bmp-2 Gene-Transferred Skeletal Muscles with Needle-Type Electrodes as Efficient and Reliable Biomaterials for Bone Regeneration.

BACKGROUND: Bone morphogenetic protein-2 (bmp-2) has a high potential to induce bone tissue formation in skeletal muscles. We developed a bone induction system in skeletal muscles using the bmp-2 gene through in vivo electroporation. Natural bone tissues with skeletal muscles can be considered potential candidates for biomaterials. However, our previous system using plate-type electrodes did not achieve a 100% success rate in inducing bone tissues in skeletal muscles. In this study, we aimed to enhance the efficiency of bone tissue formation in skeletal muscles by using a non-viral bmp-2 gene expression plasmid vector (pCAGGS-bmp-2) and needle-type electrodes. METHODS: We injected the bmp-2 gene with pCAGGS-bmp-2 into the skeletal muscles of rats' legs and immediately placed needle-type electrodes there. Skeletal tissues were then observed on the 21st day after gene transfer using soft X-ray and histological analyses. RESULTS: The use of needle-type electrodes resulted in a 100% success rate in inducing bone tissues in skeletal muscles. In contrast, the plate-type electrodes only exhibited a 33% success rate. Thus, needle-type electrodes can be more efficient and reliable for transferring the bmp-2 gene to skeletal muscles, making them potential biomaterials for repairing bone defects.

Circulatory C-type natriuretic peptide reduces mucopolysaccharidosis-associated craniofacial hypoplasia in vivo.

Skeletal alterations in the head and neck region, such as midfacial hypoplasia, foramen magnum stenosis and spinal canal stenosis, are commonly observed in patients with mucopolysaccharidosis (MPS). However, enzyme replacement therapy (ERT), one of the major treatment approaches for MPS, shows limited efficacy for skeletal conditions. In this study, we analysed the craniofacial morphology of mice with MPS type VII, and investigated the underlying mechanisms promoting jaw deformities in these animals. Furthermore, we investigated the effects of C-type natriuretic peptide (CNP), a potent endochondral ossification promoter, on growth impairment of the craniofacial region in MPS VII mice when administered alone or in combination with ERT. MPS VII mice exhibited midfacial hypoplasia caused by impaired endochondral ossification, and histological analysis revealed increased number of swelling cells in the resting zone of the spheno-occipital synchondrosis (SOS), an important growth centre for craniomaxillofacial skeletogenesis. We crossed MPS VII mice with transgenic mice in which CNP was expressed in the liver under the control of the human serum amyloid-P component promoter, resulting in elevated levels of circulatory CNP. The maxillofacial morphological abnormalities associated with MPS VII were ameliorated by CNP expression, and further prevented by a combination of CNP and ERT. Histological analysis showed that ERT decreased the swelling cell number, and CNP treatment increased the width of the proliferative and hypertrophic zones of the SOS. Furthermore, the foramen magnum and spinal stenoses observed in MPS VII mice were significantly alleviated by CNP and ERT combination. These results demonstrate the therapeutic potential of CNP, which can be used to enhance ERT outcome for MPS VII-associated head and neck abnormalities.

Effect of C-type natriuretic peptide on craniofacial skeletogenesis in mice during the pubertal growth spurt.

