Development of Rat Caries-Induced Pulpitis Model for Vital Pulp Therapy.

Rodent animal models for vital pulp therapy are commonly used in dental research because their tooth anatomy and cellular processes are similar to the anatomy and processes in humans. However, most studies have been conducted using uninfected sound teeth, which makes it difficult to adequately assess the inflammatory shift after vital pulp therapy. In the present study, we aimed to establish a caries-induced pulpitis model based on the conventional rat caries model and then evaluate inflammatory changes during the wound-healing process after pulp capping in a model of reversible pulpitis induced by carious infection. To establish the caries-induced pulpitis model, the pulpal inflammatory status was investigated at different stages of caries progression by immunostaining targeted to specific inflammatory biomarkers. Immunohistochemical staining revealed that both Toll-like receptor 2 and proliferating cell nuclear antigen were expressed in moderate and severe caries-stimulated pulp, indicating that an immune reaction occurred at both stages of caries progression. M2 macrophages were predominant in moderate caries-stimulated pulp, whereas M1 macrophages were predominant in the severe caries-stimulated pulp. Pulp capping in teeth with moderate caries (i.e., teeth with reversible pulpitis) led to complete tertiary dentin formation within 28 d after treatment. Impaired wound healing was observed in teeth with severe caries (i.e., teeth with irreversible pulpitis). During the wound-healing process in reversible pulpitis after pulp capping, M2 macrophages were predominant at all time points; their proliferative capacity was upregulated in the early stage of wound healing compared with healthy pulp. In conclusion, we successfully established a caries-induced pulpitis model for studies of vital pulp therapy. M2 macrophages have an important role in the early stages of the wound-healing process in reversible pulpitis.

Novel Functional Peptide for Next-Generation Vital Pulp Therapy.

Although vital pulp therapy should be performed by promoting the wound-healing capacity of dental pulp, existing pulp-capping materials were not developed with a focus on the pulpal repair process. In previous investigations of wound healing in dental pulp, we found that organic dentin matrix components (DMCs) were degraded by matrix metalloproteinase-20, and DMC degradation products containing protein S100A7 (S100A7) and protein S100A8 (S100A8) promoted the pulpal wound-healing process. However, the direct use of recombinant proteins as pulp-capping materials may cause clinical problems or lead to high medical costs. Thus, we hypothesized that functional peptides derived from recombinant proteins could solve the problems associated with direct use of such proteins. In this study, we identified functional peptides derived from the protein S100 family and investigated their effects on dental pulp tissue. We first performed amino acid sequence alignments of protein S100 family members from several mammalian sources, then identified candidate peptides. Next, we used a peptide array method that involved human dental pulp stem cells (hDPSCs) to evaluate the mineralization-inducing ability of each peptide. Our results supported the selection of 4 candidate functional peptides derived from proteins S100A8 and S100A9. Direct pulp-capping experiments in a rat model demonstrated that 1 S100A8-derived peptide induced greater tertiary dentin formation compared with the other peptides. To investigate the mechanism underlying this induction effect, we performed liquid chromatography-tandem mass spectrometry analysis using hDPSCs and the S100A8-derived peptide; the results suggested that this peptide promotes tertiary dentin formation by inhibiting inflammatory responses. In addition, this peptide was located in a hairpin region on the surface of S100A8 and could function by direct interaction with other molecules. In summary, this study demonstrated that a S100A8-derived functional peptide promoted wound healing in dental pulp; our findings provide insights for the development of next-generation biological vital pulp therapies.

Effects of fermented soymilk with Lactobacillus casei Shirota on skin condition and the gut microbiota: a randomised clinical pilot trial.

