Anti-leukemic activity of microRNA-26a in a chronic lymphocytic leukemia mouse model.

Dysregulation of microRNAs (miRNAs) plays an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). The Eµ-TCL1 transgenic mouse develops a form of leukemia that is similar to the aggressive type of human B-CLL, and this valuable model has been widely used for testing novel therapeutic approaches. Here, we adopted this model to investigate the potential effects of miR-26a, miR-130an and antimiR-155 in CLL therapy. Improved delivery of miRNA molecules into CLL cells was obtained by developing a novel system based on lipid nanoparticles conjugated with an anti-CD38 monoclonal antibody. This methodology has proven to be highly effective in delivering miRNA molecules into leukemic cells. Short- and long-term experiments showed that miR-26a, miR-130a and anti-miR-155 increased apoptosis after in vitro and in vivo treatment. Of this miRNA panel, miR-26a was the most effective in reducing leukemic cell expansion. Following long-term treatment, apoptosis was readily detectable by analyzing cleavage of PARP and caspase-7. These effects could be directly attributed to miR-26a, as confirmed by significant downregulation of its proven targets, namely cyclin-dependent kinase 6 and Mcl1. The results of this study are relevant to two distinct areas. The first is related to the design of a technical strategy and to the selection of CD38 as a molecular target on CLL cells, both consenting efficient and specific intracellular transfer of miRNA. The original scientific finding inferred from the above approach is that miR-26a can elicit in vivo anti-leukemic activities mediated by increased apoptosis.

TCL1 transgenic mouse model as a tool for the study of therapeutic targets and microenvironment in human B-cell chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a B-cell malignancy with a mature phenotype. In spite of its relatively indolent nature, no radical cure is as yet available. CLL is not associated with either a unique cytogenetic or a molecular defect, which might have been a potential therapeutic target. Instead, several factors are involved in disease development, such as environmental signals which interact with genetic abnormalities to promote survival, proliferation and an immune surveillance escape. Among these, PI3-Kinase signal pathway alterations are nowadays considered to be clearly important. The TCL1 gene, an AKT co-activator, is the cause of a mature T-cell leukemia, as well as being highly expressed in all B-CLL. A TCL1 transgenic mouse which reproduces leukemia with a distinct immunophenotype and similar to the course of the human B-CLL was developed several years ago and is widely used by many groups. This is a review of the CLL biology arising from work of many independent investigators who have used TCL1 transgenic mouse model focusing on pathogenetic, microenviroment and therapeutic targets.

The Tcl1 oncogene defines secondary hair germ cells differentiation at catagen-telogen transition and affects stem-cell marker CD34 expression.

Overexpression of the TCL1 gene family plays a role in the onset of T-cell leukemias in mice and in humans. The Tcl1 gene is tightly regulated during early embryogenesis in which it participates in embryonic stem (ES)-cells proliferation and during lymphoid differentiation. Here, we provide evidences that Tcl1 is also important in mouse hair follicle (HF) and skin homeostasis. We found that Tcl1(-/-) adult mice exhibit hair loss, leading to alopecia with extensive skin lesions. By analysing Tcl1 expression in the wild-type (wt) skin through different stages of hair differentiation, we observe high levels in the secondary hair germ (HG) cells and hair bulges, during early anagen and catagen-telogen transition phases. The loss of Tcl1 does not result in apparent skin morphological defects during embryonic development and at birth, but its absence causes a reduction of proliferation in anagen HFs. Importantly, we show the that absence of Tcl1 induces a significant loss of the stem-cell marker CD34 (but not alpha6-integrin) expression in the bulge cells, which is necessary to maintain stem-cell characteristics. Therefore, our findings indicate that Tcl1 gene(s) might have important roles in hair formation, by its involvement in cycling and self-renewal of transient amplifying (TA) and stem-cell (SC) populations.

Effect of different masticatory functional and mechanical demands on the structural adaptation of the mandibular alveolar bone in young growing rats.

