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Amelogenins are enamel matrix proteins currently used to treat bone defects in periodontal surgery. Recent studies have highlighted the relevance of amelogenin-derived peptides, named LRAP, TRAP, SP, and C11, in bone tissue engineering. Interestingly, these peptides seem to maintain or even improve the biological activity of the full-length protein, which has received attention in the field of bone regeneration. In this article, the authors combined a systematic and a narrative review. The former is focused on the existing scientific evidence on LRAP, TRAP, SP, and C11’s ability to induce the production of mineralized extracellular matrix, while the latter is concentrated on the structure and function of amelogenin and amelogenin-derived peptides. Overall, the collected data suggest that LRAP and SP are able to induce stromal stem cell differentiation towards osteoblastic phenotypes; specifically, SP seems to be more reliable in bone regenerative approaches due to its osteoinduction and the absence of immunogenicity. However, even if some evidence is convincing, the limited number of studies and the scarcity of in vivo studies force us to wait for further investigations before drawing a solid final statement on the real potential of amelogenin-derived peptides in bone tissue engineering.

Background: Heat necrosis due to motorized drilling during anterior cruciate ligament (ACL) reconstruction could be a factor in delayed healing at the bone–tendon graft interface. Hypothesis: The process of osteointegration could be enhanced using manual drilling. It reduces the invasiveness of mechanical-thermal stress normally caused by the traditional motorized drill bit. Study Design: Controlled laboratory study. Methods: ACL reconstruction using semitendinosus tendon autografts was performed in 28 skeletally mature female New Zealand white rabbits, which were randomly divided into 3 groups. In group A (n = 12), the tunnels were drilled using a motorized device; in group B (n = 12), the tunnels were drilled using a manual drill bit; and group C (n = 4) served as a control with sham surgical procedures. The healing process in the tunnels was assessed histologically at 2, 4, 8, and 12 weeks and graded according to the Tendon–Bone Tunnel Healing (TBTH) scoring system. In addition, another 25 rabbits were used for biomechanical testing. The structural properties of the femur–ACL graft–tibia complex, from animals sacrificed at 8 weeks postoperatively, were determined using uniaxial tests. Stiffness (N/mm) and ultimate load to failure (N) were determined from the resulting load-elongation curves. Results: The time course investigation showed that manual drilling (group B) had a higher TBTH score and improved mechanical behavior, reflecting better organized collagen fiber continuity at the bone–fibrous tissue interface, better integration between the graft and bone, and early mineralized chondrocyte-like tissue formation at all the time points analyzed with a maximum difference at 4 weeks (TBTH score: 5.4 [group A] vs 12.3 [group B]; P < .001). Stiffness (23.1 ± 8.2 vs 17.8 ± 6.3 N/mm, respectively) and ultimate load to failure (91.8 ± 60.4 vs 55.0 ± 18.0 N, respectively) were significantly enhanced in the specimens treated with manual drilling compared with motorized drilling ( P < .05 for both). Conclusion: The use of manual drilling during ACL reconstruction resulted in better tendon-to-bone healing during the crucial early weeks. Manual drilling was able to improve the biological and mechanical properties of bone–hamstring tendon graft healing and was able to restore postoperative graft function more quickly. Tunnel drilling results in bone loss and deficient tendon-bone healing, and heat necrosis after tunnel enlargement may cause mechanical stress, contributing to a delay in healing. Manual drilling preserved the bone stock inside the tunnel, reduced heat necrosis, and offered a better microenvironment for faster healing at the interface. Clinical Relevance: Based on study results, manual drilling could be used successfully in human ACL reconstruction, but further clinical studies are needed. A clinical alternative, called the original “all-inside” technique, has been developed for ACL reconstruction. In this technique, the femoral and tibial tunnels are manually drilled only halfway through the bone for graft fixation, reducing bone loss. Data from this study suggest that hamstring tendon–to–bone healing can be improved using a manual drilling technique to form femoral and tibial tunnels.

BACKGROUND: Biomechanical behaviour evaluation of a suture is an important information for the surgeon to choose the best technique to perform. OBJECTIVE: To assess the biomechanical behavior of the native and mechanically sutured bronchi. METHODS: Ten bronchi were harvested from slaughtered pigs and then randomly separated in two groups, a control intact group and a sutured group where specimens were cut in half and sutured, to evaluate mechanical properties during a tensile test using a loading frame machine. In addition optoelectric motion tracking system was used to evaluate suture profile motion during the test. RESULTS: Significant differences ( p< 0.05) were found between the two groups for the parameters investigated. The control group showed a higher maximal stress resistance and stiffness than the suture group, while elongation at rupture was increased in the sutured group. All the sutures broke in symmetric manner, as the mean of the side difference of the sutured specimens was 0.93 ± 0.80 mm at rupture. CONCLUSIONS: Biomechanical behaviour of native and sutured bronchi was evaluated, giving highly reproducible parameters regarding mechanical properties that may help clinicians and bioengineers to rationalize the choice for a particular suture material or suture technique, increasing surgical outcomes.

