Alessandro Rubinacci

Gene therapy augments the efficacy of hematopoietic cell transplantation and fully corrects mucopolysaccharidosis type I phenotype in the mouse model

Type I mucopolysaccharidosis (MPS I) is a lysosomal storage disorder caused by the deficiency of α-L-iduronidase, which results in glycosaminoglycan accumulation in tissues. Clinical manifestations include skeletal dysplasia, joint stiffness, visual and auditory defects, cardiac insufficiency, hepatosplenomegaly, and mental retardation (the last being present exclusively in the severe Hurler variant). The available treatments, enzyme-replacement therapy and hematopoietic stem cell (HSC) transplantation, can ameliorate most disease manifestations, but their outcome on skeletal and brain disease could be further improved. We demonstrate here that HSC gene therapy, based on lentiviral vectors, completely corrects disease manifestations in the mouse model. Of note, the therapeutic benefit provided by gene therapy on critical MPS I manifestations, such as neurologic and skeletal disease, greatly exceeds that exerted by HSC transplantation, the standard of care treatment for Hurler patients. Interestingly, therapeutic efficacy of HSC gene therapy is strictly dependent on the achievement of supranormal enzyme activity in the hematopoietic system of transplanted mice, which allows enzyme delivery to the brain and skeleton for disease correction. Overall, our data provide evidence of an efficacious treatment for MPS I Hurler patients, warranting future development toward clinical testing.

Vitamin D levels and risk of multiple sclerosis in patients with clinically isolated syndromes

Background: Growing evidence suggests that vitamin D deficiency may be one of the most important environmental factors for the development of multiple sclerosis (MS). Objectives: The objectives of this paper are to evaluate serum 25-hydroxyvitamin D (25(OH)D) levels in patients with clinically isolated syndromes (CIS) and to examine whether they are related to MS risk. Methods: This is a retrospective study of 100 CIS patients hospitalized from 2000 to 2009 at San Raffaele Hospital, Milan, Italy. We evaluated baseline 25(OH)D level as well as clinical, brain magnetic resonance imaging (MRI) and cerebrospinal fluid (CSF) data. Results: A total of 52% of CIS patients had vitamin D deficiency (25(OH)D < 50 nmol/l). During follow-up (median: 7.17 years), 55 patients developed clinically definite MS (CDMS). Patients with very low (< 10th percentile) and low (< 25th percentile) 25(OH)D levels were particularly at risk of CDMS (HRs (95% CIs): 2.12 (0.91–4.96) and 1.61 (0.85–3.03), respectively), while no further reduction in the HRs of disease was observed at high levels of 25(OH)D. This association was even stronger after adjustment for additional risk factors for CDMS development (HRs (95% CIs) for 25(OH)D levels < 10th and 25th percentiles: 3.34 (1.32–8.45) and 2.04 (0.96–4.36), respectively). Conclusion: Low serum vitamin D is associated with increased MS risk in patients with CIS.

Ovariectomy Sensitizes Rat Cortical Bone to Whole-Body Vibration

This study was designed to determine the modulatory effect of estrogen on mechanical stimulation in bone. Trabecular and cortical bone compartments of ovariectomized rats exposed to whole-body vibration of different amplitudes were evaluated by peripheral quantitative computed tomographic (pQCT) analysis and histomorphometry and compared to controls not exposed to vibration. Rats underwent whole-body vibration (20xa0minutes/day, 5xa0days/week) on a vibration platform for 2xa0months. The control rats were placed on the platform without vibration for the same time. We divided rats into six groups: a sham control (SHAM); a sham vibrated (SHAM-V) at 30 Hz, 0.6xa0g; a SHAM-V at 30 Hz, 3g; an ovariectomized control (OVX); an ovariectomized vibrated (OVX-V) at 30 Hz, 0.6xa0g; and an OVX-V at 30 Hz, 3g. In vivo, pQCT analyses of the tibiae were performed at the start of the experiment and after 4xa0and 8xa0weeks. After 8xa0weeks the tibiae were excised for histomorphometric and for in vitro pQCT analyses. In the SHAM-V group, vibration had no effect upon the different bone parameters. In the OVX-V group, vibration induced a significant increase compared to the OVX group of the cortical and medullary areas (Pxa0<xa00.01) and of the periosteal (Pxa0<xa00.01) and endosteal (Pxa0<xa00.05) perimeters at the 3xa0g vibration. The strain strength index increased in the OVX-V group significantly (Pxa0<xa00.01) at the higher vibration. The results showed that low-amplitude, high-frequency whole-body vibration is anabolic to bone in OVX animals. The osteogenic potential is limited to the modeling of the bone cortex and depends on the amplitude of the vibration.

Urokinase plasminogen activator receptor affects bone homeostasis by regulating osteoblast and osteoclast function.