The influence of masticatory functional and mechanical demands on the structural adaptation of the alveolar bone has not been investigated in both animals and humans. The effect of two experimental factors, the insertion of a bite-opening appliance and the alteration of food consistency, was investigated in young growing rats, with a particular emphasis on three-dimensional (3D) bone microstructure. Thirty-six male albino rats were divided into two equal groups, fed with either the standard hard diet or soft diet, at the age of 4 weeks. After 2 weeks, half of the animals in both groups had their upper molars fitted with an upper posterior bite block, an appliance similar to those used in clinical orthodontics. The remaining animals served as a control. After another 4 weeks, the animals were sacrificed, and their left hemimandibles were excised. Bone mineral density (BMD) and bone microstructure parameters of the alveolar process were subsequently measured, using dual-energy X-ray absorptiometry (DXA) and micro-computed tomography (micro-CT). The alveolar process width was also measured. Both experimental factors led to significant shape and structure modification of the mandibular alveolar bone in the growing rat. The bite block applied a continuous light force, which was associated with an inhibition of alveolar process vertical growth and a significant increase of cortical thickness. Soft diet and the consequent reduction of the intermittent forces applied to the alveolar bone during mastication resulted in a reduction of bone mineral density, accompanied by decreased trabecular bone volume and thickness. This rat model could prove to be a useful tool for the in vivo investigation of the role of muscular forces on the shape and structure adaptation of bone.

Ubiquitin dependent proteolysis is activated in apoptotic fibroblasts in culture.

The ubiquitin mediated pathway constitutes an early response in cultured cells where apoptosis, assessed by internucleosomal specific DNA fragmentation, was induced by serum withdrawal. Data demonstrate that nuclear ubiquitin proteolytic system, but not cytoplasmic, is activated. This activation is paralleled by a substantial chromatin de-condensation. We suggest that chromatin relaxation is causative of the fragmentation since it exposes the DNA to nucleolytic attack. Finally, maintenance of homeostasis and induction of apoptosis seem to undergo a parallel contemporary pathway with a possible mutual feedback.

Effects of masticatory muscle function and bite-raising on mandibular morphology in the growing rat.

The aim of this series of investigations was to study the effect of masticatory muscle function on the growth pattern and on the internal structure of the mandible during growth. The muscular and dentoskeletal growth adaptation to prolonged bite-raising and the role of the functional state of the masticatory muscles in this adaptation were also to be elucidated. Differences in masticatory muscle function were induced in young rats by altering the consistency of the diet. Bite-raising was produced by the insertion of posterior bite blocks. Morphometric analysis of the internal bone structures of the mandible was performed on microradiographs, and videodensitometric analysis was performed on lateral radiographs and microradiographs. The effect of muscle function and bite-raising on mandibular growth was studied on a series of lateral cephalograms, superimposed on bone markers. Muscle belly, sarcomere and aponeurosis length adaptation to bite-raising was studied in situ with a digital caliper and under a microscope after fluorescent vital staining of the deep masseter muscle. A soft diet altered the pattern of growth of the mandible and reduced bone growth in the angular region. Transversal dimensions and cross-sectional area of the dentoalveolar process were smaller. Bone mass in areas possibly subjected to direct loads or bending forces was smaller. This was due to either less trabecular bone or thinner cortical bone. Only a few sites showed lower bone density. Posterior bite-blocks affected the size of the mandible as well as its growth pattern, and intruded lower molars. The soft diet influenced the effect of bite-blocks and caused less intrusion of upper molars and less inhibition of bone growth at the angular process. The deep masseter muscle adapted to bite-raising by elongation of the aponeurosis, but less in rats on a soft diet. Changes in masticatory muscle function affected the growth of the mandible in both the sagittal and transversal plane. Reduced loads on molars and condyle and smaller bending forces in other regions of the mandible possibly reduced the levels of stimulation of the osteocyte network and osteoblasts, thus inducing less trabecular bone and cortical bone formation in specific areas. In rats fed the soft diet, smaller increase in bone density represented an adaptation process in areas characterised by a lower bone apposition rate. The forces produced by the passive stretching of the masseter muscle affected the skeletal growth pattern and dental eruption. Weaker forces possibly produced by passive stretching of hypofunctional muscles resulted in more eruption of the upper molars and less inhibition of periosteal bone apposition in the angular region. Length adaptation in the masseter muscle through lengthening of the aponeurosis and dentofacial growth adaptation possibly decreased passive forces applied to teeth and skeletal structures, particularly in rats with higher functional demands. This may have caused a gradually decreasing effect of the appliance.