OBJECTIVE: This study aimed to determine the effect of C-type natriuretic peptide (CNP) overexpression on craniofacial growth during the pubertal growth period in mice. DESIGN: Six-week-old C57BL/6 mice were injected with pLIVE-Empty vectors (Control mice) and pLIVE-NPPC vectors (CNP mice) using the hydrodynamic method. Morphological analyses were performed at the age of 12 weeks. RESULTS: Micro-computed tomography (µCT) images showed significant (p < 0.05) hyperplasia in the maxilla along the sagittal plane (CNP mice: 13.754 mm, Control mice: 13.215 mm). Further, the images revealed significant bone overgrowth in the sagittal direction in the sphenoid (CNP mice: 6.936 mm, Control mice: 6.411 mm) and occipital (CNP mice: 4.051 mm, Control mice: 3.784 mm) bones in the CNP mice compared with that in the Control mice. Compared with SAP-Nppc-Tg mice in previous studies, although there was no effect on nose length and nasal bone length, the effect was sufficient to improve craniofacial hypogrowth. Furthermore, CNP promoted sagittal cranial growth by increasing the thickness of the spheno-occipital synchondrosis in organ cultures and nasal septal cartilage in micromass cultures, which were derived from 6-week-old mice. CONCLUSIONS: We have previously shown that the elevated blood levels of CNP from the neonatal period affect midfacial skeletogenesis by promoting endochondral ossification using mice (SAP-Nppc-Tg mice). The overexpression of CNP, even in 6-weeks-old mice, promoted growth in the sagittal direction within the maxillary region. These findings indicate the therapeutic potential of CNP for the treatment of midfacial hypoplasia during the pubertal growth spurt.

Periodontal Tissue as a Biomaterial for Hard-Tissue Regeneration following bmp-2 Gene Transfer.

The application of periodontal tissue in regenerative medicine has gained increasing interest since it has a high potential to induce hard-tissue regeneration, and is easy to handle and graft to other areas of the oral cavity or tissues. Additionally, bone morphogenetic protein-2 (BMP-2) has a high potential to induce the differentiation of mesenchymal stem cells into osteogenic cells. We previously developed a system for a gene transfer to the periodontal tissues in animal models. In this study, we aimed to reveal the potential and efficiency of periodontal tissue as a biomaterial for hard-tissue regeneration following a bmp-2 gene transfer. A non-viral expression vector carrying bmp-2 was injected into the palate of the periodontal tissues of Wistar rats, followed by electroporation. The periodontal tissues were analyzed through bone morphometric analyses, including mineral apposition rate (MAR) determination and collagen micro-arrangement, which is a bone quality parameter, before and after a gene transfer. The MAR was significantly higher 3-6 d after the gene transfer than that before the gene transfer. Collagen orientation was normally maintained even after the bmp-2 gene transfer, suggesting that the bmp-2 gene transfer has no adverse effects on bone quality. Our results suggest that periodontal tissue electroporated with bmp-2 could be a novel biomaterial candidate for hard-tissue regeneration therapy.

Low bone mineral density due to secondary hyperparathyroidism in the GlatmTg(CAG-A4GALT) mouse model of Fabry disease.

Low bone mineral density (BMD)-diagnosed as osteoporosis or osteopenia-has been reported as a new characteristic feature of Fabry disease; however, the mechanism underlying the development of low BMD is unknown. We previously revealed that a mouse model of Fabry disease [GlatmTg(CAG-A4GALT)] exhibits impaired functioning of medullary thick ascending limb (mTAL), leading to insufficient Ca2+ reabsorption and hypercalciuria. Here, we investigated bone metabolism in GlatmTg(CAG-A4GALT) mice without marked glomerular or proximal tubular damage. Low BMD was detected by 20 weeks of age via micro-X-ray-computed tomography. Bone histomorphometry revealed that low BMD results by accelerated bone resorption and osteomalacia. Plasma parathyroid hormone levels increased in response to low blood Ca2+-not plasma fibroblast growth factor 23 (FGF-23) elevation-by 5 weeks of age and showed progressively increased phosphaturic action. Secondary hyperparathyroidism developed by 20 weeks of age and caused hyperphosphatemia, which increased plasma FGF-23 levels with phosphaturic action. The expression of 1α-hydroxylase [synthesis of 1α,25(OH)2D3] in the kidney did not decrease, but that of 24-hydroxylase [degradation of 1α,25(OH)2D3] decreased. Vitamin D deficiency was ruled out as the cause of osteomalacia, as plasma 1α,25(OH)2D3 and 25(OH)D3 levels were maintained. Results demonstrate that secondary hyperparathyroidism due to mTAL impairment causes accelerated bone resorption and osteomalacia due to hyperphosphaturia and hypercalciuria, leading to low BMD in Fabry model mice.

