Non-specific Alkaline Phosphatase Activity Research Articles

1,25-Dihydroxyvitamin D3 and extracellular calcium promote mineral deposition via NPP1 activity in a mature osteoblast cell line MLO-A5

While vitamin D supplementation is common, the anabolic mechanisms that improve bone status are poorly understood. Under standard mineralising conditions including media ionised calcium of 1.1 mM, 1,25-dihydroxyvitamin D3 (1,25D) enhanced differentiation and mineral deposition by the mature osteoblast/pre-osteocyte cell line, MLO-A5. This effect was markedly increased with a higher ionised calcium level (1.5 mM). Gene expression analyses revealed that 1,25D-induced mineral deposition was associated with induction of Enpp1 mRNA, coding for nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) and NPP1 protein levels. Since MLO-A5 cells express abundant alkaline phosphatase that was not further modified by 1,25D treatment or exposure to increased calcium, this finding suggested that the NPP1 production of pyrophosphate (PPi) may provide alkaline phosphatase with substrate for the generation of inorganic phosphate (Pi). Consistent with this, co-treatment with Enpp1 siRNA or a NPP1 inhibitor, PPADS, abrogated 1,25D-induced mineral deposition. These data demonstrate that 1,25D stimulates osteoblast differentiation and mineral deposition, and interacts with the extracellular calcium concentration. 1,25D regulates Enpp1 expression, which presumably, in the context of adequate tissue non-specific alkaline phosphatase activity, provides Pi to stimulate mineralisation. Our findings suggest a mechanism by which vitamin D with adequate dietary calcium can improve bone mineral status.

In vitro Response of Human Mesenchymal Stromal Cells to Titanium Coated Peek Films and Their Suitability for Magnetic Resonance Imaging

Medical imaging is an important tool for the post-operative checkup of an accurate position of an implant as well as for monitoring the integration in the adjacent tissue that may influence the success of a medical device. Unfortunately, the possibility to use imaging methods is associated with the implant material and all the established metallic materials for surgery do not show a proper “imaging compatibility”. The present study is a combined investigation of the in vitro response to human mesenchymal stromal cells (hMSC) and magnetic resonance imaging (MRI) compatibility of the potential material combination polyetheretherketone/titanium (PEEK/Ti) for medical devices. Because of the advantageous imaging properties and the mechanical and chemical stability, PEEK becomes more and more an alternative to common metallic implant materials like titanium or cobalt‒chrome. However, PEEK is a bioinert material having a limited ability for direct bone incorporation. Due to its excellent biocompatibility, Ti was chosen as coating material to enhance the cellular response. The result is a combination with advantageous properties: the magnetic susceptibility and elastic modulus close to bone, corrosion resistance and mechanical flexibility of PEEK and the excellent biocompatibility of titanium. The appearance of metal-related artifact was discussed in electrical resistivity and magnetic susceptibility. Therefore, two titanium coatings have been investigated: a complete coating and a structured surface avoiding surface conductivity. To determine the in vitro biocompatibility, the cell responses were assessed in terms of the overall morphology of the hMSC and their cell area distribution, proliferation, osteogenic differentiation and mineral deposition. The cellular stress was evaluated by the prostaglandin E2 level. The bonded materials both produced no disturbing artifacts in magnetic resonance imaging. Compared to the pure PEEK material, the titanium coated specimens showed an enhanced biocompatibility, which is indicated by a higher cell number, larger activity of the enzyme tissue non-specific alkaline phosphatase and therefore a greater amount of deposited calcium and phosphate. The results on bare PEEK are accompanied with a higher cellular stress level, which is indicated by prostaglandin E2.