Several clinical studies have shown that isoflavones and Lactobacillus casei Shirota (LcS) have beneficial effects on skin condition and the gut microbiota, respectively. Thus, we investigated the effects of consecutive intake of fermented soymilk (FSM) with LcS on skin condition and the gut microbiota, as well as isoflavone bioavailability, in a randomised, double-blind, placebo-controlled trial as a pilot study. Sixty healthy premenopausal Japanese women received FSM containing a moderate level of isoflavone aglycones and a probiotic LcS, or soymilk (SM) containing neither of them, twice a day for 8 weeks. Skin condition was assessed by a subjective questionnaire for face and morphological analysis of the stratum corneum on the inner forearm. Faecal microbiota and urinary isoflavone were analysed by 16S rRNA gene amplicon sequencing and high-performance liquid chromatography tandem mass spectrometry, respectively. Both the FSM and SM groups had improved skin condition as assessed from scores of overall satisfaction, dryness, moisture, elasticity, coarseness, pigmentation and/or stratum corneum morphology, as well as significantly increased levels of urinary isoflavones during the intake period compared with the pre-intake period, although there were no significant differences between the two groups. There was a significant positive correlation between urinary isoflavone levels and skin questionnaire scores. In contrast, the relative abundance levels of Lactobacillaceae significantly increased and those of Bifidobacteriaceae tended to increase during the intake period compared with the pre-intake period. For the after-intake period they only decreased significantly in the FSM group. The levels of Enterobacteriaceae and Porphyromonadaceae significantly decreased during the intake period in the FSM group. These findings suggest that daily intake of FSM, as well as SM, provides health benefits that improve skin condition via increased levels of isoflavone absorption in the body, and that only FSM beneficially modifies the gut microbiota in premenopausal healthy women.

Discontinuation of leisure time impact-loading exercise is related to reduction of a calcaneus quantitative ultrasound parameter in young adult Japanese females: a 3-year follow-up study.

UNLABELLED: A 3-year follow-up study on 334 young Japanese females enrolled in a university at the age of 18 years revealed that discontinuation of leisure time impact-loading exercises performed in junior high and/or high school was associated with increased risk of reduction in calcaneus osteo-sono assessment index (OSI). INTRODUCTION: Bone strength rapidly increases during puberty and reaches its peak by the end of adolescence. The aim of this study was to determine the lifestyle factors that influence the maintenance of calcaneus OSI in young adult females around the time when peak bone mass is attained. METHODS: Annual health checkups including OSI measurements, anthropometrics, lifestyle analysis, and blood examination were performed 4 times on 334 Japanese females enrolled in a university at the age of 18 years. According to the slope of OSI change during the 3-year follow-up, the subjects were grouped into two categories: OSI loss (the lowest tertile) and OSI gain/stable (the second and third tertiles). RESULTS: At the baseline assessment, the OSI loss group had higher OSI and height and an earlier menarche age than the OSI gain/stable group. Performing leisure time impact-loading exercise in junior high and/or high school but discontinuing it at university was associated with increased risk of OSI loss, independent of OSI, height and weight at the age of 18 years, weight change during follow-up, age of menarche, energy-adjusted nutrient intake, and alcohol drinking; the odds ratios were 4.1-4.9 compared with those performing impact-loading exercise at university. In particular, duration, frequency, and subjective intensity of impact-loading exercise during high school were positively associated with OSI loss. CONCLUSION: Discontinuation of leisure time impact-loading exercises performed during late adolescence is associated with an increased risk of OSI loss in young adult females during the 3-year follow-up period.

The role of osteocytes in bone resorption during orthodontic tooth movement.

We investigated the roles of osteocytes in osteoclastic bone resorption during orthodontic tooth movement using the transgenic mice in which osteocytes can be specifically ablated. Because these transgenic mice express the receptor for diphtheria toxin on the cell surfaces of osteocytes, the injection of diphtheria toxin can ablate their osteocytes in vivo. Injection of diphtheria toxin into the transgenic mice significantly increased the number of ablated osteocytes in alveolar bone compared with that in wild-type mice with or without diphtheria toxin injection. Increased numbers of ablated osteocytes were observed from day 4 to day 12 after the injection in alveolar bones as well as in cortical bone of the tibiae. We applied the orthodontic force 4 days after the injection of diphtheria toxin, and the distance of tooth movement on day 12 was significantly smaller in transgenic mice than that in control mice. The numbers of osteoclasts and the quantity of eroded bone surface at the compression site were significantly reduced in the transgenic mice injected with diphtheria toxin than in control mice. These results provide in vivo demonstration of osteocyte involvement in osteoclastic bone resorption during orthodontic tooth movement.

Application of MRI movie for observation of articulatory movement during a fricative /s/ and a plosive /t/.