Fgfr1 conditional-knockout in neural crest cells induces heterotopic chondrogenesis and osteogenesis in mouse frontal bones.

Most facial bones, including frontal bones, are derived from neural crest cells through intramembranous ossification. Fibroblast growth factor receptor 1 (Fgfr1) plays a pivotal role in craniofacial bone development, and loss of Fgfr1 leads to cleft palate and facial cleft defects in newborn mice. However, the potential role of the Fgfr1 gene in neural crest cell-mediated craniofacial development remains unclear. To investigate the role of Fgfr1 in neural crest cells, we analyzed Wnt1-Cre;Fgfr1flox/flox mice. Our results show that specific knockout of Fgfr1 in neural crest cells induced heterotopic chondrogenesis and osteogenesis at the interface of the anterior portions of frontal bones. We observed that heterotopic bone formation continued through postnatal day 28, whereas heterotopic chondrogenesis lasted only through the embryonic period. In summary, our results indicate that loss of Fgfr1 in neural crest cells leads to heterotopic chondrogenesis and osteogenesis.

Analysis of mineral apposition rates during alveolar bone regeneration over three weeks following transfer of BMP-2/7 gene via in vivo electroporation

Alveolar bone is not spontaneously regenerated following trauma or periodontitis. We previously proposed an animal model for new alveolar bone regeneration therapy based on the non-viral BMP-2/7 gene expression vector and in vivo electroporation, which induced the formation of new alveolar bone over the course of a week. Here, we analysed alveolar bone during a period of three weeks following gene transfer to periodontal tissue. Non-viral plasmid vector pCAGGS-BMP-2/7 or pCAGGS control was injected into palatal periodontal tissue of the first molar of the rat maxilla and immediately electroporated with 32 pulses of 50 V for 50 msec. Over the following three weeks, rats were double bone-stained by calcein and tetracycline every three days and mineral apposition rates (MAR) were measured. Double bone-staining revealed that MAR of alveolar bone was as similar level three days before BMP-2/7 gene transfer as three days after gene transfer. However, from 3 to 6 days, 6 to 9 days, 9 to 12 days, 12 to 15 days, 15 to 18 days, and 18 to 20 days after, MARs were significantly higher than prior to gene transfer. Our proposed gene therapy for alveolar bone regeneration combining non-viral BMP-2/7 gene expression vector and in vivo electroporation could increase alveolar bone regeneration potential in the targeted area for up to three weeks.

Non-surgical model for alveolar bone regeneration by bone morphogenetic protein-2/7 gene therapy.

BACKGROUND: Alveolar bone is a critical tissue for tooth retention; however, once alveolar bone is lost, it may not spontaneously regenerate. Currently, bone grafts or artificial bone is commonly used for alveolar bone regeneration therapy. However, these therapies require surgical procedures, which present risks, particularly in elderly patients. Therefore, development of alveolar bone regeneration techniques that do not require surgical procedures is critical. It is well known that stem cells present in the periosteal and periodontal ligament may be induced to differentiate into osteogenic cells. This study hypothesizes that transfer of the bone morphogenetic protein-2/7 (BMP-2/7) gene into periodontal tissues via in vivo electroporation induces exogenous BMP production and causes stem cells in periodontal tissues to differentiate into osteogenic cells, enabling generation of new alveolar bone. METHOD: The BMP-2/7 gene expression vector was introduced via electroporation into the target site in periodontal tissues of the first molar of rat maxillae. RESULTS: Exogenous BMP-2 and -7 were detected in the target areas, and growth of new alveolar bone tissue was observed 5 days after gene transfer. On day 7, new alveolar bone tissues were found to connect to the original bone tissues. Moreover, mineral apposition rates of the alveolar bone after BMP-2/7 gene transfer were significantly higher than those in the control group after LacZ gene transfer. CONCLUSION: The present findings indicate that a combination of the BMP-2/7 non-viral vector and in vivo electroporation represents a promising non-surgical option for alveolar bone regeneration therapy.