Nerve growth factor and its receptors, TrkA and p75NTR, are involved in inflammation and airways diseases, but their role in chronic obstructive pulmonary disease is still unclear and not well investigated. our data indicate the stage dependent variation of nerve growth factor and its receptors in chronic obstructive pulmonary disease progression. In fact, for the first time, this study evaluates the presence of nerve growth factor and its receptors in serum and in peripheral blood mononuclear cells of patients with different stages of chronic obstructive pulmonary disease compared to healthy subjects, non-smoker and current smoker. Serum monocyte chemoattractant protein-1, tumor necrosis factor-α, interleukin-10 and forced expiratory volume in 1 s were also analyzed. Compared to healthy subjects, chronic obstructive pulmonary disease patients presented a staging-dependent increase in serum nerve growth factor, negatively correlated to forced expiratory volume in 1 s and positively to monocyte chemoattractant Protein-1. The percentage of p75NTR+ peripheral blood mononuclear cells increased in early stages of chronic obstructive pulmonary disease (I–II), while TrKA+ peripheral blood mononuclear cells increased in late stages (III–IV). Our data demonstrate the involvement and modulation of nerve growth factor and its receptors in chronic obstructive pulmonary disease and in its staging.

Herbal medicines have been recently employed in research and clinical studies for the potential treatment of behavioral and psychological symptoms associated with Alzheimer's Disease (AD) and other types of dementia. The present study investigates the effect of trans-crocetin, an active constituent of Crocus sativus L., to restore in vitro the reduced ability of AD patients' monocytes to degrade amyloid-β(1-42) (Aβ42). CD14+ monocytes from 22 sporadic AD patients with moderate cognitive impairment were isolated; then, the role of trans-crocetin, purified from saffron extracts, was evaluated in terms of Aβ42 degradation rate through flow cytometry, as well as expression of cathepsin B by Western blotting. We observed that low micromolar doses of trans-crocetin enhanced Aβ42 degradation in AD monocytes through the upregulation of the lysosomal protease cathepsin B. CA074Me, a potent and selective cathepsin B inhibitor, counteracted such trans-crocetin-induced effect. These data suggest that the carotenoid trans-crocetin improves in vitro the clearance of Aβ42 through the involvement of cathepsin B, and this could be of value in developing a new anti-amyloid strategy in AD.

In recent decades, tissue engineering strategies have been proposed for the treatment of musculoskeletal diseases and bone fractures to overcome the limitations of the traditional surgical approaches based on allografts and autografts. In this work we report the development of a composite porous poly(DL-lactide-co-glycolide) scaffold suitable for bone regeneration. Scaffolds were produced by thermal sintering of porous microparticles. Next, in order to improve cell adhesion to the scaffold and subsequent proliferation, the scaffolds were coated with the osteoconductive biopolymers chitosan and sodium alginate, in a process that exploited electrostatic interactions between the positively charged biopolymers and the negatively charged PLGA scaffold. The resulting scaffolds were characterized in terms of porosity, degradation rate, mechanical properties, biocompatibility and suitability for bone regeneration. They were found to have an overall porosity of approximate to 85% and a degradation half time of approximate to 2weeks, considered suitable to support de novo bone matrix deposition from mesenchymal stem cells. Histology confirmed the ability of the scaffold to sustain adipose-derived mesenchymal stem cell adhesion, infiltration, proliferation and osteo-differentiation. Histological staining of calcium and microanalysis confirmed the presence of calcium phosphate in the scaffold sections.