The uPAR and its ligand uPA are expressed by both osteoblasts and osteoclasts. Their function in bone remodeling is unknown. We report that uPAR‐lacking mice display increased BMD, increased osteogenic potential of osteoblasts, decreased osteoclasts formation, and altered cytoskeletal reorganization in mature osteoclasts.

Bone marrow as an alternative site for islet transplantation

The liver is the current site for pancreatic islet transplantation, but has many drawbacks due to immunologic and nonimmunologic factors. We asked whether pancreatic islets could be engrafted in the bone marrow (BM), an easily accessible and widely distributed transplant site that may lack the limitations seen in the liver. Syngeneic islets engrafted efficiently in the BM of C57BL/6 mice rendered diabetic by streptozocin treatment. For more than 1 year after transplantation, these animals showed parameters of glucose metabolism that were similar to those of nondiabetic mice. Islets in BM had a higher probability to reach euglycemia than islets in liver (2.4-fold increase, P = .02), showed a compact morphology with a conserved ratio between alpha and beta cells, and affected bone structure only very marginally. Islets in BM did not compromise hematopoietic activity, even when it was strongly induced in response to a BM aplasia-inducing infection with lymphocytic choriomeningitis virus. In conclusion, BM is an attractive and safe alternative site for pancreatic islet transplantation. The results of our study open a research line with potentially significant clinical impact, not only for the treatment of diabetes, but also for other diseases amenable to treatment with cellular transplantation.

Effects of calcitonin gene-related peptide and amylin on human osteoblast-like cells proliferation.

Expression of mRNA for calcitonin gene-related peptide (CGRP) and CGRP receptor has been detected in osteoblasts indicating that CGRP could play a role in bone metabolism. In the present study, we evaluated the effect of CGRP on primary culture of human osteoblast-like cells proliferation. The peptide was able to stimulate [3H]thymidine incorporation in human osteoblast-like cells with a maximal effect at 10(-8) M. The proliferating activity of CGRP was not inhibited by the two antagonists, CGRP-(8-37) or amylin-(8-37), whereas amylin fragment antagonized the proliferating activity of amylin. In human osteoblast-like cells CGRP, but not amylin, was able to stimulate adenylyl cyclase activity and this effect was completely antagonized only by CGRP-(8-37) and not by amylin-(8-37). These data suggest that the CGRP induced stimulation of cAMP is not involved in the peptide proliferating effect in human osteoblast-like cells and that in this cell population there are receptor subtypes for CGRP, distinct from that of amylin.

ADA-deficient SCID is associated with a specific microenvironment and bone phenotype characterized by RANKL/OPG imbalance and osteoblast insufficiency.

Adenosine deaminase (ADA) deficiency is a disorder of the purine metabolism leading to combined immunodeficiency and systemic alterations, including skeletal abnormalities. We report that ADA deficiency in mice causes a specific bone phenotype characterized by alterations of structural properties and impaired mechanical competence. These alterations are the combined result of an imbalanced receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoprotegerin axis, causing decreased osteoclastogenesis and an intrinsic defect of osteoblast function with subsequent low bone formation. In vitro, osteoblasts lacking ADA displayed an altered transcriptional profile and growth reduction. Furthermore, the bone marrow microenvironment of ADA-deficient mice showed a reduced capacity to support in vitro and in vivo hematopoiesis. Treatment of ADA-deficient neonatal mice with enzyme replacement therapy, bone marrow transplantation, or gene therapy resulted in full recovery of the altered bone parameters. Remarkably, untreated ADA-severe combined immunodeficiency patients showed a similar imbalance in RANKL/osteoprotegerin levels alongside severe growth retardation. Gene therapy with ADA-transduced hematopoietic stem cells increased serum RANKL levels and childrens growth. Our results indicate that the ADA metabolism represents a crucial modulatory factor of bone cell activities and remodeling.

In vivo leptin expression in cartilage and bone cells of growing rats and adult humans

The present investigation was carried out to analyse, immunohistochemically, in vivo leptin expression in cartilage and bone cells, the latter restricted to the elements of the osteogenic system (stromal cells, osteoblasts, osteocytes, bone lining cells). Observations were performed on the first lumbar vertebra, tibia and femur of four rats and on the humerus, femur and acromion of four patients. Histological sections of paraffin‐embedded bone samples were immunostained using antibody to leptin. The results showed that, in growing rat bone, leptin is expressed in chondrocytes and stromal cells, but not in osteoblasts; bone lining cells were not found in the microscopic fields examined. In adult human bone, leptin is expressed in chondrocytes, stromal cells and bone lining cells; osteoblasts were not found in the microscopic fields examined. Osteocytes were found to be leptin positive only occasionally and focally in both rat and human bone. The in vivo findings reported show, for the first time, that leptin appears to be expressed only in the cells of the osteogenic lineage (stromal cells, bone lining cells, osteocytes) that, with respect to osteoblasts, are permanent and inactive, i.e. in those cells that according to our terminology constitute the bone basic cellular system (BBCS). Because the BBCS seems to be primarily involved in sensing and integrating mechanical strains and biochemical factors and then in triggering and driving bone formation and/or bone resorption, it appears that leptin seems to be mainly involved in modulating the initial phases of bone modelling and remodelling processes.