Morphological and Functional Analyses of the Tight Junction in the Palatal Epithelium of Mouse.

Tight junction (TJ) is one of the cell-cell junctions and known to have the barrier and fence functions between adjacent cells in both simple and stratified epithelia. We examined the distribution pattern, constitutive proteins, and permeability of TJ in the stratified squamous epithelium of the palatal mucosa of mice. Ultrastructural observations based on the ultrathin section and freeze-fracture methods revealed that poorly developed TJs are located at the upper layer of the stratum granulosum. The positive immunofluorescence of occludin (OCD), claudin (CLD)-1 and -4 were localized among the upper layer of the stratum granulosum showing a dot-like distribution pattern. And CLD-1 and -4 were localized among the stratum spinosum and the lower part of stratum granulosum additionally showed a positive reaction along the cell profiles. Western blotting of TJ constitutive proteins showed OCD, CLD-1, -2, -4, and -5 bands. The permeability test using biotin as a tracer revealed both the areas where biotin passed through beyond OCD positive points and the areas where biotin stopped at OCD positive points. These results show that poor TJs localize at the upper layer of the stratum granulosum of the palatal epithelium, and the TJs are leaky and include at least CLD-1 and -4.

Determination of cell fate in skeletal muscle following BMP gene transfer by in vivo electroporation.

We previously developed a novel method for gene transfer, which combined a non-viral gene expression vector with transcutaneous in vivo electroporation. We applied this method to transfer the bone morphogenetic protein (BMP) gene and induce ectopic bone formation in rat skeletal muscles. At present, it remains unclear which types of cells can differentiate into osteogenic cells after BMP gene transfer by in vivo electroporation. Two types of stem cells in skeletal muscle can differentiate into osteogenic cells: muscle-derived stem cells, and bone marrow-derived stem cells in the blood. In the present study, we transferred the BMP gene into rat skeletal muscles. We then stained tissues for several muscle-derived stem cell markers (e.g., Pax7, M-cadherin), muscle regeneration-related markers (e.g., Myod1, myogenin), and an inflammatory cell marker (CD68) to follow cell differentiation over time. Our results indicate that, in the absence of BMP, the cell population undergoes muscle regeneration, whereas in its presence, it can differentiate into osteogenic cells. Commitment towards either muscle regeneration or induction of ectopic bone formation appears to occur five to seven days after BMP gene transfer.

Tensile stress stimulates the expression of osteogenic cytokines/growth factors and matricellular proteins in the mouse cranial suture at the site of osteoblast differentiation.

Mechanical stress promotes osteoblast proliferation and differentiation from mesenchymal stem cells (MSCs). Although numerous growth factors and cytokines are known to regulate this process, information regarding the differentiation of mechanically stimulated osteoblasts from MSCs in in vivo microenvironment is limited. To determine the significant factors involved in this process, we performed a global analysis of differentially expressed genes, in response to tensile stress, in the mouse cranial suture wherein osteoblasts differentiate from MSCs. We found that the gene expression levels of several components involved in bone morphogenetic protein, Wnt, and epithelial growth factor signalings were elevated with tensile stress. Moreover gene expression of some extracellular matrices (ECMs), such as cysteine rich protein 61 (Cyr61)/CCN1 and galectin-9, were upregulated. These ECMs have the ability to modulate the activities of cytokines and are known as matricellular proteins. Cyr61/CCN1 expression was prominently increased in the fibroblastic cells and preosteoblasts in the suture. Thus, for the first time we demonstrated the mechanical stimulation of Cyr61/CCN1 expression in osteogenic cells in an ex vivo system. These results suggest the importance of matricellular proteins along with the cytokine-mediated signaling for the mechanical regulation of MSC proliferation and differentiation into osteoblastic cell lineage in vivo.

