Conditional Alpl Ablation Phenocopies Dental Defects of Hypophosphatasia.

Loss-of-function mutations in ALPL result in hypophosphatasia (HPP), an inborn error of metabolism that causes defective skeletal and dental mineralization. ALPL encodes tissue-nonspecific alkaline phosphatase, an enzyme expressed in bone, teeth, liver, and kidney that hydrolyzes the mineralization inhibitor inorganic pyrophosphate. As Alpl-null mice die before weaning, we aimed to generate mouse models of late-onset HPP with extended life spans by engineering a floxed Alpl allele, allowing for conditional gene ablation (conditional knockout [cKO]) when crossed with Cre recombinase transgenic mice. The authors hypothesized that targeted deletion of Alpl in osteoblasts and selected dental cells ( Col1a1-cKO) or deletion in chondrocytes, osteoblasts, and craniofacial mesenchyme ( Prx1-cKO) would phenocopy skeletal and dental manifestations of late-onset HPP. Col1a1-cKO and Prx1-cKO mice were viable and fertile, and they did not manifest the epileptic seizures characteristic of the Alpl-/- model of severe infantile HPP. Both cKO models featured normal postnatal body weight but significant reduction as compared with wild type mice by 8 to 12 wk. Plasma alkaline phosphatase for both cKO models at 24 wk was reduced by approximately 75% as compared with controls. Radiography revealed profound skeletal defects in cKO mice, including rachitic changes, hypomineralized long bones, deformations, and signs of fractures. Microcomputed tomography confirmed quantitative differences in cortical and trabecular bone, including decreased cortical thickness and mineral density. Col1a1-cKO mice exhibited classic signs of HPP dentoalveolar disease, including short molar roots with thin dentin, lack of acellular cementum, and osteoid accumulation in alveolar bone. Prx1-cKO mice exhibited the same array of periodontal defects but featured less affected molar dentin. Both cKO models exhibited reduced alveolar bone height and 4-fold increased numbers of osteoclast-like cells versus wild type at 24 wk, consistent with HPP-associated periodontal disease. These novel models of late-onset HPP can inform on long-term skeletal and dental manifestations and will provide essential tools to further studies of etiopathologies and therapeutic interventions.

Role of PHOSPHO1 in Periodontal Development and Function.

The tooth root and periodontal apparatus, including the acellular and cellular cementum, periodontal ligament (PDL), and alveolar bone, are critical for tooth function. Cementum and bone mineralization is regulated by factors including enzymes and extracellular matrix proteins that promote or inhibit hydroxyapatite crystal growth. Orphan Phosphatase 1 (Phospho1, PHOSPHO1) is a phosphatase expressed by chondrocytes, osteoblasts, and odontoblasts that functions in skeletal and dentin mineralization by initiating deposition of hydroxyapatite inside membrane-limited matrix vesicles. The role of PHOSPHO1 in periodontal formation remains unknown and we aimed to determine its functional importance in these tissues. We hypothesized that the enzyme would regulate proper mineralization of the periodontal apparatus. Spatiotemporal expression of PHOSPHO1 was mapped during periodontal development, and Phospho1(-/-) mice were analyzed using histology, immunohistochemistry, in situ hybridization, radiography, and micro-computed tomography. The Phospho1 gene and PHOSPHO1 protein were expressed by active alveolar bone osteoblasts and cementoblasts during cellular cementum formation. In Phospho1(-/-) mice, acellular cementum formation and mineralization were unaffected, whereas cellular cementum deposition increased although it displayed delayed mineralization and cementoid. Phospho1(-/-) mice featured disturbances in alveolar bone mineralization, shown by accumulation of unmineralized osteoid matrix and interglobular patterns of protein deposition. Parallel to other skeletal sites, deposition of mineral-regulating protein osteopontin (OPN) was increased in alveolar bone in Phospho1(-/-) mice. In contrast to the skeleton, genetic ablation of Spp1, the gene encoding OPN, did not ameliorate dentoalveolar defects in Phospho1(-/-) mice. Despite alveolar bone mineralization defects, periodontal attachment and function appeared undisturbed in Phospho1(-/-) mice, with normal PDL architecture and no evidence of bone loss over time. This study highlights the role of PHOSPHO1 in mineralization of alveolar bone and cellular cementum, further revealing that acellular cementum formation is not substantially regulated by PHOSPHO1 and likely does not rely on matrix vesicle-mediated initiation of mineralization.

Bioluminescence-enhanced immunoassays: an ultrasensitive method.

Ultrasensitive bioluminescence immunoassays for the determination of peptides and proteins are illustrated in this paper with the enzyme immunological determination of total IgE in human serum. The usable range of standard curves is from 5 pg to 5000 pg per liter. The intra- and interassay coefficients of variation of this test are in the same range as obtained for chromogenic enzyme immunoassays.

Carotid artery: percutaneous transbrachial selective arteriography with a 4-F catheter.

Bilateral selective common carotid artery catheterization was attempted in 72 patients by means of percutaneous placement of a 4-F catheter from a right brachial artery puncture site in the antecubital fossa. The success rate was high (95.8%) and the complication rate low (6.9%), and there were no instances of brachial artery spasm or thrombosis. The experience, while small, suggests that selective common carotid arteriography can be done safely and efficaciously from the right brachial artery approach.