Innervation of histamine neurons in the caudal part of the arcuate nucleus of hypothalamus and their activation in response to food deprivation under scheduled feeding.

It has been well established that histaminergic neurons innervate densely the anterior hypothalamus and regulate several functions through the histamine H1 receptor (H1R). However, the physiological function of the histaminergic neurons in other regions including the posterior hypothalamus has not been fully investigated. Recently, we have found a selective c-Fos expression in the caudal part of the arcuate nucleus of the hypothalamus (cARC) by food deprivation under scheduled feeding in rats. In this study, we histochemically examined the correlation of this c-Fos expression with the activation of histaminergic neurons in this region using an anti-H1R antibody. Strong H1R immunoreactivity was observed in the perikarya of the c-Fos positive cells. Abundant histamine-containing fibers were also found in the cARC and in the area between the cARC and the tuberomammillary nucleus (TM), where the histaminergic neuronal cell bodies are exclusively distributed. Our morphological observations suggest that c-Fos expression in the cARC by food deprivation under scheduled feeding is caused by the activation of histaminergic neurons projected from the TM.

Suppressive effects of 1,4-dihydroxy-2-naphthoic acid administration on bone resorption.

SUMMARY: The main component of the metabolic by-products of fermentation by Propionibacterium freudenreichii ET-3 is 1,4-dihydroxy-2-naphthoic acid (DHNA), which has a naphthoquinone skeleton, as in vitamin K2. This study showed that DHNA improved bone mass reduction with osteoporosis model mice caused by FK506. INTRODUCTION: Growth of the intestinal bacterium Lactobacillus bifidus is specifically facilitated by DHNA. The present study used osteoporosis model mice to investigate the effects of DHNA on bone remodeling. METHODS: FK506, an immunosuppressant, was used to prepare osteoporosis model mice. Thirty mice were divided into three groups: FK group, FK+DHNA group, and control group. In the FK group, FK506 was administered to induce bone mass reduction. In the FK-DHNA group, FK506 and DHNA were administered concurrently to observe improvements in bone mass reduction. To ascertain systemic and local effects of DHNA, we investigated systemic pathological changes in colon, kidney function and cytokine dynamics, and morphological and organic changes in bone and osteoclast dynamics as assessed by culture experiments. RESULTS: Compared to the FK group without DHNA, colon damage and kidney dysfunction were milder for FK+DHNA group, and production of inflammatory cytokines (interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha) was more suppressed. Furthermore, compared to the group without DHNA, histological analyses and radiography showed that bone resorption was suppressed for the DHNA group. Culture experiments using osteoclasts from murine bone marrow showed osteoclast suppression for the DHNA group compared to the group without DHNA. CONCLUSION: These results show that DHNA has some effects for improving bone mass reduction caused by FK506.

Expression of human beta-defensin -1, -2, and -3 in non-inflamed pseudocyst, mucoceles.

OBJECTIVES AND DESIGN: The expressions of human beta defensin-1 (HBD-1), -2 (HBD-2) and -3 (HBD-3) in non-inflamed pseudocysts such as mucoceles were investigated immunohistochemically in this study. MATERIALS AND METHODS: Mucocele specimens were obtained from 21 patients. The expression of HBDs was studied immunohistochemically by using antibodies directed against HBD-1, -2, and -3. Statistical analyses were carried out on serial sections stained with antibodies. RESULTS: Cells expressing HBDs were found in mucoceles. The expression of HBD-2 was observed in floating cells in all the specimens, whereas HBD-1 and HBD-3-expressing cells were detected in 93% and 73% of the mucoceles, respectively. The HBD-2 signal was the most intense and the HBD-3 signal intensity was weaker than that of HBD-1. HBDs were expressed in neutrophils and in other floating cells. Interestingly, the signal intensity and the population of positive cells located close to the centers of cysts were higher than those located in the peripheral areas of cysts. CONCLUSION: The expression of HBDs was found even in non-inflamed pseudocysts such as mucoceles. These results suggest that an unknown mechanism not involved in biophylaxis for the expression of HBDs may exist.

Expression of osteoclast differentiation factor and osteoclastogenesis inhibitory factor in rat osteoporosis induced by immunosuppressant FK506.