Effect of renal impairment on cognitive function during a 3-year follow up in elderly patients with type 2 diabetes: Association with microinflammation.

AIMS/INTRODUCTION: We investigated the effect of renal impairment on cognitive function during a 3-year follow up in elderly type 2 diabetic patients, and an association with microinflammation. MATERIALS AND METHODS: Four cognitive function tests - Mini-Mental State Examination (MMSE), word recall, Digit Symbol Substitution (DSS) and Stroop Color Word - were carried out in 67 patients. Renal impairment was defined as the presence of albuminuria and a decline in estimated glomerular filtration (eGFR) <60 mL/min/1.73 m(2). Inflammatory markers, such as highly sensitive C-reactive protein (hs-CRP), tumor necrotizing factor-α (TNF-α), interleukin (IL)-1ß and IL-6, were measured at baseline. RESULTS: At baseline, cognitive decline was found in patients with renal impairment. The DSS test was independently associated with eGFR decline, whereas MMSE tended to be associated with albuminuria after adjusting for confounding factors. Regarding changes in cognitive function and renal impairment, changes in urinary albumin to creatinine ratios were strongly and independently associated with changes in word recall scores. In patients with persistent eGFR decline, there was a tendency toward a greater decrease in MMSE and DSS scores, whereas in those with newly detected albuminuria, there was a tendency toward a greater decrease in word recall scores. Increased baseline levels of hs-CRP, TNF-α and IL-6 were associated with renal impairment and cognitive function, especially DSS tests, respectively. However, the increased levels were not independent predictors for cognitive decline. CONCLUSIONS: The present study showed a reciprocal relationship between cognitive decline and renal impairment, especially progression of albuminuria. Thus, monitoring treatment using renal biomarkers will be important for preserving both renal and cognitive function.

Obvious optic disc swelling in a patient with cryopyrin-associated periodic syndrome.

Cryopyrin-associated periodic syndrome (CAPS) is a group of rare hereditary autoinflammatory diseases caused by mutations of the NLRP3 gene, and leads to excessive production of the proinflammatory cytokine, interleukin-lß. A 35-year-old male presented with recurrent symptoms of urticarial-like rash, periodic fever, arthralgia, headache, and eye redness. His best-corrected visual acuity was 1.0 OD and 0.9 OS. Slit-lamp examination showed conjunctival and episcleral injection in both eyes. Ophthalmoscopy revealed obvious bilateral optic disc swelling and retinal vascular sheathing around the optic discs. Spectral domain optical coherence tomography also showed obvious optic disc swelling. Steroid and nonsteroidal anti-inflammatory drugs did not improve these symptoms. Genetic testing detected a heterozygous mutation of c.907G>A. Thus, the patient was genetically confirmed with CAPS. Visual acuity did not decrease for 3 years, although the optic discs became white in color. CAPS should therefore be distinguished from other disorders when examining optic disc swelling and/or uveitis patients with urticarial-like rash and periodic fever.

Differential expression patterns of the tight junction-associated proteins occludin and claudins in secretory and mature ameloblasts in mouse incisor.

Tight junctions (TJs) function primarily as a barrier against paracellular transport between epithelial cells and are composed mainly of occludin (OLD) and claudins (CLDs). The CLD family consists of 24 members that show tissue- or cell-specific expression. Ameloblasts, which originate from the oral epithelium, form enamel, and enamel proteins and minerals are transported across the ameloblastic layer during amelogenesis. We immunohistochemically examined the distribution patterns of TJs in ameloblasts by observing the expression patterns of OLD and CLDs (CLD-1 to CLD-10). Secretory ameloblasts contained OLD and CLD-1, -8, and -9 at the distal end of the cell. In mature ameloblasts, OLD and CLD-1, -6, -7, -8, -9, and -10 were present mainly at both the distal and proximal ends of the cell, regardless of whether the ameloblasts were ruffle-ended or smooth-ended. Mature ameloblasts in which only the proximal ends were stained for OLD and CLDs were also found. These results indicate that the expression patterns of CLDs and the distribution patterns of TJs change drastically between the secretory and mature ameloblast stages, suggesting that these patterns reflect the different functions of these cells, specifically in the transport of proteins and ions for enamel formation.

