Immunolocalization of Enzymes/Membrane Transporters Related to Bone Mineralization in the Metaphyses of the Long Bones of Parathyroid-Hormone-Administered Mice.

The present study aimed to demonstrate the immunolocalization and/or gene expressions of the enzymes and membrane transporters involved in bone mineralization after the intermittent administration of parathyroid hormone (PTH). The study especially focused on TNALP, ENPP1, and PHOSPHO1, which are involved in matrix vesicle-mediated mineralization, as well as PHEX and the SIBLING family, which regulate mineralization deep inside bone. Six-week-old male mice were subcutaneously injected with 20 µg/kg/day of human PTH (1-34) two times per day (n = 6) or four times per day (n = 6) for two weeks. Additionally, control mice (n = 6) received a vehicle. Consistently with an increase in the volume of the femoral trabeculae, the mineral appositional rate increased after PTH administration. The areas positive for PHOSPHO1, TNALP, and ENPP1 in the femoral metaphyses expanded, and the gene expressions assessed by real-time PCR were elevated in PTH-administered specimens when compared with the findings in control specimens. The immunoreactivity and/or gene expressions of PHEX and the SIBLING family (MEPE, osteopontin, and DMP1) significantly increased after PTH administration. For example, MEPE immunoreactivity was evident in some osteocytes in PTH-administered specimens but was hardly observed in control specimens. In contrast, mRNA encoding cathepsin B was significantly reduced. Therefore, the bone matrix deep inside might be further mineralized by PHEX/SIBLING family after PTH administration. In summary, it is likely that PTH accelerates mineralization to maintain a balance with elevated matrix synthesis, presumably by mediating TNALP/ENPP1 cooperation and stimulating PHEX/SIBLING family expression.

Comparative immunolocalization of tissue nonspecific alkaline phosphatase and ectonucleotide pyrophosphatase/phosphodiesterase 1 in murine bone.

OBJECTIVE: This study aimed to demonstrate the immunolocalization and gene expression of tissue nonspecific alkaline phosphatase (TNALP) and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) in osteoblasts, preosteoblasts, and osteocytes of murine bone to provide clues for a better understanding of the supply of phosphate ions (Pi) during bone mineralization. METHODS: Six-week-old male C57BL/6J mice (n = 6) were fixed with a paraformaldehyde solution, and the right femora were extracted for immunodetection of TNALP and ENPP1, while the left tibiae were used for reverse transcription polymerase chain reaction to evaluate Tnalp and Enpp1 gene expression. RESULTS: TNALP was intensely localized on the basolateral cell membranes of mature osteoblasts and preosteoblastic cells. There was little immunoreactivity of TNALP on the secretory surface of the osteoblasts and no TNALP reactivity in the osteocytes. In contrast, ENPP1 was observed throughout the cytoplasm of mature osteoblasts and osteocytes embedded in bone but was not observed in preosteoblasts. Together, despite the fact that the osteoid is a site of matrix vesicle-mediated mineralization, ENPP1, which inhibits mineralization by providing pyrophosphates, was localized in close proximity of the osteoid, whereas TNALP, which facilitates mineralization by providing Pi, was relatively distant from the osteoid. CONCLUSION: It seems likely that the differential localization of TNALP and ENPP1 around the osteoid observed at the microscopic level may provide preferential micro-circumstance for a balanced concentration of Pi and pyrophosphate for bone mineralization.

Central nervous system relapse of Whipple's disease.

A 50-year-old man presented with a 12 kg weight loss in 8 months. Upper gastrointestinal endoscopy findings showed strong erosion and diffuse bleeding in the duodenum. Histopathological findings showed PAS staining-positive macrophages consistent with Whipple's disease. He was treated with trimethoprim-sulfamethoxazole. His condition initially improved. However, during his 6-year course of treatment he developed a central nervous system relapse. Tropheryma whipplei DNA was detected by a polymerase chain reaction in his cerebrospinal fluid. This relapse was successfully treated with ceftriaxone sodium (CTRX). We considered that as initial therapy for Whipple's disease, it would be important to administer CTRX for at least a few months, due to its high translatability to CSF.