Heritability and Demographic Analyses in the Large Isolated Population of Val Borbera Suggest Advantages in Mapping Complex Traits Genes

Background Isolated populations are a useful resource for mapping complex traits due to shared stable environment, reduced genetic complexity and extended Linkage Disequilibrium (LD) compared to the general population. Here we describe a large genetic isolate from the North West Apennines, the mountain range that runs through Italy from the North West Alps to the South. Methodology/Principal Findings The study involved 1,803 people living in 7 villages of the upper Borbera Valley. For this large population cohort, data from genealogy reconstruction, medical questionnaires, blood, anthropometric and bone status QUS parameters were evaluated. Demographic and epidemiological analyses indicated a substantial genetic component contributing to each trait variation as well as overlapping genetic determinants and family clustering for some traits. Conclusions/Significance The data provide evidence for significant heritability of medical relevant traits that will be important in mapping quantitative traits. We suggest that this population isolate is suitable to identify rare variants associated with complex phenotypes that may be difficult to study in larger but more heterogeneous populations.

Is there an impact of birth weight and early life nutrition on bone mineral content in preterm born infants and children

Low birth weight and early life nutrition have a strong impact on the skeleton of infants appropriate for gestational age (AGA), as shown by single photon absorptiometry (1,2) and radiography (3 ) . The mineralization defect of bone matrix depends mostly on the mineral and vitamin D formulation of the diet (4). While the immediate effects of low birth weight and of early life nutrition on bone mineralization are accepted widely, there is poor agreement on their late effects. According to Helin et al. (3, preterm infants continue to show lower bone mineral content (BMC) than their agematched controls, up to the ages of 4 to 16 years; on the contrary, Horsman et al. (6) claim that the mineralization defect of the preterm AGA infant is quickly overcome by a phase of rapid mineral accretion, occurring between 40 and 60 weeks’ postconception. Moreover, at the postconceptional age of 56 weeks, AGA infants fed their own mother’s milk were found to have similar growth patterns but a different bone mineralization rate compared with preterm infants fed a standard cow’s milk-based formula (7). The aim of the present study was to assess if low birth weight and early life nutrition in AGA infants have late effects on BMC before adolescence. Eighty-two preterm-born infants and children aged 2 months to 12 years were admitted to the study after oral informed parental consent. The children were recruited from an urban community of Caucasian, middle-class, well educated and well nourished people. For each subject, date of birth, birth weight and feeding history from birth to weaning were retrieved from the clinical records and by mothers’ recall. BMC and bone width (BW) at the distal third of the right radius were measured by single photon absorptiometry (Norland bone mineral analyser 2780) during a clinic health visit. On the same occasion, body weight, height and head circumference were recorded also. In order to set up a reference group, BMC and BW values were also measured in a small group of 24 termborn infants and children aged 5 months to 15 years, belonging to the same population as the study group and attending the Orthopaedic Department for problems not related to bone mineral metabolism. They were recruited after oral informed parental consent. BMC and BW values measured in this small group of termborn babies were compared with those reported from larger series in the literature (8-1 1). As we found that the measured BMC and BW values were within the variance limits ( f 2 SD) of the larger series published previously, the latter were accepted as reference values. The 82 subjects (42 males and 40 females) were selected according to the following characteristics: gestational age at birth between 26 and 37 weeks, appropriate weight for gestational age at birth, birth weight from 900 to 2640 g. Half of the subjects (n = 40) had been fed their mother’s milk supplemented with human milk protein (1 g/dl/day), calcium (30 mg/kg/ day) and phosphorus (20 mg/kgg/day) from birth to time of discharge from the hospital at a body weight of 2200 g. After discharge, the infants were exclusively breast fed until weaning was started at the age of four months. The second half of the subjects (n=34) received a wheypredominant cow’s milk formula designed specifically for low-birth-weight infants containing 2.0 g of protein/ dl and providing 75 kcal/dl (Humana 0, Humana Milchwerke Westfalen-Herford, Germany) from birth to discharge and thereafter a whey-predominant cow’s milk formula designed for term infants containing 1.4 g of protein/dl and providing 69 kcal/dl (Humana 1, Humana Milchwerke Westfalen-Herford, Germany). For 8 infants, feeding history was not available at the time of study. When all infants were grouped by age at which the BMC measurements were performed, it was found that birth weights were different among the classes (Table 1). The collected data were therefore analysed by covariance analysis, adjusting for the covariates (age at which the BMC measurement was performed and birth