS to sixty SBMA patients during routine follow-up of clinical evaluations. To estimate the test stability, the scale was re-administered to a subset of 39 randomly selected patients after 8 weeks. The patients underwent clinical evaluation including 6-min walk. Psychometric analysis included reliability assessment and factorial analysis. To evaluate convergent validity, correlations between SBMAFRS items and muscular force assessed by manual testing, ALSFRS total score and subscales scores, and forced vital capacity, were performed. Internal consistency as measured by Cronbach’s alpha (total scale 0.85) was high. Test–retest reliability assessed by Spearman’s rho was also high. Principal component analysis with varimax rotation yielded a four-factor solution accounting for approximately 79 % of the variance. The scale total score and subscales score were strongly correlated with respective items and subscores of the ALSFRS, with respiratory function and with the 6-min walk test. In conclusion, we performed an Italian validation of the only existing disease-specific Functional Rating Scale for SBMA patients. This scale will be a useful tool not only in the clinical practice but also as an outcome measure in upcoming clinical trials.     

Risk factors in multiple sclerosis: a population-based case–control study in Sicily. Background and methods

Incidence of multiple sclerosis (MS) has steeply increased over time during the last 30 years in the city of Catania. We carried out a population-based case–control study to evaluate the possible role of both environmental and genetic factors. From 1975 to 2004 in Catania, 367 MS patients diagnosed according to the Poser’s criteria had the onset of disease. A sample of MS patients was randomly selected from this incident cohort. Three controls matched by age and sex were randomly selected from the rosters of 14 GPs. Controls were proportionally selected according to the distribution by municipality of the target population using a multistage sampling methods. All cases and controls underwent a face-to-face interview to record information concerning environmental factors and a blood sample was taken for serological and genetic analysis. 164 MS patients (64 % women; mean age of 46.4 ± 10.7) and 481 controls (69 % women; mean age of 47.7 ± 14.8) were enrolled in the study. The distribution of the whole population and the selected controls by municipalities was similar. A blood sample was taken from 150 MS cases and from 337 controls. At the end of the enrolment, we obtained a representative sample of the MS cases and population controls avoiding possible selection bias. Participation rate was very high also concerning the collection of biological specimens.     

Foot pad skin biopsy in mouse models of hereditary neuropathy

Numerous transgenic and knockout mouse models of human hereditary neuropathies have become available over the past decade. We describe a simple, reproducible, and safe biopsy of mouse skin for histopathological evaluation of the peripheral nervous system (PNS) in models of hereditary neuropathies. We compared the diagnostic outcome between sciatic nerve and dermal nerves found in skin biopsy (SB) from the hind foot. A total of five animal models of different Charcot-Marie-Tooth neuropathies, and one model of congenital muscular dystrophy associated neuropathy were examined. In wild type mice, dermal nerve fibers were readily identified by immunohistochemistry, light, and electron microscopy and they appeared similar to myelinated fibers in sciatic nerve. In mutant mice, SB manifested myelin abnormalities similar to those observed in sciatic nerves, including hypomyelination, onion bulbs, myelin outfolding, redundant loops, and tomacula. In many strains, however, SB showed additional abnormalities--fiber loss, dense neurofilament packing with lower phosphorylation status, and axonal degeneration-undetected in sciatic nerve, possibly because SB samples distal nerves. SB, a reliable technique to investigate peripheral neuropathies in human beings, is also useful to investigate animal models of hereditary neuropathies. Our data indicate that SB may reveal distal axonal pathology in mouse models and permits sequential follow-up of the neuropathy in an individual mouse, thereby reducing the number of mice necessary to document pathology of the PNS.     

Molecular and functional characterization of allogantigen-specific anergic T cells suitable for cell therapy

Background

CD4⁺ regulatory T cells are a specialized subset of T cells that actively control immune responses. Several experimental protocols have been used to expand natural regulatory T cells and to generate adaptive type 1 regulatory T cells for regulatory T-cell-based therapies.

Design and Methods

The ability of exogenous recombinant human interleukin-10 to induce alloantigen-specific anergy in T cells was investigated and compared to that of interleukin-10 derived from tolerogenic dendritic cells, in mixed lymphocyte cultures. A detailed characterization of the effector functions of the resulting anergized T cells is reported.

Results

Interleukin-10, whether exogenous or derived from tolerogenic dendritic cells, induces a population of alloantigen-specific T cells (interleukin-10-anergized T cells) containing type 1 regulatory T cells, which are anergic and actively suppress alloantigen-specific effector T cells present within the mixed population. Interleukin-10-induced anergy is transforming growth factor-β independent, and is associated with a decreased frequency of alloantigen-specific cytotoxic T lymphocyte precursors, but interleukin-10-anergized T cells are still responsive to third-party, bacterial, and viral antigens. Tolerogenic dendritic cells are more powerful than exogenous interleukin-10 in generating type 1 regulatory T-cell precursors, and are also effective in the context of HLA-matched donors.

Conclusions

Based on these studies, we have developed an efficient and reproducible in vitro method to generate antigen-specific type 1 regulatory T-cell precursors starting from total peripheral blood cells with minimal cell manipulation and suitable for generating type 1 regulatory T cells for regulatory T-cell-based therapies.