Artificial Matrices With High‐Sulfated Glycosaminoglycans and Collagen Are Anti‐Inflammatory and Pro‐Osteogenic for Human Mesenchymal Stromal Cells

Bone healing has been described to be most efficient if the early inflammatory phase is resolved timely. When the inflammation elevates or is permanently established, bone healing becomes impaired and, moreover, bone destruction often takes place. Systemic disorders such as diabetes and bone diseases like arthritis and osteoporosis are associated with sustained inflammation and delayed bone healing. One goal of biomaterial research is the development of materials/surface modifications which support the healing process by inhibiting the inflammatory bone erosion and suppressing pro-inflammatory mediators and by that promoting the bone repair process. In the present study, the influence of artificial extracellular matrices (aECM) on the interleukin (IL)-1β-induced pro-inflammatory response of human mesenchymal stromal cells (hMSC) was studied. hMSC cultured on aECM composed of collagen I and high-sulfated glycosaminoglycan (GAG) derivatives did not secrete IL-6, IL-8, monocyte chemoattractant protein-1, and prostaglandin E2 in response to IL-1β. The activation and nuclear translocation of nuclear factor κBp65 induced by IL-1β, tumor necrosis factor-α or lipopolysaccharide was abrogated. Furthermore, these aECM promoted the osteogenic differentiation of hMSC as determined by an increased activity of tissue non-specific alkaline phosphatase (TNAP); however, the aECM had no effect on the IL-1β-induced TNAP activity. These data suggest that aECM with high-sulfated GAG derivatives suppress the formation of pro-inflammatory mediators and simultaneously promote the osteogenic differentiation of hMSC. Therefore, these aECM might offer an interesting approach as material/surface modification supporting the bone healing process.

CD73-TNAP crosstalk regulates the hypertrophic response and cardiomyocyte calcification due to α1 adrenoceptor activation.

Cluster of differentiation 73 (CD73) is an ecto-5' nucleotidase which catalyzes the conversion of AMP to adenosine. One of the many functions of adenosine is to suppress the activity of tissue nonspecific alkaline phosphatase (TNAP), an enzyme important in regulating intracellular calcification. Since myocardial calcification is associated with various cardiac disease states, we studied the individual roles and crosstalk between CD73 and TNAP in regulating myocyte responses to the α1 adrenoceptor agonist phenylephrine in terms of calcification and hypertrophy. Cultured neonatal rat cardiomyocytes were treated with 10 µM phenylephrine for 24 h in the absence or presence of the stable adenosine analog 2-chloro-adenosine, the TNAP inhibitor tetramisole or the CD73 inhibitor α,β-methylene ADP. Phenylephrine produced marked hypertrophy as evidenced by significant increases in myocyte surface area and ANP gene expression, as well as calcification determined by Alizarin Red S staining. These responses were associated with reduced CD73 gene and protein expression and CD73 activity. Conversely, TNAP expression and activity were significantly increased although both were suppressed by 2-chloro-adenosine. CD73 inhibition alone significantly reduced myocyte-derived adenosine levels by >50 %, and directly induced hypertrophy and calcification in the absence of phenylephrine. These responses and those to phenylephrine were abrogated by TNAP inhibition. We conclude that TNAP contributes to the hypertrophic effect of phenylephrine, as well as its ability to produce cardiomyocyte calcification. These responses are minimized by CD73-dependent endogenously produced adenosine.

Abstract 51: Sortilin 1 Promotes Matrix Vesicle-Mediated Vascular Microcalcification via Rab11 Trafficking and C-Terminal Phosphorylation