Simple strategy for bone regeneration with a BMP-2/7 gene expression cassette vector.

Bone morphogenetic protein (BMP) is one of the most promising candidates for bone regeneration therapy. Heterodimers of BMP family proteins, such as BMP-2/4 or BMP-2/7, are well known to have stronger osteoinduction activity than BMP homodimers. Here, we constructed a double gene cassette vector encoding BMP-2 and BMP-7, pCAGGS-BMP-2/7, and examined its potential for osteoinduction in vitro and in vivo. Expression of the pCAGGS-BMP-2/7 vector induced osteogenic differentiation in various cell lines with the same efficiency as BMP-2 and BMP-7 co-expressed from separate vectors. Moreover, the pCAGGS-BMP-2/7 vector strongly induced bone formation in rat skeletal muscle when introduced by in vivo electroporation, compared with BMP-2 or BMP-7 alone. Thus, our BMP-2/7 double gene cassette vector, or some variation of it, may be applicable for the future clinical induction of bone formation, because it does not require multiple vectors or complicated preparation.

Simultaneous gene transfer of bone morphogenetic protein (BMP) -2 and BMP-7 by in vivo electroporation induces rapid bone formation and BMP-4 expression.

BACKGROUND: Transcutaneous in vivo electroporation is expected to be an effective gene-transfer method for promoting bone regeneration using the BMP-2 plasmid vector. To promote enhanced osteoinduction using this method, we simultaneously transferred cDNAs for BMP-2 and BMP-7, as inserts in the non-viral vector pCAGGS. METHODS: First, an in vitro study was carried out to confirm the expression of BMP-2 and BMP-7 following the double-gene transfer. Next, the individual BMP-2 and BMP-7 plasmids or both together were injected into rat calf muscles, and transcutaneous electroporation was applied 8 times at 100 V, 50 msec. RESULTS: In the culture system, the simultaneous transfer of the BMP-2 and BMP-7 genes led to a much higher ALP activity in C2C12 cells than did the transfer of either gene alone. In vivo, ten days after the treatment, soft X-ray analysis showed that muscles that received both pCAGGS-BMP-2 and pCAGGS-BMP-7 had better-defined opacities than those receiving a single gene. Histological examination showed advanced ossification in calf muscles that received the double-gene transfer. BMP-4 mRNA was also expressed, and RT-PCR showed that its level increased for 3 days in a time-dependent manner in the double-gene transfer group. Immunohistochemistry confirmed that BMP-4-expressing cells resided in the matrix between muscle fibers. CONCLUSION: The simultaneous transfer of BMP-2 and BMP-7 genes using in vivo electroporation induces more rapid bone formation than the transfer of either gene alone, and the increased expression of endogenous BMP-4 suggests that the rapid ossification is related to the induction of BMP-4.

Runx3 expression during mouse tongue and palate development.

Studies of molecular mechanisms underlying the development of the mammalian oral mucosa have revealed a major involvement of transforming growth factor beta (TGF-beta) and bone morphologic protein (BMP) signaling pathways. Here, we examined the expression of a downstream target of TGF-beta and BMPs, Runx3, in oral mucosa. Runx3 is a runt-related transcription factor that acts as a gastric tumor suppressor and regulator of growth and differentiation in mammalian gastric epithelial cells. Another member of the Runx family in C. elegans, run, is involved in the development of a functional hypodermis and gut. In this report, we examined Runx3 expression using reverse transcription-polymerase chain reaction, immnunohistochemistry, and in situ hybridization and found that Runx3 is expressed in the tongue and palate epithelium of mouse embryos from embryonic day 12.5 to 16.5. The functional relationship between Runx3 and TGF-beta/BMPs signaling in tongue and palate development is discussed.