OBJECTIVE: To use an accurate method of tooth visualization in magnetic resonance imaging (MRI) movie for the observation of spatio-temporal relationships among articulators. MATERIALS AND METHODS: The subjects were two volunteers. Each subject repeated a vowel-consonant-vowel syllable (ie, /asa/; /ata/), and the run was measured using a gradient echo sequence. A custom-made clear retainer filled with the jelly form of ferric ammonium citrate was then fit onto the dental arch, and a T1-weighted turbo-spin-echo sequence was taken. Landmarks were used for superimposition of the incisor boundary onto sequential images of MRI movie. Tracings were conducted to observe the spatio-temporal relationships among articulators. RESULTS: The incisor boundary was clearly visible in the magnetic resonance images. After superimposition, the contact distance of the tongue to palate/incisor was found to be longer during /t/-articulation than during /s/-articulation. There were prominent differences in images with and without tooth superimposition in the front oral cavity. CONCLUSIONS: The method could distinctly extract a tooth boundary in MRI. Detailed configurational relationships between the tongue and tooth were observed during the production of a fricative and a plosive in MRI movie using this method.

Non-syndromic oligodontia with a novel mutation of PAX9.

Agenesis of the permanent teeth is a congenital anomaly that is frequently seen in humans. Oligodontia is a severe type of tooth agenesis involving 6 or more congenitally missing teeth, excluding the third molars. Previous studies have indicated that mutations in the homeobox gene MSX1, paired domain transcription factor PAX9, and EDA are associated with non-syndromic oligodontia. This study reports a Japanese family (eight of 14 family members affected) with non-syndromic oligodontia who preferentially lacked molar teeth. In this family, a novel frameshift mutation (321_322insG) was identified in the paired domain of PAX9. The frameshift mutation caused altered amino acids in the paired domain and premature termination of translation by 26 amino acids. When transfected into COS-7 cells, the mRNA expression of 321_322insG PAX9 was comparable with that of wild-type PAX9. However, the mRNA of 321_322insG PAX9 was more unstable than that of wild-type PAX9. This mRNA instability caused a marked decrease in protein production, as evaluated by Western blot analysis and immunostaining. These findings suggest that the 321_322insG mutation causes insufficient function of PAX9 protein and haploinsufficiency as a genetic model of familial non-syndromic oligodontia with a PAX9 mutation.

Correlation between genotype and supernumerary tooth formation in cleidocranial dysplasia.

INTRODUCTION: Cleidocranial dysplasia (CCD, MIM#119600), for which the responsible gene is RUNX2, is a genetic disorder characterized by hypoplasia or aplasia of the clavicles, patent fontanelles, and a short stature. Supernumerary teeth and delayed eruption and impaction of permanent teeth are frequently associated with CCD. Our previous study reported wide intrafamilial variation in supernumerary tooth formation associated with a mutation in the RUNT-domain of RUNX2, suggesting a low correlation between the genotype and supernumerary tooth formation. To further clarify this point, a more precise evaluation was performed. DESIGN: Gene mutational analysis of nine Japanese individuals with CCD was performed. Dental and skeletal characteristics were examined based on patient examinations and radiographs. RESULTS: Four different gene mutations, including one novel mutation in RUNX2 gene (NM_001024630), were identified. Among them, four individuals had the R225Q mutation, three siblings had the P224S mutation, and the other two individuals had different frame-shift mutations. Wide variations in supernumerary tooth formation were observed in individuals with identical gene mutations, and discordance was seen between monozygotic twins. Asymmetric supernumerary tooth formation was noted in five out of the nine individuals. CONCLUSION: Individuals with identical gene mutations showed a wide variation in the supernumerary tooth formation. Not only the genotype but also environmental factors and a complex system including epigenetics and copy number variation might regulate supernumerary tooth formation in CCD.

A Mongolian patient with hypohidrotic ectodermal dysplasia with a novel P121S variant in EDARADD.