Background: Arterial microcalcification predicts cardiovascular events and contributes to plaque rupture. We recently demonstrated that matrix vesicles (MVs) participate in the formation of microcalcifications. Yet, the precise mechanisms by which MVs regulate calcification in atheroma remain unknown. We hypothesized that sortilin 1, a protein associated with arterial calcification in GWAS, promotes calcification via its phosphorylation and recruitment to MVs. Results: We detected sortilin 1 in calcified MVs of human plaques using transmission electron microscopy-based immunogold labeling. Sortilin 1 loading into MVs increased in sortilin 1-overexpressing smooth muscle cells (SMCs), and rose further in MVs derived from calcifying SMCs. Nanoparticle tracking analysis indicated no alteration in MV release. Silencing sortilin 1 reduced tissue non-specific alkaline phosphatase (TNAP) activity within MVs (-45%; p<0.01), whereas sortilin 1 overexpression promoted TNAP activity within MVs twofold (p<0.05). Silencing Rab11 reduced sortilin 1 protein (-44%, p<0.01) as well as TNAP activity (-34%, p<0.01) and mineralization (-36%, p<0.01). Further, silencing Rab11 abolished the induction of TNAP activity by sortilin 1 overexpression. We used a mass spectrometry-based strategy to monitor sortilin 1 post-translational modifications in calcifying SMCs and identified a calcification-dependent phosphorylation within the C-terminal domain. Mutations on Ser825 and Ser819, which prevented sortilin 1 phosphorylation, 1) abolished SMC TNAP activity (-43%; p<0.05) and calcification (-72%; p<0.05), 2) mitigated TNAP activity (-36%; p<0.05) and hydroxyapatite content (-34%; p<0.05) in MVs, and 3) prevented loading of sortilin 1 into MVs. In contrast, mutations on Ser825 and Ser819 that mimic a constitutive active phosphorylation enhanced TNAP activity without affecting sortilin 1 expression. Conclusion: Sortilin 1 promotes MV-mediated vascular microcalcification via Rab11 trafficking and C-terminal phosphorylation. Our data provide mechanistic insight into the correlation between sortilin 1 and calcification and identifies sortilin 1 as a potential therapeutic target to modulate the formation of microcalcifications.

Artificial extracellular matrices with oversulfated glycosaminoglycan derivatives promote the differentiation of osteoblast-precursor cells and premature osteoblasts.

Sulfated glycosaminoglycans (GAG) are components of the bone marrow stem cell niche and to a minor extent of mature bone tissue with important functions in regulating stem cell lineage commitment and differentiation. We anticipated that artificial extracellular matrices (aECM) composed of collagen I and synthetically oversulfated GAG derivatives affect preferentially the differentiation of osteoblast-precursor cells and early osteoblasts. A set of gradually sulfated chondroitin sulfate and hyaluronan derivatives was used for the preparation of aECM. All these matrices were analysed with human bone marrow stromal cells to identify the most potent aECM and to determine the influence of the degree and position of sulfate groups and the kind of disaccharide units on the osteogenic differentiation. Oversulfated GAG derivatives with a sulfate group at the C-6 position of the N-acetylglycosamine revealed the most pronounced proosteogenic effect as determined by tissue nonspecific alkaline phosphatase activity and calcium deposition. A subset of the aECM was further analysed with different primary osteoblasts and cell lines reflecting different maturation stages to test whether the effect of sulfated GAG derivatives depends on the maturation status of the cells. It was shown that the proosteogenic effect of aECM was most prominent in early osteoblasts.

The Rachitic Tooth

Teeth are mineralized organs composed of three unique hard tissues, enamel, dentin, and cementum, and supported by the surrounding alveolar bone. Although odontogenesis differs from osteogenesis in several respects, tooth mineralization is susceptible to similar developmental failures as bone. Here we discuss conditions fitting under the umbrella of rickets, which traditionally referred to skeletal disease associated with vitamin D deficiency but has been more recently expanded to include newly identified factors involved in endocrine regulation of vitamin D, phosphate, and calcium, including phosphate-regulating endopeptidase homolog, X-linked, fibroblast growth factor 23, and dentin matrix protein 1. Systemic mineral metabolism intersects with local regulation of mineralization, and factors including tissue nonspecific alkaline phosphatase are necessary for proper mineralization, where rickets can result from loss of activity of tissue nonspecific alkaline phosphatase. Individuals suffering from rickets often bear the additional burden of a defective dentition, and transgenic mouse models have aided in understanding the nature and mechanisms involved in tooth defects, which may or may not parallel rachitic bone defects. This report reviews dental effects of the range of rachitic disorders, including discussion of etiologies of hereditary forms of rickets, a survey of resulting bone and tooth mineralization disorders, and a discussion of mechanisms, known and hypothesized, involved in the observed dental pathologies. Descriptions of human pathology are augmented by analysis of transgenic mouse models, and new interpretations are brought to bear on questions of how teeth are affected under conditions of rickets. In short, the rachitic tooth will be revealed.