Adaptation of normal and hypofunctional masseter muscle after bite-raising in growing rats.

The aim of this study was to analyze the effects of prolonged muscular elongation induced by bite-raising on the length of the muscle belly, sarcomeres and aponeurosis of the anterior deep masseter in the growing rat. Another aim was to determine the role of different functional conditions of this muscle in the adaptation process. Ninety-six young male rats were split into two groups: one was fed a hard diet and the other a soft diet to develop different functional capacities in the masticatory muscles. After 2 wk, half of the rats in both groups were fitted with an appliance that raised the bite by 2 mm. The measurements on the muscles were performed in situ. The insertion of the appliance stretched the anterior masseter muscle. After 4 wk, the vertical dentoskeletal dimension, the muscle belly, and the sarcomeres showed no difference in length among the groups. However, the aponeurosis was longer in the rats wearing the appliance compared to the controls, and among the bite block groups it was longer in the rats fed a hard diet. Length adaptation occurred in the aponeurosis. Clinically this may imply a need for reactivation of functional appliances to increase their efficiency, at a rate possibly depending on masseter muscles functional condition.

Effect of masticatory function on the internal bone structure in the mandible of the growing rat.

The influence of changes in the masticatory function on bone mass, bone density and cortical thickness was analyzed in different functional units of the mandible of the growing rat. Young male albino rats were fed either a hard diet or a soft diet for 4 wk. Undecalcified coronal sections were selected from five mandibular regions and microradiographic images of the specimens alongside an aluminium stepwedge were obtained. Bone mass and density were measured at selected sites with a computer-aided image analysis system. Cortical thickness was measured on diagrams. The reduced masticatory function in the soft diet group produced different adaptive responses in the tested regions of the mandible. Some regions showed reduced cortical thickness (underneath the incisor and lateral to the first molar and some locations of the ramus), a few showed reduced bone density (medial to the first molar and in the ramus), and only one showed both (in the ramus). Reduced bone mass was associated mostly with thinner cortical bone rather than lower cortical bone density. Changes in cortical thickness and changes in bone density may be two different mechanisms for adjusting local mechanical properties in the mandible of the rat. Masticatory muscle function is a determinant for the amount and density of cortical and trabecular bone and may possibly influence results of orthodontic tooth movement and its possible relapse.

Craniofacial manifestations in the Marfan syndrome: palatal dimensions and a comparative cephalometric analysis.

The purpose of this study was to determine how the craniofacial morphology, evaluated from dental casts and lateral cephalograms, in individuals affected by the Marfan syndrome diverge from healthy control groups. The high and narrow palatal vault as well as maxillary and mandibular retrognathy were strongly correlated to the syndrome. About 70% of the Marfan syndrome patients (n = 76) had been referred for orthodontic treatment, mostly because of crowded teeth or extreme maxillary overjet. In 36%, the orthodontic treatment was carried out before diagnosis or suspicion about the Marfan syndrome. In comparison to healthy orthodontic patients (n = 86), selected because of presence of high and narrow palatal vaults, crowding of teeth, extreme maxillary overjet, and open bite were much more prevalent in the Marfan syndrome patients than in the orthodontic control group.

Effects of masticatory muscle function on bone mass in the mandible of the growing rat.

The effect of altered masticatory muscle function on bone mass at different sites in the rat mandible was studied using aluminium as a reference substance in computer-assisted image analysis. Forty-two growing male rats were divided into three groups, of which one group was killed at the beginning of the experiment to provide baseline values. Of the remaining two, one received a hard diet (control group) and the other a soft diet. After 28 days the rats were killed and the mandibles were excised. Lateral radiographs were taken of the mandible halves together with an aluminium stepwedge and then analysed by an image analysis system. Radiographic bone mass was measured in the transverse plane as "aluminium-equivalent thickness' in millimetres. Standardized areas were chosen to investigate regions with different functional demands. Significant differences were found between the groups in the alveolar bone of the molars and the incisor, as well as in the condylar costa and the condylar process. Significant differences were also found in some areas related to muscle insertion, such as the anterior lower border of the ramus, whereas no statistically significant differences could be found in others, such as the posterior lower border of the angular process. In conclusion, altered masticatory function influences the amount of bone mass in certain parts of mandible, where changes in applied bending forces have possibly altered the bone strain.