INTRODUCTION: Hypohidrotic ectodermal dysplasia is a genetic disorder characterized by diminished or a lack of sweating, congenital missing teeth, and sparse or absent hair. Three genes, EDA, EDAR, and EDARADD, all related to tumor necrosis factor signaling, have been reported as responsible genes for this disorder. Among them, the largest numbers of mutations have been identified in EDA, and only two mutations identified in EDARADD. MATERIALS AND METHODS: DNA analysis of EDA, EDAR, and EDARADD was performed on a Mongolian patient by polymerase chain reaction-direct sequencing. RESULTS: The 5-year-old Mongolian individual had no erupted deciduous or permanent teeth. A panoramic radiograph showed only one tooth in the right mandible. His hair and eyebrows were sparse, but he did not have a short stature. He showed diminished sweating. The nails of his fingers and toes were normal. Based on these conditions, he was diagnosed with hypohidrotic ectodermal dysplasia. There was no gene mutation of EDA or EDAR. A novel heterozygous variant (P121S; c.361C>T) was identified in the death domain of EDARADD (NM_080738). No other member of his family was affected, and this variant was not identified in his parents or maternal grandparents. CONCLUSION: This study reports an individual affected with hypohidrotic ectodermal dysplasia with a novel heterozygous P121S variant in the death domain of EDARADD.

Control of cellular adhesiveness in an alginate-based hydrogel by varying peroxidase and H(2)O(2) concentrations during gelation.

An aqueous solution of alginate possessing phenolic hydroxyl (Alg-Ph) groups is gellable via a horseradish peroxidase (HRP)-catalyzed oxidative crosslinking reaction between Ph groups, consuming H(2)O(2) as an electron acceptor. This study evaluates the effect of H(2)O(2) and HRP concentrations on cellular adhesiveness and proliferation on the resultant enzymatically crosslinked Alg-Ph gels. After 4h of seeding, 81.1% of L929 fibroblast cells adhere to an Alg-Ph hydrogel prepared with 1 U ml(-1) HRP and 1mM H(2)O(2). Increasing the concentration of H(2)O(2) to 15 mM decreases the percentage of adhering cells to 28.4%. The cellular adhesion at this H(2)O(2) concentration is increased to 82.6% by increasing the HRP concentration to 10 U ml(-1). The cells adhering to the Alg-Ph hydrogels with higher cellular adhesiveness establish a confluent monolayer during 168 h of culture. A cell sheet can then be harvested within 5 min of immersion in a medium containing alginate lyase at 1.0 mg ml(-1). The harvested cell sheet re-adhere, and the cells contained in the sheet proliferate after being transferred to another cell culture dish.

Acceleration of palatal wound healing in Smad3-deficient mice.

Wound healing is a well-orchestrated complex process leading to the repair of injured tissues. It is suggested that transforming growth factor (TGF)-beta/Smad3 signaling is involved in wound healing. The purpose of this study was to investigate the role of TGF-beta/Smad3 signaling in palatal wound healing in Smad3-deficient (Smad3(-/-)) mice. Histological examination showed that wound closure was accelerated by the proliferation of epithelium and dermal cells in Smad3(-/-) mice compared with wild-type (WT) mice. Macrophage/monocyte infiltration at wounded regions in Smad3(-/-) mice was decreased in parallel with the diminished production of TGF-beta1, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1alpha compared with WT mice. Fibrocytes, expressing hematopoietic surface marker and fibroblast products, were recruited and produced alpha-smooth-muscle actin in WT mice, but were not observed in Smad3(-/-) mice. These results suggest that TGF-beta/Smad3 signaling may play an important role in the regulation of palatal wound healing.

In vivo application of amelogenin suppresses root resorption.

Amelogenin is recognized as an enamel protein associated with enamel formation. Besides this well-known function, remarkable root resorption has been seen in amelogenin-null mutant mice. Moreover, in vitro culture studies showed that amelogenin suppressed osteoclast differentiation. These studies raised the hypothesis that amelogenin can inhibit root resorption by reducing odontoclast number. To examine this hypothesis, we applied porcine amelogenins in a rat root resorption model, in which maxillary first molars were replanted after being air-dried. Compared with untreated and carrier-treated tooth roots, the application dramatically reduced the odontoclast number on root surfaces and inhibited cementum and root dentin resorption. Amelogenin significantly reduced the number of human odontoclastic cells in culture. It also inhibited RANKL expression in mouse bone marrow cell cultures. All these findings support our hypothesis that amelogenin application suppresses root resorption by inhibiting odontoclast number, and suggest that this is mediated by the regulation of RANKL expression.

Short-term changes in temporomandibular joint function in subjects with cleft lip and palate treated with maxillary distraction osteogenesis.