Enzymatic, antimicrobial and toxicity studies of the aqueous extract of Ananas comosus (pineapple) crown leaf

Ethnopharmacological relevanceVarious parts of the plant pineapple (Ananas comosus) are used in traditional medicine worldwide for treatment of a number of diseases and disorders. In folk medicine, pineapple leaf extract was used as an antimicrobial, vermicide, purgative, emmenagoogue, abortifacient, anti-oedema and anti-inflammatory agent. Compared to the fruit and stem extracts of pineapple, information about its leaf extract is limited. The potential of pineapple crown leaf extract as an ethno-medicine has been evaluated in terms of its enzymatic activities related to wound healing, antimicrobial property and toxicity. Materials and methodsMajor protein components of the extract were revealed by 2-D gel electrophoresis followed by MS/MS analysis. Zymography, DQ-gelatin assay were performed to demonstrate proteolytic, fibrinolytic, gelatinase and collagenase activities. DNase and RNase activities were revealed from agarose gel electrophoresis. Antimicrobial activity was evaluated spectrophotometrically from growth inhibition. Sprague-Dawley rat model was used to measure acute and sub-acute toxicity of the extract by analyzing blood markers. ResultThe extract contains several proteins that were clustered under native condition. Proteomic studies indicated presence of fruit bromelain as major protein constituent of the extract. It showed nonspecific protease activity, gelatinolytic, collagenase, fibrinolytic, acid and alkaline phosphatase, peroxidase, DNase and RNase activities along with considerable anti-microbial property. The leaf extract did not induce any toxicity in rats after oral administration of acute and sub-acute doses. ConclusionPineapple leaf extract is nontoxic, contains enzymes related to damage tissue repairing, wound healing and possibly prevents secondary infections from microbial organisms.

Chemical nanoroughening of Ti40Nb surfaces and its effect on human mesenchymal stromal cell response

Samples of low modulus beta-type Ti40Nb and cp2-Ti were chemically treated with 98% H2 SO4 + 30% H2 O2 (vol. ratio 1:1) solution. Surface analytical studies conducted with HR-SEM, AFM, and XPS identified a characteristic nanoroughness of the alloy surface related with a network of nanopits of ∼25 nm diameter. This is very similar to that obtained for cp2-Ti. The treatment enhances the oxide layer growth compared to mechanically ground states and causes a strong enrichment of Nb2 O5 relative to TiO2 on the alloy surface. The in vitro analyses clearly indicated that the chemical treatment accelerates the adhesion and spreading of human mesenchymal stromal cells (hMSC), increases the metabolic activity, and the enzyme activity of tissue non-specific alkaline phosphatase (TNAP). Surface structures which were generated mimic the cytoplasmic projections of the cells on the nanoscale. Those effects are more pronounced for the Ti40Nb alloy than for cp2-Ti. The relation between alloy surface topography and chemistry and cell functions is discussed.

Over‐sulfated chondroitin sulfate Derivatives induce osteogenic differentiation of hMSC independent of BMP‐2 and TGF‐β1 signalling