Immunosuppressant drugs are currently required by transplant recipients for the remainder of their lives, despite the many adverse effects associated with these therapies. Acute osteoporosis is one such effect, and a reproducible osteoporosis model has been established through the administration of the immunosuppressant drug FK506 in rats. The cause of this osteoporosis has been shown to be abnormal osteoclast proliferation, altering the process of bone remodeling. However, the reasons why FK506 induces osteoclast proliferation and whether this process is mediated by cytokine changes or an increase in bone resorption factors have been unclear. An investigation was therefore conducted focusing on the recent discoveries of osteoclast differentiation factor (ODF) and osteoclastogenesis inhibitory factor (OCIF). These factors led to elucidation of the osteoclast differentiation-maturation mechanism. An osteoporosis model was produced in rats utilizing intramuscular FK506 injection (1 mg/kg) for 28 consecutive days. Trabecular bone resorption was observed inferior to enchondral ossification in the FK506 group, and tartrate resistant acid phosphatase (TRAP) staining revealed a clear increase in osteoclasts at the site of enchondral ossification, relative to the control group. Real-time PCR and in situ hybridization (ISH) demonstrated minimal differences in OCIF expression between control and the treatment groups. However, Real-time PCR revealed clearly increased ODF expression in the treatment group. ODF expression was also shown to be increased in the treatment group using ISH. This was histologically consistent with a region of osteoclast proliferation inferior to enchondral ossification. The results of this study support the hypothesis that FK506-mediated osteoporosis occurs by action of the drug on osteoclasts, promoting expression of ODF messenger ribonucleic acid (mRNA) and thus prompting osteoclast differentiation and maturation.

Pathological change of articular cartilage in the mandibular head treated with immunosuppressant FK 506.

While several reports have documented immunosuppressant-induced osteoporosis, the exact mechanism of the pathological change of the joint remains to be clarified. In the present study, we have demonstrated the pathological change of the articular cartilage in the mandibular head of five Sprague-Dawley rats administered with the immunosuppressant FK 506 for 28 days. Three-dimensional micro-computed tomography of the mandibular heads in treated rats showed a significant decrease in trabecular bone volume compared to control rats. Histological observation revealed atrophic change of the articular cartilage. Immunohistological observation using anti-proliferative cell nuclear antibody (PCNA), type I, II, and type X collagen antibodies showed significantly decreased proliferation and differentiation of chondrocytes in the articular cartilage compared with the control group (p<0.05). Tartrate-resistant acid phosphatase (TRAP) staining revealed no significant difference in the numbers of osteoclasts at the chondro-osseous junction. Thus, FK 506 administration inhibited chondrogenic cell proliferation and differentiation and might cause osteoporotic change of subcartilage trabecular bone that subsequently forms in the mandibular head.

Treatment of a plunging ranula with fenestration and continuous pressure.

We present a new method of fenestration and continuous pressure as a simple, effective and uninvasive procedure for the treatment of plunging ranulas. We have recently used in four female patients, aged 10-29 years old. After treatment, the patients remained symptom-free and assessment by magnetic resonance imaging (MRI) showed regression of the ranula in all cases. The procedure resulted in satisfactory healing and we advocate it as a simple and effective treatment that is better for patients than conventional treatment.

Immunohistochemical study on expression of alpha-defensin and beta-defensin-2 in human buccal epithelia with candidiasis.

OBJECTIVES AND DESIGN: It has been previously reported that alpha-defensin (HNPs) and beta-defensin-2 (HBD-2) peptides with antifungal and cytotoxic activities can be detected in oral carcinomas and the saliva of patients with oral carcinomas. The present study investigated the presence of HNPs and HBD-2 in oral epithelia with candidiasis. MATERIALS AND METHODS: Tissue sections (4 microm) were prepared from biopsy and surgically removed specimens diagnosed as oral candidiasis (n = 10). The sections were examined immunohistochemically with antibodies directed against HNPs and HBD-2. RESULTS: Tissue sections of oral candidiasis were immunostained with antidefensin antibodies. Neutrophils in the inflamed lamina propria were positively immunostained with anti-HNPs antibody. The cytoplasm of cells in the upper spinous layer, in the lower spinous layer and in the parakeratinized layer of buccal epithelia with candidiasis was immunostained intensely with anti-HBD-2 antibody. In contrast, the expression of HBD-2 in the normal spinous layer was much weaker than that in oral candidiasis. No signals of HNPs were found in normal buccal epithelium. CONCLUSION: Buccal specimens from individuals with oral candidiasis show greater levels of expression of both HNPs and HBD-2. There might be a dual protection manner by defensins against fungal inflammation in infected buccal epithelia locally. Generally, HBD-2 signals have been found everywhere in the buccal epithelium; however, in an infected area, the signal intensity of HBD-2 has increased. HNPs signals have not been found in the normal buccal epithelium; however, HNPs signals have increased when the infection occurred.