OBJECTIVES: To investigate the short-term effects of maxillary distraction osteogenesis (DO) on temporomandibular joint (TMJ) function in 21 subjects with cleft lip and palate (CLP). Design - Morphological changes in the maxillofacial region were measured using lateral cephalometric radiographs taken immediately before (pre-DO) and after DO (post-DO) and 1 year after DO (1-year follow-up). A questionnaire was evaluated using a visual analog scale. A chi-square test was used to compare the prevalence of TMJ symptoms between pre-DO and 1-year follow-up. The Spearman correlation coefficient was used to determine the correlation between changes in cephalometric variables and TMJ symptoms in association with maxillary DO. Statistical significance was set at p < 0.05. Results - The ANB (anteroposterior relationship of the maxilla with the mandible) angle and the mandibular plane angle at pre-DO, post-DO, and 1-year follow-up were -4.3 degrees , +5.8 degrees , +4.3 degrees and 32.1 degrees , 33.5 degrees , 33.6 degrees , respectively. The average amounts of anterior and downward movement of the maxilla at post-DO and 1-year follow-up were 8.3, -1.3 and 0.9, 1.1 mm, respectively. The prevalence of TMJ symptoms showed no significant increase in association with maxillary DO. Moreover, there was no significant correlation between changes in cephalometric variables and TMJ symptoms. Conclusion - These results suggest that there was no short-term (i.e., up to 1 year after DO) effect of maxillary DO on TMJ function in subjects with CLP.

The efficiency variation method for 4pibeta-gamma coincidence counting by ink-jet printing.

In order to vary the counting efficiencies in the 4pibeta-gamma coincidence extrapolation technique, a radioactive source was coated directly with varying amounts of an electrical conducting pigment using an ink-jet printer. This method can be used to efficiently prepare the multiple sources needed to generate efficiency extrapolation curves, and was successfully applied to the standardization of a (54)Mn source.

Atelocollagen-mediated local and systemic applications of myostatin-targeting siRNA increase skeletal muscle mass.

RNA interference (RNAi) offers a novel therapeutic strategy based on the highly specific and efficient silencing of a target gene. Since it relies on small interfering RNAs (siRNAs), a major issue is the delivery of therapeutically active siRNAs into the target tissue/target cells in vivo. For safety reasons, strategies based on vector delivery may be of only limited clinical use. The more desirable approach is to directly apply active siRNAs in vivo. Here, we report the effectiveness of in vivo siRNA delivery into skeletal muscles of normal or diseased mice through nanoparticle formation of chemically unmodified siRNAs with atelocollagen (ATCOL). ATCOL-mediated local application of siRNA targeting myostatin, a negative regulator of skeletal muscle growth, in mouse skeletal muscles or intravenously, caused a marked increase in the muscle mass within a few weeks after application. These results imply that ATCOL-mediated application of siRNAs is a powerful tool for future therapeutic use for diseases including muscular atrophy.

Diversity of supernumerary tooth formation in siblings with cleidocranial dysplasia having identical mutation in RUNX2 : possible involvement of non-genetic or epigenetic regulation.

INTRODUCTION: Cleidocranial dysplasia (CCD, MIM #119600) is an autosomal-dominant disorder characterized by hypoplasia or aplasia of clavicles, patent fontanelles and short stature. The responsible gene has been identified as RUNX2. CCD is also accompanied by characteristic dental abnormalities, e.g. supernumerary teeth, delayed eruption and impaction of permanent teeth. Intrafamilial variations of skeletal abnormalities are reported but those of dental abnormalities are obscure. To clarify this point, a precise examination of the dental features of CCD siblings having identical mutation was performed. DESIGN: Gene mutational analysis of three Japanese CCD siblings and their father was performed. Skeletal and dental characteristics were examined by the inquiry and radiographs. RESULTS: Three siblings uniformly showed patent fontanelles and short stature. They and their father had a novel missense mutation in the RUNT-domain (P210S) of RUNX2. The siblings were completely discordant for the dental characteristics with the position and number of supernumerary teeth being completely different. The youngest, a 12-year-old boy, had six supernumerary teeth, which appeared symmetrically around the maxillary canines and mandibular premolars. The second, a 15-year-old girl, had four supernumerary teeth which appeared around the mandibular incisors. The oldest, a 17-year-old boy, had 11 supernumerary teeth, which were symmetrically around the mandibular lateral dentition and asymmetrically around the maxillary incisors and premolars. CONCLUSION: The present study suggests the involvement of non-genetic or epigenetic regulation in supernumerary tooth formation in CCD.