Natural glycosaminoglycans (GAGs) and chemically modified GAG derivatives are known to support osteogenic differentiation of mesenchymal stromal cells (MSC). This effect has mainly been described to be mediated by increasing the effectiveness of bone anabolic growth factors such as bone morphogenetic proteins (BMPs) due to the binding and presentation of the growth factor or by modulating its signal transduction pathway. In the present study, the influence of chondroitin sulfate (CS) and two chemically over-sulfated CS derivatives on osteogenic differentiation of human mesenchymal stromal cells (hMSC) and on BMP-2 and transforming growth factor β1 (TGF-β1) signalling was investigated. Over-sulfated CS derivatives induced an increase of tissue non-specific alkaline phosphatase (TNAP) activity and calcium deposition, whereas collagen synthesis was slightly decreased. The BMP-2-induced Smad1/5 activation was inhibited in the presence of over-sulfated CS derivatives leading to a loss of BMP-2-induced TNAP activity and calcium deposition. In contrast, the TGF-β1-induced activation of Smad2/3 and collagen synthesis were not affected by the over-sulfated CS derivatives. BMP-2 and TGF-β1 did not activate the extracellular signal-regulated kinase 1/2 or mitogen-activated protein kinase p38 in hMSC. These data suggest that over-sulfated CS derivatives themselves are able to induce osteogenic differentiation, probably independent of BMP-2 and TGF-β1 signalling, and offer therefore an interesting approach for the improvement of bone healing.

Characterization of Different Subpopulations from Bone Marrow-Derived Mesenchymal Stromal Cells by Alkaline Phosphatase Expression

Multiple surface markers have been utilized for the enrichment of bone marrow mesenchymal stromal cells (MSCs) and to define primitive stem cells. We classified human bone marrow-derived MSC populations according to tissue nonspecific alkaline phosphatase (TNAP) activity. TNAP expression varied among unexpanded primary MSCs, and its level was not related to colony-forming activity or putative surface markers, such as CD105 and CD29, donor age, or gender. TNAP levels were increased in larger cells, and a colony-forming unit-fibroblast assay revealed that the colony size was decreased during in vitro expansion. TNAP-positive (TNAP+) MSCs showed limited multipotential capacity, whereas TNAP-negative (TNAP-) MSCs retained the differentiation potential into 3 lineages (osteogenic-, adipogenic-, and chondrogenic differentiation). High degree of calcium mineralization and high level of osteogenic-related gene expression (osteopontin, dlx5, and cbfa1) were found in TNAP+ cells. In contrast, during chondrogenic differentiation, type II collagen was successfully induced in TNAP- cells, but not in TNAP+ cells. TNAP+ cells showed high levels of the hypertrophic markers, type X collagen and cbfa1. Mesenchymal stem cell antigen-1 (MSCA-1) is identical to TNAP. Therefore, TNAP+ cells were sorted by using antibody targeting MSCA-1. MSCA-1-positive cells sorted for TNAP+ cells exhibited low proliferation rates. Expression of cell cycle-related genes (cyclin A2, CDK2, and CDK4) and pluripotency marker genes (rex1 and nanog) were higher in TNAP- MSC than in TNAP+ MSC. Therefore, TNAP- cells can be described as more primitive bone marrow-derived cells and TNAP levels in MSCs can be used to predict chondrocyte hypertrophy or osteogenic capacity.

Respiratory mechanics in an infant with perinatal lethal hypophosphatasia treated with human recombinant enzyme replacement therapy

Hypophosphatasia is a rare autosomal recessive disorder caused by deficient activity of tissue nonspecific alkaline phosphatase (TNSALP) and characterized by defective bone mineralization. In the perinatal lethal form, respiratory complications due to rachitic deformities of the thoracic cage and associated hypoplastic lungs are present. ENB-0040 is a bone-targeted human recombinant TNSALP fusion protein that aims to restore skeletal mineralization. The goal of this study was to characterize pulmonary and thoracic cage mechanics in an infant with the perinatal lethal form of hypophosphatasia under enzyme replacement therapy. Pulmonary function testing was performed on a preterm, 8-week-old patient with hypophosphatasia who was mechanically ventilated since birth because of severe chest wall insufficiency. The measurements consisted of respiratory impulse oscillation measurements (resistance and reactance), ventilatory mechanics (compliance and resistance), and thoracoabdominal motion (TAM) analysis. At baseline, chest wall compliance was 50% of normal, and the TAM indicated predominantly abdominal displacement. After 12 weeks of treatment, a consistent decrease in ventilator requirements and improvement in lung function and chest wall mechanics were observed and correlated with thoracic cage radiologic findings. Measurable changes in chest wall dynamics and respiratory mechanics using noninvasive technology were useful for respiratory management and therapeutic guidance of ENB-0040 treatment in this patient.