Cellular origin of endochondral ossification from grafted periosteum.

Grafted periosteum is known to have potential for heterotopic bone formation by endochondral ossification. Although osteochondrogenic cells have been thought to originate from the osteogenic layer in grafted periosteum, no histological report has yet demonstrated this. The present study was designed to elucidate the origin of chondrogenesis preceding bone formation in grafted periosteum. Periostea harvested from young Japanese white rabbits' tibiae were grafted into suprahyoid muscles and examined radiographically and histologically at postoperative days 1, 7, 9, 14, 21, and 35. Normal periostea and tibial graft site were also examined. Surgical harvesting of the periosteum split and damaged its osteogenic layer but retained the fibrous layer intact. Most of the osteoblasts remained on the tibial bone surface, and only few cells of the osteogenic layer were present in grafted tissue. By the seventh day after grafting, the fibrous layer had thickened. The fibroblastic cells in the fibrous layer had significantly increased in number (P < 0.01) and were positively stained for proliferating cell nuclear antigen. These cells exhibited alkaline phosphatase activity at day 9. The differentiated chondrocytes had formed cartilage at postoperative day 14. Cells in the osteogenic layer appeared necrotic and subsequently disappeared. Following postoperative day 21, cartilage was replaced by trabecular bone. Bone formation was completed by 35 days. An X-ray analysis at this time also revealed new bone formation. These findings indicate that grafted periosteum forms bone by endochondral ossification and that the cells of the fibrous layer play essential roles in chondrogenesis that precedes such bone formation.

Prefabricated bone graft induced from grafted periosteum for the repair of jaw defects: an experimental study in rabbits.

PURPOSE: The purpose of the study was to determine whether a prefabricated graft of new bone induced from periosteum grafted into muscle was an effective material for the repair of jaw defects. MATERIAL AND METHODS: Artificial mandibular jaw defects in young Japanese rabbits were covered either with free grafted periosteum (n = 5) or a prefabricated graft of newly formed bone induced from periosteum, which was first grafted into the floor of the mouth, and placed as a revascularized muscle-pedicled bone flap (n = 5). Bone formation in jaw defects was examined radiographically and histologically 28 days after grafting into defects. RESULTS: Bone formation was confirmed radiographically and histologically in both groups. However, the free grafted periosteum formed thin bone and fibrous tissue existed between the new and the original bone. In contrast, more active bone formation was observed with the prefabricated graft. This grafted new bone developed further and fused to the mandible. Blood vessels surrounding the new bone were observed histologically. CONCLUSION: These experimental findings suggested that prefabricated bone grafts induced from periosteum grafts are potentially useful for correction of jaw defects.

Human alpha-and beta-defensin immunoreactivity in oral mucoepidermoid carcinomas.

The purpose of this study was to demonstrate the immunohistochemical localization and distribution of human alpha- and beta-defensins, peptides with antimicrobial activity, in oral mucoepidermoid carcinoma tissue. Tissue samples were embedded in paraffin and alpha- and beta-defensins were immunostained by the streptavidin-biotin coupled peroxidase method. Cancer cells that constituted the ducts, as well as neutrophils, were positively immunostained with the anti-alpha-defensin antibody (HNPs). On the other hand, epidermoid cells and intermediate cells were intensely stained with the anti-beta-defensin-2 (HBD-2) antibody. Mucous-secreting cells were clearly not immunostained with the anti-HBD-2 antibody. The epithelial hyperplasia region adjacent to the tumor tissues was also positively immunostained with the anti-HBD-2 antibody.

Identification of a stop codon mutation in the CBFA1 runt domain from a patient with cleidocranial dysplasia and cleft lip.

We examined a patient with cleidocranial dysplasia (CCD) and cleft lip and found a new stop codon mutation in CBFA1. This mutation was a heterozygous C-to-T transition in exon 3 of CBFA1. This nucleotide change converts a CAA codon to a TAA (stop) codon at amino acid position Gln195 in the runt domain of CBFA1.