Objective Nitric oxide (NO) production and Ca 2+ homeostasis are key determinants for the control of many cell functions. NO is known to be a mediator of Ca 2+ homeostasis in a highly complex and cell-specific manner and although Ca 2+ homeostasis has been explored in human oral cancer cells, the exact mechanisms are not completely understood. In this study we investigated the impact of exogenous NO on [Ca 2+ ] c homeostasis in PE/CA-PJ15 cells. Design Cells were treated with S-nitrosocysteine as NO-donor and the determinations of cytosolic Ca 2+ concentrations were performed using FURA-2 AM. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) and oligomycin were used to challenge mitochondrial functionality, whereas thapsigargin (TG) and La 3+ were employed to perturb intracellular calcium levels. Results NO derived from S-nitrosocysteine (CySNO) induced a dose-dependent reduction of cytosolic calcium [Ca 2+ ] c whereas oxy-haemoglobin (oxyHb) completely counteracted this effect. Subsequently, we assessed possible relationships between NO and cellular structures responsible for Ca 2+ homeostasis. We found that uncoupling of mitochondrial respiration with carbonyl-cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) and oligomycin strongly reduced the effect of NO on [Ca 2+ ] c . Moreover, we found that during this mitochondrial energetic deficit, the effect of NO on [Ca 2+ ] c was also reduced in the presence of La 3+ or thapsigargin. Conclusions NO induces a concentration-dependent [Ca 2+ ] c reduction in PE/CA-PJ15 human oral cancer cells and potentiates mitochondrial Ca 2+ buffering in the presence of TG or La 3+ . Further, we show that exogenous NO deregulates Ca 2+ homeostasis in PE/CA-PJ15 cells with fully energized mitochondria.

The synthesis and characterization of new biodegradable polymeric NPs loaded with bovine serum albumin marked with fluorescein isothiocyanate (FITC-BSA) is reported. The protein is encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) NPs by the double emulsion method with subsequent solvent evaporation. The NPs display a spherical shape with a narrow size distribution and no aggregation is observed after drying. Steady-state and time-resolved fluorescence measurements appear to be a sensitive method to investigate the protein environment on the nanometer-scale. Finally, FITC-BSA-loaded NPs are rapidly internalized in stem cells. Interestingly, 25% cells were slightly positive after 28 days.

Osteoarthritis (OA) of the knee is the most common form of non-traumatic joint disease. Previous studies have shown the involvement of β-NGF and its receptors TrKA and p75NTR in OA-related pain, but their role in its pathogenesis is still unclear. The aim of our study was to investigate the amount of β-NGF and the expression levels of its receptors on cells isolated from synovial fluid and blood from OA patients who had undergone total knee arthroplasty, in order to check any possible correlation with the disease staging. Our results show a progressive stage-related increase of β-NGF and its receptors both in serum and synovial fluid. Furthermore, with respect to control subjects, OA patients show an increased amount of inflammatory monocytes along with an increased expression of β-NGF, TrKA and p75NTR. In conclusion, our study suggests a stage-related modulation of β-NGF and its receptors in the inflammatory process of OA.

In an attempt to investigate whether the genetic defect in the HEXA and HEXB genes (which causes the absence of the lysosomal β-N-acetyl-hexosaminidase), are related to the wide inflammation in GM2 gangliosidoses (Tay-Sachs and Sandhoff disease), we have chosen the dendritic cells (DCs) as a study model. Using the RNA interference approach, we generated an in vitro model of HEXs knock-down immunogenic DCs (i-DCs) from CD34(+)-haemopoietic stem cells (CD34(+)-HSCs), thus mimicking the Tay-Sachs (HEXA-/-) and Sandhoff (HEXB-/-) cells. We showed that the absence of β-N-acetyl-hexosaminidase activity does not alter the differentiation of i-DCs from HSCs, but it is critical for the activation of CD4(+)T cells because knock-down of HEXA or HEXB gene causes a loss of function of i-DCs. Notably, the silencing of the HEXA gene had a stronger immune inhibitory effect, thereby indicating a major involvement of β-N-acetyl-hexosaminidase A isoenzyme within this mechanism.

This study investigated how the design of surface topography may stimulate stem cell differentiation towards a neural lineage. To this end, hydrogenated amorphous carbon (a-C:H) groove topographies with width/spacing ridges ranging from 80/40μm, 40/30μm and 30/20μm and depth of 24 nm were used as a single mechanotransducer stimulus to generate neural cells from human bone marrow mesenchymal stem cells (hBM-MSCs) in vitro. As comparative experiments, soluble brain-derived neurotrophic factor (BDNF) was used as additional biochemical inducer agent. Despite simultaneous presence of a-C:H micropatterned nanoridges and soluble BDNF resulted in the highest percentage of neuronal-like differentiated cells our findings demonstrate that the surface topography with micropatterned nanoridge width/spacing of 40/30μm (single stimulus) induced hBM-MSCs to acquire neuronal characteristics in the absence of differentiating agents. On the other hand, the alternative a-C:H ridge dimensions tested failed to induce stem cell differentiation towards neuronal properties, thereby suggesting the occurrence of a mechanotransducer effect exerted by optimal nano/microstructure dimensions on the hBM-MSCs responses.