Oral manifestations of patients with hypophosphatasia

Abstract Hypophosphatasia is a rare inherited disorder characterized by defective bone mineralization and deficiency of tissue non-specific alkaline phosphatase (TNSALP) activity. The disease is caused by mutations in the liver/bone/kidney alkaline phosphatase gene ( ALPL ) encoding TNSALP. As for dental manifestations, premature loss of deciduous teeth due to disturbed cementum formation is well known. However, few reports of multiple cases have been presented. The oral manifestations of patients diagnosed with hypophosphatasia were analyzed by collecting clinical records of cases from a nationwide survey of pediatric dentistry clinics affiliated with 29 university dental hospitals in Japan. We inquired regarding the number of cases and clinical findings of diagnosed patients. We obtained information for 9 children diagnosed with hypophosphatasia from our university and 10 from 6 other universities. The main oral manifestation was early exfoliation of deciduous teeth, which was found in 15 of the 19 cases. Early exfoliation of mandibular deciduous anterior teeth was recognized in 14, whereas there were no cases of early exfoliation of a permanent tooth. The main oral finding of hypophosphatasia was early exfoliation of deciduous teeth, predominantly in the mandibular anterior region of children aged 1 to 4 years old.

Prosthetic rehabilitation of a young patient with Hypophosphatasia - A review and case report

Hypophosphatasia is a congenital disease characterized by deficiency of serum and tissue non-specific alkaline phosphatase activity. The disease occurs due to mutations in the liver/bone/kidney alkaline phosphatase gene. Six clinical forms of hypophosphatasia are recognized. Systemic symptoms of the disease are respiratory complications, premature craniosynostosis, widespread demineralization and rachitic changes in the metaphases, stress fractures, chondrocalcinosis and osteoarthropathy. Characteristic dental symptoms include premature loss of deciduous teeth, premature exfoliation of fully rooted permanent teeth, severe dental caries and alveolar bone loss. This clinical report describes the prosthetic rehabilitation of a sixteen year old female patient with hypophosphatasia with partial anodontia and no occlusion. Patient was managed clinically by saving her remaining teeth, fabricating a maxillary overdenture and mandibular cast partial denture. This not only helped her functionally in biting and chewing food and achieving a good occlusion, but also restored her confidence and self image by improving the aesthetics.

Response of human bone marrow stromal cells, MG‐63, and SaOS‐2 to titanium‐based dental implant surfaces with different topography and surface energy

Osseointegration is dependent on different parameters of the implant surface like surface roughness and physicochemical properties. In vitro studies using a wide variety of surface parameters and cell lines make it difficult to address the influence of a single parameter. With this study the influence of surface topography and energy on different osteoblast derived cell lines, namely MG-63 and SaOS-2 and of human mesenchymal stromal cells (hMSC) were investigated. Cells were cultured on polished (POL) and sandblasted/hot acid etched (SBA) titanium surfaces which were partly alkaline treated (SBA NaOH). Cell morphology, metabolic activity, tissue non-specific alkaline phosphatase (TNAP) activity and prostaglandin E(2) (PGE(2) ) formation were determined. Impaired spreading was found on both SBA surfaces. Proliferation after 4 and 7days increased on POL compared to both SBA surfaces. TNAP activity of hMSC and MG-63 was increased on POL compared to both SBA surfaces whereas SaOS-2 did not discriminate between the three surfaces. PGE(2) formation of hMSC and MG-63 was on both SBA surfaces after 2days significantly higher than on POL. The results of this study show that surface roughness has a distinct influence on proliferation and differentiation of osteoblasts. However, variations in physicochemical properties seem to have little influence under the used experimental conditions. It is suggested that more sever and long-lasting modifications of surface chemistry would have an influence on osteoblastic cells.