Presence of human beta-defensin-2 in oral squamous cell carcinoma.

The purpose of this study was to demonstrate immunohistochemically the localization and distribution of human beta-defensin-2 (HBD-2), a peptide with antimicrobial activity, in oral carcinoma tissues. Tissue samples were embedded in paraffin, and HBD-2 was immunostained by the streptavidin-biotin coupled peroxidase method. Cancer cells in the cornified region of well differentiated squamous cell carcinomas were stained intensely. Stained cancer cells detected by anti-HBD-2 antibody were scattered among the cells of the non-cornified region.

Detection of human alpha-defensin-1, an antimicrobial peptide, in the fluid of jaw cysts.

OBJECTIVE: The purpose of this study was to isolate and to identify an antimicrobial peptide, human alpha-defensin-1 (HNP-1) in jaw cyst fluid. STUDY DESIGN: We collected jaw cyst fluid from 22 patients with various jaw cysts and analyzed the peptide components of them by reversed-phase high-performance liquid chromatography (HPLC). HNP-1 in the jaw cyst fluid was identified by the amino acid sequence and the molecular weight. The concentration of HNP-1 in the fluid of various jaw cysts was quantified by HPLC. RESULTS: HNP-1 in the jaw cyst fluid was isolated, purified, and identified. The concentrations of HNP-1 in the jaw cyst fluid ranged from 27.0 to 3725.4 microg/mL. CONCLUSION: HNP-1 was detected in the fluid of various types of jaw cysts.

Immunohistochemical staining of human alpha-defensin-1 (HNP-1), in the submandibular glands of patients with oral carcinomas.

The purpose of this study was the immunohistochemical localization and distribution of HNP-1 in the submandibular glands of patients with oral carcinomas. Tissue sections were embedded in paraffin, and HNP-1 was immunostained by the streptavidin-biotin coupled peroxidase method. Striated duct cells in the submandibular glands were stained with anti-defensin antibody. Neutrophils and capillary intimal cells were also stained. Defensins (HNPs) are peptides that occur in neutrophils and protect against bacteria and tumor cells. Human alpha-defensin-1 (HNP-1) is such a peptide, possessing both antimicrobial and cytotoxic activities. The presence of HNP-1 in striated duct cells in the submandibular glands of oral cancer patients, suggests a likely role in tumor immunity, for this peptide.

Defensin-1, an antimicrobial peptide present in the saliva of patients with oral diseases.

OBJECTIVES AND DESIGN: A preceding paper has noted a detection of defensin-1 (HNP-1), a peptide with antimicrobial and cytotoxic properties, in the saliva of patients with oral squamous cell carcinoma. The present study deals with the presence of HNP-1 in the saliva of patients with various oral diseases. METHODS: Whole saliva samples were obtained from the patients. HNP-1 in the saliva was isolated and purified by HPLC and the amino acid sequence of the peptide was determined. The molecular weight of HNP-1 was measured by mass spectrometry. The concentration of HNP-1 in saliva was determined by comparing the height of eluted HNP-1 with that of a synthetic HNP-1 standard. RESULTS: The concentrations of HNP-1 in the saliva of patients with oral lichen planus (n = 5), leukoplakia (n = 4), and glossitis associated with iron deficiency (n = 4) were 8.3 +/- 4.3 micrograms ml-1, 13.2 +/- 7.9 micrograms ml-1, and 11.4 +/- 4.9 micrograms ml-1, (mean +/- s.d.), respectively. These concentrations were significantly higher than those in healthy subjects (0.8 microgram ml-1) (P < 0.01). In contrast, salivary HNP-1 concentrations in patients with glossodynia (n = 4) and oral discomfort (n = 4) were similar to those in healthy subjects. CONCLUSIONS: Since HNP-1 is a non-specific defensive peptide present in neutrophils, it may play an important role in the protection against diseases such as oral lichen planus, leukoplakia, and glossitis associated with iron deficiency.

Levels of human defensin-1, an antimicrobial peptide, in saliva of patients with oral inflammation.