The term “chondropenia” indicates the early stage of degenerative cartilage disease, and it has been identified by carefully monitoring early-stage osteoarthritis (OA). Not only is it the loss of articular cartilage volume, but it is also a rearrangement of biomechanical, ultrastructural, biochemical and molecular properties typical of healthy cartilage tissue. Diagnosing OA at an early stage or an advanced stage is valuable in terms of clinical and therapeutic outcome. In fact degenerative phenomena are supported by a complex biochemical cascade which unbalances the extracellular matrix homeostasis, closely regulated by chondrocytes. In the first stage an intense inflammatory reaction is triggered: pro-catabolic cytokines such as IL-1β and TNF-α triggering matrix metalloproteases and aggrecanase (ADAMT-4 and 5), responsible for the early loss of ultrastructural components, such as type II collagen and aggrecan. In addition nitric oxide and reactive oxygen species modulate the physiopathology of the condral matrix inducing apoptosis of chondrocytes through a mitochondria-dependent pathway. In addition, “Lonely Death”: chondrocytes, are confined within a dense, avascular extracellular matrix capsule, and can trigger a genetically induced apoptosis and necrosis. The degenerative process starts from a central point and then spreads in a centrifugal manner in depth and in adjacent areas, eventually covering the whole joint; chondropenia represents a journey from the first clinically detectable time-point until it can be characterized as frank osteoarthritis. Currently, there are no instruments sensitive enough which allow a timely diagnosis of chondropenia. Innovative magnetic resonance imaging techniques, such as T2 mapping, can be effective and a sensitive diagnostic instrument for quantifying cartilage volume and proteoglycan content. However, avant-garde biophysical techniques, such as mechanical indenters, ultrasound and biochemical markers (uCTX-II), are rational and scientific tools applicable to the clinical and therapeutic management of early degenerative cartilage disease. The objective of this review on chondropenia is to present a state of the art and innovative concepts.

Background: Alzheimer’s disease (AD) is characterized by cognitive decline and hallmark neuropathologies, including b-amyloid (Ab). Therapeutic strategies for AD focus on reducing the production or deposition of Ab. Canines develop Ab neuropathology and cognitive decline with age similar to AD patients, making them a useful model for testing potential therapies. Immunization with Ab1-42 (IMM)in aged canines significantly decreases brain Ab and maintains executive function. However, behavioral enrichment (BEH) improves cognition without reducing brain Ab. We hypothesized that IMM combined with BEH would provide larger cognitive benefits and further reduce neuropathology, as compared to controls or individual IMM and BEH treatments alone. Methods: Aged beagles (10.513.6 y) were placed into groups: control (Alum adjuvant only), fibrillar Ab1-42 + Alum vaccine, BEH with Alum, and combination treatment (IMM+BEH). Animals were treated for 18 months. Cognition was measured throughout the study using various learning and memory tasks. Serum IgG antibody titers, cerebral spinal fluid (CSF) Ab levels, and insoluble brain Ab (formic acid extracted) were measured by ELISA. Ab plaque load (6E10, anti-Ab 1-42, Pyro Glu3), CAA (congo red), and microhemorrhages (prussian blue) were measured by immunohistochemistry. Results: Anti-Ab1-42 IgG responses in IMM animals increased significantly and were maintained. BEH significantly increased CSF Ab1-40, while no systematic effects were seen on Ab1-42 or total Ab. IMM significantly reduced both insoluble Ab1-40 and 1-42, as well as all forms of measured plaque load. An overall reduction in 6E10, and Ab 1-42 plaque load due to BEH was also seen. A significant additive affect from BEH and IMM was seen in clearance of Ab 1-42 plaque load. Conclusions: IMM successfully induced an immune response in treated animals and was maintained. IMM significantly reduced all forms of Ab pathology. Additionally, BEH increased CSFAb 1-40 and reduced 6E10 and Ab 1-42 plaque loads. A significant additive effect from BEH and IMM was seen in clearance of Ab 142 plaque load. While changes in cognition, CAA and microhemorrhages remain to be analyzed, it is expected that levels of CAA and microhemorrhages will be lower in IMM animals, and IMM+BEH will show greatest cognitive benefits.