Cell-specific effects of TNF-α and IL-1β on alkaline phosphatase: implication for syndesmophyte formation and vascular calcification

Tumor necrosis factor (TNF)-α and interleukin (IL)-1β stimulate tissue non-specific alkaline phosphatase (TNAP) activity and mineralization in cultures of vascular smooth muscle cells (VSMCs). They are, therefore, considered as stimulators of vascular calcification in the context of atherosclerosis and diabetes type 2. In contrast, although ankylosing spondylitis (AS) leads to the formation of syndesmophytes, which are ectopic ossifications from entheses (where ligaments, tendons and capsules are attached to bone), anti-TNF-α therapies fail to block bone formation in this disease. In this context, our aims were to compare the effects of TNF-α and IL-1β on TNAP activity and mineralization in entheseal cells and VSMCs. Organotypic cultures of mouse ankle entheses were treated or not with TNF-α and IL-1β for 5 days. Micro-computed tomography was performed to determine trabecular bone parameters, and histology to assess TNAP activity and mineralization. Human mesenchymal stem cells cultured in pellets in chondrogenic conditions and human VSMCs were also used to determine the effects of cytokines on TNAP activity and expression, measured by quantitative PCR. In organotypic cultures, TNF-α and IL-1β significantly reduced the tibia BV/TV ratio. They also inhibited TNAP activity in entheseal chondrocytes in situ, and in mouse and human chondrocytes in vitro. In contrast, TNF-α stimulated TNAP expression and activity in human VSMCs. These differences were likely due to cell-specific effects of peroxisome proliferator-activated receptor γ (PPARγ), which is inhibited by TNF-α. Indeed, in human chondrocytes and VSMCs, the PPARγ inhibitor GW-9662 displayed the same opposite effects as TNF-α on TNAP expression. In conclusion, whereas TNF-α and IL-1β stimulate TNAP activity in VSMCs, they inhibit it in entheseal cells in situ and on chondrocytes in vitro. The identification of PPARγ as a likely mediator of cytokine effects deserves consideration for future research on the mechanisms of ectopic ossification.

Synthesis and evaluation of thiophenyl derivatives as inhibitors of alkaline phosphatase

Pathological calcifications induced by deposition of basic phosphate crystals or hydroxyapatite (HA) on soft tissues are a large family of diseases comprising of ankylosing spondylitis (AS), end-stage osteoarthritis (OA) and vascular calcification. High activity of tissue non-specific alkaline phosphatase (TNAP) is a hallmark of pathological calcifications induced by HA deposition. The use of TNAP inhibitor is a possible therapeutic option to address calcific diseases produced by HA deposition on soft tissues. We report the synthesis of a series of thiopheno-imidazo[2,1-b]thiazole derivatives which were evaluated as potential inhibitors of TNAP displaying a large range of IC(50) at pH 10.4 (from 42±13 μM to more than 800 μM).

Layer-specific activity of tissue non-specific alkaline phosphatase in the human neocortex

The ectoenzyme tissue non-specific alkaline phosphatase (TNAP) is mostly known for its role in bone mineralization. However, in the severe form of hypophosphatasia, TNAP deficiency also results in epileptic seizures, suggesting a role of this enzyme in brain functions. Accordingly, TNAP activity was shown in the neuropil of the cerebral cortex in diverse mammalian species. However in spite of its clinical significance, the neuronal localization of TNAP has not been investigated in the human brain. By using enzyme histochemistry, we found an unprecedented pattern of TNAP activity appearing as an uninterrupted layer across diverse occipital-, frontal- and temporal lobe areas of the human cerebral cortex. This marked TNAP-active band was localized infragranulary in layer 5 as defined by quantitative comparisons on parallel sections stained by various techniques to reveal the laminar pattern. On the contrary, TNAP activity was localized in layer 4 of the primary visual and somatosensory cortices, which is consistent with earlier observations on other species. This result suggests that the expression of TNAP in the thalamo-recipient granular layer is an evolutionary conserved feature of the sensory cortex. The observations of the present study also suggest that diverse neurocognitive functions share a common cerebral cortical mechanism depending on TNAP activity in layer 5. In summary, the present data point on the distinctive role of layer 5 in cortical computation and neurological disorders caused by TNAP dysfunctions in the human brain.