OBJECTIVE: The purpose of this study was to investigate the presence of an antimicrobial peptide, human defensin-1, in the saliva of patients with oral inflammation. STUDY DESIGN: Whole saliva samples were collected from patients with oral inflammation and from healthy volunteers. Human defensin-1 in saliva was isolated and purified by reversed-phase high-performance liquid chromatography. The amino acid sequence and molecular weight of defensin-1 were determined. The concentration of defensin-1 in saliva was quantified by high-performance liquid chromatography. Serum C-reactive protein concentration was measured by particle-enhanced turbidimetric immunoassay. RESULTS: The salivary defensin-1 concentration was significantly higher in patients with oral inflammation than in healthy volunteers; furthermore, in patients with oral inflammation, the concentration was significantly higher before treatment than after treatment. In the patients with oral inflammation, there was a strong positive correlation between salivary defensin-1 concentration and serum C-reactive protein concentration. CONCLUSIONS: The findings suggest that defensin-1 in saliva may be a convenient marker of inflammation associated with oral disease.

Experimental study of bone formation from autogenous periosteal graft following insulin-like growth factor I administration.

The present study evaluates the effect of insulin-like growth factor I (IGF-I) administration on bone formation following grafted periosteum. Twenty-four Japanese white rabbits were randomly assigned into one IGF-I administered group (1 mg for 14 days) and a second control group. Grafted periosteum taken from the tibia was placed under the submandibular muscles. At 14, 21, and 28 days following the operation, the grafted periosteum was extirpated and examined. Throughout all stages of the experiment, active bone formation was confirmed histologically and radiographically in both control and experimental groups. In addition, a micro-CT scan was used to observe three-dimensional micro structures in newly formed bone and to measure the trabecular bone thickness as a marker of bone development. As a result, a significant increase in bone formation in the IGF-I group was observed when compared with the findings in the control group. Trabecular bone thickness in the IGF-I group was significantly greater when compared with the control group at 14 days and 21 days following grafting (P < 0.01). At 28 days following grafting, there were no significant differences, suggesting that administration of IGF-I may play an important role in inducing bone formation from grafted periosteum in the early stages.

Presence of defensin in epithelial Langerhans cells adjacent to oral carcinomas and precancerous lesions.

We aimed to immunohistochemically study the localization of defensin (HNPs), a family of peptides with antimicrobial and cytotoxic activity, in oral tumor tissue. Therefore, tissue sections were embedded in paraffin, and defensin was immunostained by the streptavidin-biotin coupled peroxidase method. Langerhans cells were confirmed by indirect immunostaining with anti-S-100 protein polyclonal antibody. Melanocytes were stained with Fontana-Masson's stain. Neutrophils and intimal cells were stained by anti-defensin antibody. Langerhans cells in normal epithelium or dysplasic epithelium adjacent to squamous cell carcinoma and precancerous lesion were also stained. Defensins (HNPs) are nonspecific peptides that occur in neutrophils and protect against bacteria, fungi, and tumor cells. Since defensins are also found in epithelial Langerhans cells adjacent to tumor tissue, these peptides most likely have a role in tumor immunity.

Defensin-1, a peptide detected in the saliva of oral squamous cell carcinoma patients.

The saliva of patients with oral squamous cell carcinoma was subjected to reversed-phase HPLC, and a peak (P 14) was eluted with a linear gradient of acetonitrile. The molecular weight of P14 was 3,442 daltons by mass spectrometry. The results of amino acid sequencing by Edman degradation and homology studies indicated that P14 was consistent with a peptide, human alpha-defensin 1 (HNP-1) which belongs to the defensin family. The concentration of HNP-1 in the saliva of 6 squamous cell carcinoma patients before surgery and 11 after surgery was 12.3 +/- 8.6 and 6.5 +/- 5.9 micrograms/ml (mean +/- S.D.), respectively. In contrast, minimal amounts of HNP-1 were found in the saliva of adenocarcinoma patients and healthy volunteers. The concentration of salivary HNP-1 correlated positively with serum squamous cell carcinoma related antigen (SCC-Ag)level (r = 0.879, p < 0.01).

Metaphyseal fibrous defect (nonossifying fibroma) in the mandible. A case report.

A 7-year-old boy presented in whom a metaphyseal fibrous defect was diagnosed. The lesion was enucleated under local anesthesia. The postoperative course was uneventful and there have been no signs of recurrence as of 14 months after operation. Although rare, metaphyseal fibrous defect should be included in the differential diagnosis of tumors arising in the jaws.