Alzheimer's disease (AD), the most common form of dementia in elderly individuals, is characterized by neurofibrillary tangles, extracellular amyloid-β (Aβ) plaques and neuroinflammation. New evidence has shown that the lysosomal system might be a crossroad in which etiological factors in AD pathogenesis converge. This study shows that several lysosomal enzymes, including Cathepsin B, D, S, β-Galactosidase, α-Mannosidase, and β-Hexosaminidase, were less expressed in monocytes and lymphocytes from patients with a clinical diagnosis of AD dementia compared with cells from healthy controls. In vitro experiments of gain and loss of function suggest that down-regulation is a direct consequence of miR-128 up-regulation found in AD-related cells. The present study also demonstrates that miR-128 inhibition in monocytes from AD patients improves Aβ(1-42) degradation. These results could contribute to clarify the molecular mechanisms that affect the imbalanced Aβ production/clearance involved in the pathogenesis of AD.

The synthesis and characterization of new biodegradable polymeric NPs loaded with bovine serum albumin marked with fluorescein isothiocyanate (FITC-BSA) is reported. The protein is encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) NPs by the double emulsion method with subsequent solvent evaporation. The NPs display a spherical shape with a narrow size distribution and no aggregation is observed after drying. Steady-state and time-resolved fluorescence measurements appear to be a sensitive method to investigate the protein environment on the nanometer-scale. Finally, FITC-BSA-loaded NPs are rapidly internalized in stem cells. Interestingly, 25% cells were slightly positive after 28 days.

Background: The elucidation of mechanistic aspects of relapsing–remitting multiple sclerosis (RRMS) pathogenesis may offer valuable insights into diagnostic decisions and medical treatment. Results: Two lysosomal proteases, cathepsins S and D (CatS and CatD), display an exclusive pattern of expression in CD34+ hematopoietic stem cells (HSCs) from peripheral blood of acute MS (A-MS) patients ( n = 20). While both enzymes normally exist as precursor forms in the HSCs of healthy individuals ( n = 30), the same cells from A-MS patients consistently exhibit mature enzymes. Further, mature cathepsins are expressed at lower rates in stable MS subjects (S-MS, n = 15) and revert to precursor proteins after interferon-β1a treatment ( n = 5). Mature CatD and CatS were induced in HSCs of healthy donors that were either co-cultured with PBMCs of A-MS patients or exposed to their plasma, suggesting a functional involvement of soluble agents. Following HSC exposure to several cytokines known to be implicated in MS, and based on relative cytokine levels displayed in A-MS, S-MS and control individuals, we identified IL-16 as a specific cell signaling factor associated with cathepsin processing. Conclusions: These data point to an evident correlation between CatS and CatD expression and MS clinical stage, and define a biochemical trait in HSCs with functional, medical, and diagnostic relevance.

Background NO is a key molecule involved in the regulation of cell survival, proliferation and differentiation in many cell types. In this study we investigated the contribution of NO during the differentiation of human peripheral blood hemopoietic stem cells (CD34 + HSCs) toward immunogenic dendritic cells (i-DCs). Methods We depleted autocrine NO production, using N G -monomethyl- l -arginine monoacetate ( l -NMMA) and paracrine NO , using oxy-hemoglobin (HbO 2 ) as a NO scavenger during in vitro differentiation of CD34 + HSCs to i-DCs. We monitored the NO level, cell proliferation, phenotype and differentiation potential. Results We found that the depletion of paracrine or autocrine NO correlated with (I) an active proliferation state at the end of differentiation, when control cells were not proliferating; (II) a significant reduction in the expression levels of differentiative markers (CD1a and HLA-DR) with a parallel high expression of the CD34 marker (III) with a retrieved clonogenic ability compared to control cells. Conclusions On the whole, our data indicate that the depletion of NO during the commitment stage blocks CD34 + HSC differentiation into i-DCs and maintains an undifferentiated, highly proliferating cell population, indicating/revealing a novel role for NO in the commitment of CD34 + HSCs into i-DCs. General significance The essential finding of the present study is that NO , produced in HSCs by NOS enzymes, may act as autocrine and paracrine effectors regulating the in vitro differentiation process of CD34 + -HSCs toward i-DCs.