Enzymatic study of tonsil tissue alkaline and acid phosphatase in children with recurrent tonsillitis and tonsil hypertrophy

Objective Indications for tonsillectomy in recurrent tonsillitis are defined according to the number of episodes of acute bacterial infections in a year. However, little is known about the tonsil immune competence status in patients presenting with recurrent tonsillitis with either hypertrophied or atrophied tonsils, or in patients presenting with obstructive sleep apnoea. In this study we examined the tonsil immune status in children with 3–5 acute recurrent infections a year and in children with obstructive sleep apnoea by comparing the activity of tonsil and adenoid tissue nonspecific alkaline and acid phosphatase. Methods Specific activity of tonsil and adenoid tissue nonspecific alkaline and acid phosphatase was investigated in children who underwent tonsillectomy and adenoidectomy for recurrent infection (72 children) and for obstructive sleep apnoea (10 children). Tissue enzyme activities were measured using p-nitrophenylphosphate as a substrate. Tissue samples were examined by the haematoxylin–eosin histological technique. Statistical analyses were performed using SPSS v. 16 software. Results The tissue nonspecific alkaline phosphatase activity was similar in hypertrophied tonsils in the recurrent infection group and in the obstructive sleep apnoea group (3.437 ± 1.226 and 3.978 ± 0.762 U/mg of protein, respectively). The enzyme activity in both hypertrophied tonsil groups was significantly higher as compared to atrophied tonsils in the recurrent tonsillitis group, p = 0.021 and p = 0.006, respectively. The enzyme activity was significantly higher in the adenoids compared to the tonsils from all three groups. Contrary to this, no significant differences were noticed for tonsil and adenoid acid phosphatase activities among the groups. Conclusion Similar acid phosphatase activity in all three groups implies that all three groups have preserved antigen presenting cell activity. In patients with hypertrophied tonsils similar tissue nonspecific alkaline phosphatase activity suggests preserved B cell tonsil immune activity, regardless of the pathology. Patients with atrophied tonsils had significantly lower alkaline phosphatase activity, indicating relative tonsil B cell immune deficiency. Thus, different immunological status in patients presenting with hypertrophied vs. atrophied tonsils could point to a different underlying pathophysiologic mechanism of the disease.

Synthesis and evaluation of benzo[ b]thiophene derivatives as inhibitors of alkaline phosphatases

Presence of basic calcium phosphate in knee joints of osteoarthritis patients could be prevented by inhibiting tissue non-specific alkaline phosphatase (TNAP) activity. Levamisole or the L stereoisomer of tetramisole (a known TNAP inhibitor) has been used as a treatment for curing rheumatoid arthritis but its therapeutical use is limited due to side effects. We report the synthesis and the TNAP inhibition property of benzo[ b]thiophene derivatives, among which benzothiopheno-tetramisole and benzothiopheno-2,3-dehydrotetramisole, which could be involved in a drug therapy for osteoarthritis. Two water soluble racemic benzothiopheno-tetramisole and -2,3-dehydrotetramisole with apparent inhibition constants K i = 85 ± 6 μM and 135 ± 3 μM ( n = 3) comparable to that of enantiomeric levamisole 93 ± 4 μM were found. Several novel derivatives showed more pronounced inhibition properties towards intestinal alkaline phosphatase than TNAP.