Human BMP-2 gene transfer using transcutaneous in vivo electroporation induced both intramembranous and endochondral ossification.

It has been generally accepted that bone morphogenetic protein-2 (BMP-2) can induce osteogenesis in skeletal muscles via endochondral ossification. However, it is not clear how the ossification process occurs after the BMP-2 gene transfer to skeletal muscles in rats using in vivo electroporation. In this study, we evaluated the ossification process by BMP-2 gene transfer using in vivo electroporation. The gastrocnemius muscles of Wistar rats were injected with human BMP-2 gene expression vector (pCAGGS-BMP-2), followed by electroporation under the condition of 100 V, 50 msec per 1 sec, x8. Light and electron microscopic and radiographic analyses were performed at 1, 3, 5, 7, and 10 days after treatment. At 7 days, no sign of cartilage and/or bone formation was detected. However, at 10 days after in vivo electroporation, soft X-ray analysis revealed small lucent areas around the plasmid-injected region. Clusters of both cartilage tissues, leading to endochondral ossification and intramembranous bones of various sizes, were observed between muscle fibers. RT-PCR detected osteocalcin mRNA, showing bone formation at 10 days. Our findings strongly suggest that BMP-2 gene transfer using in vivo electroporation induces not only endochondral ossification but also intramembranous ossification.

Involvement of reactive oxygen intermediate in the enhanced expression of virulence-associated genes of Listeria monocytogenes inside activated macrophages.

Listeriolysin O encoded by 1,587 bp hly is the essential virulence factor of Listeria monocytogenes that replicates in the cytosolic space after escaping from phagosome of macrophages. By using murine macrophage-like J774.1 cells with or without activation by IFN-gamma plus LPS, the expression of both hly and its positive regulator prfA was monitored by means of RT-PCR. In activated J774.1 cells, the level of hly expression was enhanced although the multiplication of bacteria was significantly suppressed. The elevated expression of hly inside activated macrophage was abolished by addition of SOD and catalase, suggesting that reactive oxygen intermediates contribute to the upregulation of prfA and hly transcriptions. Moreover, we found that exposure of L. monocytogenes to H2O2 dramatically enhanced the expression of both prfA and hly mRNAs. Spontaneous ONOO- generator, SIN-1, also promoted the transcription to a certain level. These results suggested that oxygen radicals generated in activated macrophages provide a positive signal for up-regulation of virulence genes in L. monocytogenes.

Ultrastructural and immunohistochemical studies of medullary bone calcification, with special reference to sulphated glycosaminoglycans.

Histochemical, immunohistochemical and electron energy-loss spectroscopic studies were performed to examine the relationship between sulphated glycosaminoglycans and medullary bone calcification using oestrogen-injected male Japanese quail. Sulphated glycosaminoglycans, detected by high iron diamine (HID) or HID-thiocarbohydrazide-silver protein (HID-TCH-SP) methods, were distributed throughout the matrix of medullary bone, some periphery and extending tips of the trabeculae stained weakly, and the globular structures at osteoid areas were exclusively positive for HID-TCH-SP stain. Immunohistochemistry identified keratan sulphate located in the globular structures at osteoid areas and calcified matrix, but chondroitin-4 sulphate and chondroitin-6 sulphate were not detected in the matrix. Using electron spectroscopic imaging, sulphur was determined to be localized in the globular structures. These results demonstrate that medullary bone matrix accumulates keratan sulphate in the globular structures, which are the foci for calcification, and eventually in the calcified areas. This suggests that keratan sulphate containing sulphur is maintained in the calcified matrix. These results indicate a unique process of calcification exists in medullary bone.