In this study, we investigated whether multipotent (human-bone-marrow-derived mesenchymal stem cells [hBM-MSCs]) and pluripotent stem cells (murine-induced pluripotent stem cells [iPSCs] and murine embryonic stem cells [ESCs]) respond to nanocomposite fibrous mats of poly(L-lactic acid) (PLLA) loaded with 1 or 8 wt % of calcium-deficient nanohydroxyapatite (d-HAp). Remarkably, the dispersion of different amounts of d-HAp to PLLA produced a set of materials (PLLA/d-HAp) with similar architectures and tunable mechanical properties. After 3 weeks of culture in the absence of soluble osteogenic factors, we observed the expression of osteogenic markers, including the deposition of bone matrix proteins, in multi/pluripotent cells only grown on PLLA/d-HAp nanocomposites, whereas the osteogenic differentiation was absent on stem-cell-neat PLLA cultures. Interestingly, this phenomenon was confined only in hBM-MSCs, murine iPSCs, and ESCs grown on direct contact with the PLLA/d-HAp mats. Altogether, these results indicate that the osteogenic differentiation effect of these electrospun PLLA/d-HAp nanocomposites was independent of the stem cell type and highlight the direct interaction of stem cell-polymeric nanocomposite and the mechanical properties acquired by the PLLA/d-HAp nanocomposites as key steps for the differentiation process.

Multiple epidemiological studies have shown that individuals affected by type-2 diabetes mellitus (T2DM) carry a 2-to-5-fold higher risk of developing Alzheimer's disease (AD) when compared to non-diabetic subjects. Thus, biochemical parameters that can be easily and routinely assessed for high-confidence evaluation of diabetic conditions leading to AD (AD-T2DM) are regarded as efficient tools aimed at early diagnosis and, in turn, timely AD treatment. In this regard, the activity of lysosomal glycohydrolases may of use, in light of the implication of these enzymes in early events that underlie AD pathology and an overt correlation, in diabetes, between altered metabolic homeostasis, abnormal glycohydrolase secretion in body fluids, and occurrence of diabetic complications. Based on marked up-regulation previously shown in a peripheral, cell-based model of AD, we selected β-Galactosidase, β-Hexosaminidase, and α-Mannosidase to discriminate T2DM from AD-T2DM subjects. A screen of 109, 114, and 116 patients with T2DM, AD and AD-T2DM, respectively, was performed by testing enzyme activities in both blood plasma and peripheral blood mononuclear cells. Compared to age-matched, healthy controls (n = 122), β-Galactosidase and β-Hexosaminidase activities markedly diverged across the three groups, whereas virtually unchanged values were observed for α-Mannosidase. In particular, plasma β-Galactosidase and β-Hexosaminidase levels were higher in patients with AD-T2DM compared to those with T2DM, suggesting different mechanisms leading to enzyme secretion. Statistical analyses based on ROC curves showed that both β-Galactosidase and β-Hexosaminidase activities, either intracellular or plasma-secreted, may be used to discriminate AD patients from controls and AD-T2DM from T2DM patients.

The identity of biochemical players which underpin the commitment of CD34(+) hematopoietic stem cells to immunogenic or tolerogenic dendritic cells is largely unknown. To explore this issue, we employed a previously established cell-based system amenable to shift dendritic cell differentiation from the immunogenic into the tolerogenic pathway upon supplementation with a conventional cytokine cocktail containing thrombopoietin (TPO) and IL-16. We show that stringent regulation of cathepsins S and D, two proteases involved in antigen presentation, is crucial to engage cell commitment to either route. In response to TPO+IL-16-dependent signaling, both cathepsins undergo earlier maturation and down-regulation. Additionally, cystatin C orchestrates cathepsin S expression through a tight but reversible interaction that, based on a screen of adult stem cells from disparate origins, CD14(+) cells, primary fibroblasts and the MCF7 cell line, appears unique to CD34(+) stem cells from peripheral and cord blood. As shown by CD4(+) T cell proliferation in mixed-lymphocyte reactions, cell commitment to either pathway is disrupted upon cathepsin knockdown by RNAi. Surprisingly, similar effects were also observed upon gene overexpression, which prompts atypically accelerated maturation of cathepsins S and D in cells of the immunogenic pathway, similar to the tolerogenic route. Furthermore, RNAi studies revealed that cystatin C is a proteolytic target of cathepsin D and has a direct, causal impact on cell differentiation. Together, these findings uncover a novel biochemical cluster that is subject to time-controlled and rigorously balanced expression to mediate specific stem cell commitment at the crossroads towards tolerance or immunity.

The effectiveness of therapeutic treatment based on regenerative medicine for degenerative diseases (i.e., neurodegenerative or cardiac diseases) requires tools allowing the visualization and analysis of the three-dimensional (3D) distribution of target drugs within the tissue. Here, we present a new computational procedure able to overcome the limitations of visual analysis emerging by the examination of a molecular signal within images of serial tissue/organ sections by using the conventional techniques. Together with the 3D anatomical reconstitution of the tissue/organ, our framework allows the detection of signals of different origins (e.g., marked generic molecules, colorimetric, or fluorimetric substrates for enzymes; microRNA; recombinant protein). Remarkably, the application does not require the employment of specific tracking reagents for the imaging analysis. We report two different representative applications: the first shows the reconstruction of a 3D model of mouse brain with the analysis of the distribution of theβ-Galactosidase, the second shows the reconstruction of a 3D mouse heart with the measurement of the cardiac volume.

The interaction between stem cells and biomaterials with nanoscale topography represents a main route in the roadmap for tissue engineering-based strategies. In this study, we explored the interface between human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and hydrogenated amorphous carbon (a-C:H) film designed with uniform, groove, or grid nanopatterns. In either case, hBM-MSCs preserved growth rate and multi-differentiation properties, suggesting that the films were biocompatible and suitable for stem cell culture. hBM-MSCs responded to different nanopattern designs with specific changes of microtubule organization. In particular, the grid pattern induced a square-localized distribution of alpha-tubulin/actin fibers, whereas the groove pattern exerted a more dynamic effect, associated with microtubule alignment and elongation.

RNA interference technology is an ideal strategy to elucidate the mechanisms associated with humanCD34+hematopoietic stem cell differentiation into dendritic cells. Simple manipulations in vitro can unequivocally yield alloreactive or tolerogenic populations, suggesting key implications of biochemical players that might emerge as therapeutic targets for cancer or graft-versus-host disease. To knockdown proteins typically involved in the biology of dendritic cells, we employed an siRNA delivery system based on the cationic liposome DOTAP as the carrier. Freshly-isolatedCD34+cells were transfected with siRNA for cathepsin S with negligible cytotoxicity and transfection rates (>60%) comparable to the efficiency shown by lentiviral vectors. Further, cathepsin S knockdown was performed during both cell commitment and through the entire 14-day differentiation process with repeated transfection rounds that had no effect per se on cell development. Tested in parallel, other commercially-available chemical reagents failed to meet acceptable standards. In addition to safe and practical handling, a direct advantage of DOTAP over viral-mediated techniques is that transient silencing effects can be dynamically appraised through the recovery of targeted proteins. Thus, our findings identify DOTAP as an excellent reagent for gene silencing in resting and differentiatingCD34+cells, suggesting a potential for applications in related preclinical models.

In this work we showed that genotype-related patterns of hexosaminidase activity, isoenzyme composition, gene expression and ganglioside metabolism observed during embryonic and postnatal brain development are recapitulated during the progressive stages of neural precursor cell (NPC) differentiation to mature glia and neurons in vitro. Further, by comparing NPCs and their differentiated progeny established from Tay-Sachs (TS) and Sandhoff (SD) animal models with the wild-type counterparts, we studied the events linking the accumulation of undegraded substrates to hexosaminidase activity. We showed that similarly to what observed in brain tissues in TS NPCs and progeny, the stored GM2 was partially converted by sialidase to GA2, which can be then degraded in the lysosomes to its components. The latter can be used in a salvage pathway for the formation of GM3. Interestingly, results obtained from ganglioside feeding assays and from measurement of lysosomal sialidase activity suggest that a similar pathway might work also in the SD model. © 2009 International Society for Neurochemistry.

Background: The determination of cellular β-galactocerebrosidase activity is an established procedure to diagnose Krabbe disease and monitor the efficacy of gene/stem cell-based therapeutic approaches aimed at restoring defective enzymatic activity in patients or disease models. Current biochemical assays for β-galactocerebrosidase show high specificity but generally require large protein amounts from scanty sources such as hematopoietic or neural stem cells. We developed a novel assay based on the hypothesis that specific measurements of β-galactocerebrosidase activity can be performed following complete inhibition of β-galactosidase activity. Methods: We performed the assay using 2–7.5 μg of sample proteins with the artificial fluorogenic substrate 4-methylumbelliferone-β-galactopyranoside (1.5 mmol/L) resuspended in 0.1/0.2 mol/L citrate/phosphate buffer, pH 4.0, and AgNO3. Reactions were incubated for 30 min at 37 °C. Fluorescence of liberated 4-methylumbelliferone was measured on a spectrofluorometer (λex 360 nm, λem 446 nm). Results: AgNO3 was a competitive inhibitor of β-galactosidase [inhibition constant (Ki) = 0.12 μmol/L] and completely inhibited β-galactosidase activity when used at a concentration of 11 μmol/L. Under this condition, the β-galactocerebrosidase activity was preserved and could be specifically and accurately measured. The assay can detect β-galactocerebrosidase activity in as little as 2 μg cell protein extract or 7.5 μg tissue. Assay validation was performed using (a) brain tissues from wild-type and twitcher mice and (b) murine GALC−/− hematopoietic stem cells and neural precursor cells transduced by GALC-lentiviral vectors. Conclusions: The procedure is straightforward, rapid, and reproducible. Within a clinical context, our method unequivocally discriminated cells from healthy subjects and Krabbe patients and is therefore suitable for diagnostic applications.

Aldo Orlacchio, Carlo Massini, Roberto Tiribuzi, Egidia Costanzi, Georgia Makrypidi, Filippo Mattoli, Zaira Esposito, Clarice Patrono, Marialuisa Miele, Mariangela Maiotti, Giuseppe Sancesario, Mauro Zampolini, Antonio Orlacchio, Sabata Martino, Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Universita di Perugia, Perugia, Italy; Laboratorio di Neurogenetica, CERC-IRCCS Santa Lucia, Rome, Italy; Dipartimento di Neuroscienze, Universita di Roma ‘Tor Vergata’, Rome, Italy; Struttura Complessa di Neurologia e Neuroriabilitazione, Ospedale Nuovo San Giovanni Battista, ASL3, Foligno (PG), Italy. Contact email: orly@unipg.it

The lysosomal enzyme di-N-acetylchitobiase hydrolyzes N-acetylglucosamine from the reducing-end of the N,N' diacetylchitobiose core of N-linked-oligosaccharides. The presence of chitobiase in the tissues of different species is probably responsible for differences in the structure of oligosaccharides accumulated in the lysosomal storage disease beta-mannosidosis. The disease has so far been described in humans, cats, cattle and goats. Low chitobiase activity has been observed in the tissues of ruminants and it has been hypothesized that in cattle this low level of expression is due to evolutionary changes in the promoter region. A cDNA encoding the mouse chitobiase has been isolated, sequenced and its identity confirmed by expression in COS-7 cells. Comparison of the mouse genomic sequence with the cDNA sequence revealed the presence of seven exons within the chitobiase gene. The gene spans about 15 kb and a single transcription initiation site was determined by 5'RACE. Chitobiase is differentially and ubiquitously expressed in mouse tissues as demonstrated by qRT-PCR analysis. Chitobiase is differentially expressed at lower levels in bovine tissues. In two bovine tissues (heart and muscle) mRNA was not detectable. Mouse and bovine promoters have been isolated and sequenced and their activities compared. The activity of the bovine promoter is very low and might explain the low activity of chitobiase observed in cattle.

The production of active Arylsulfatase A is a key step in the development of enzyme replacement therapy for Metachromatic Leukodystrophy. To obtain large amounts of purified Arylsulfatase A for therapeutic use, we combined a retroviral expression system with a versatile and rapid purification protocol that can easily and reliably be adapted to high-throughput applications. The purification method consists of an initial ion-exchange DEAE-cellulose chromatography step followed by immuno-affinity purification using a polyclonal antibody against a 29-mer peptide of the Arylsulfatase A sequence. Immuno-adsorbed protein was eluted with a combination of acidic pH and an optimal concentration of the 29-mer peptide. This protocol reproducibly yielded approximately 100 μg of >99% pure human Arylsulfatase A, corresponding to 152 mU of enzyme activity, per liter of culture medium with properties similar to those of human non-recombinant protein.

Sulphamidase is a lysosomal enzyme necessary for the degradation of heparan sulphate. The deficiency of this hydrolase causes a disorder known as mucopolysaccharidosis type IIIA, characterized by a profound neurological deterioration. Human and mouse exon–intron structures were reported without any characterization of their promoter regions [DNA Res. 3 (1996) 269; Mamm. Genome 11 (2000) 436]. The promoter region was isolated and characterized to understand the factors affecting the expression of mouse sulphamidase. The 5′-flanking region was shown to contain a GC-rich region and putative binding sites for the transcription factors SRY, MZF1 and Nkx-2.5 with no TATA or CAAT boxes present. The 5′ region had promoter activity to drive luciferase gene expression in transfected COS cells. The transcription initiation site of mouse sulphamidase was mapped to a single adenine residue 355 bases upstream of ATG codon. Northern blot analysis revealed differential expression of a major transcript of 4.5 kb in all tissues examined. Finally, the 3′-untranslated region of the mouse sulphamidase gene was isolated and found to be longer than the region identified in the human gene.