Development of the fast neutron standard using a Be({alpha},n) reaction at the National Metrology Institute of Japan.

This paper describes the 8-MeV neutron field where the neutrons are generated in the (9)Be(alpha,n)(12)C reaction by bombardment of a beryllium target with a 2.4-MeV (4)He(+) beam from a Van de Graaff accelerator. The neutron field is being prepared for a new national standard on neutron fluence in Japan. Absolute measurement of the neutron fluence was taken using a proton recoil neutron detector, consisting of a silicon surface barrier detector with a polyethylene radiator. Neutron spectra were measured using a newly developed recoil proton spectrometer and a liquid organic scintillation detector. The gamma rays existing in the field were also characterised using a liquid organic scintillation detector. The ambient dose equivalents of the gamma rays were estimated to be <100 microSv at the neutron fluence of 10(7) neutrons cm(-2).

Identification of genes related to mechanical stress in human periodontal ligament cells using microarray analysis.

BACKGROUND AND OBJECTIVE: Differential expression of genes in human periodontal ligament (PDL) under mechanical stress, such as orthodontic force, is thought to be involved in the remodeling of PDL cells and periodontal tissues. However, little is known about the genes expressed in PDL cells under mechanical stress. MATERIAL AND METHODS: We employed microarray analysis to assess, in a comprehensive manner, the gene expression profiles in PDL cells compressed by a static force using an in vitro three-dimensional culture system. Six genes were selected and validated by quantitative real-time polymerase chain reaction analysis, consistent with the microarray data. RESULTS: The microarray data revealed that 108 of 30,000 genes tested were differentially expressed by mechanical force loading. Among them, 85 genes were up-regulated by mechanical stress, while 23 genes were down-regulated, judging by the thresholds of a two-fold increase/decrease compared with the controls. Thirty-two of the up-regulated and eight of the down-regulated genes, well-characterized in protein function, were involved in numerous biological processes including cell communication, cell signaling, cell cycle, stress response, and calcium release. However, several genes differentially expressed in our microarray data have not been well defined as stress-response molecules. CONCLUSION: Our microarray is the first to show the gene profile in PDL cells caused by mechanical stress; however, further studies to clarify the physiological function of these molecules in PDL cells are required.

Leukotrienes mediate 5-hydroxytryptamine-induced plasma extravasation in the rat knee joint via CysLT-type receptors.

OBJECTIVE AND DESIGN: The aim of this study was to investigate whether leukotrienes synthesized by 5-lipoxygenase (5-LO) and acting via leukotriene (LT) receptors contribute to 5-hydroxytryptamine (5-HT)-induced knee joint plasma extravasation (PE). MATERIALS AND METHODS: Knee joints of rats under anesthesia were perfused with 5-HT and synovial vascular Evans Blue dye leakage was measured spectrophotometrically. A series of 5-LO inhibitors and LT receptor antagonists were investigated for their ability to inhibit 5-HT-induced synovial PE. RESULTS: Inhibitors of 5-LO (NDGA and REV 5901) significantly attenuated 5-HT-induced plasma extravasation. MK 571, LY 171883, BAY u9773 (CysLT receptor antagonists) and REV 5901 (a CysLT receptor antagonist and a 5-LO inhibitor) were equally effective in inhibiting 5-HT-induced PE, indicating that leukotrienes mediate 5-HT-induced PE via CysLT receptors. In contrast, antagonists selective for LTB(4) receptors (BLT(1) and BLT(2) receptors) failed to reduce 5-HT-induced PE. CONCLUSIONS: These results demonstrate that leukotrienes, specifically cysteinyl-leukotrienes contribute to synovial plasma extravasation and suggest that leukotrienes act downstream of 5-HT in the inflammatory cascade.

Recombinant monoclonal thyrotropin-stimulation blocking antibody (TSBAb) established from peripheral lymphocytes of a hypothyroid patient with primary myxedema.

Anti-TSH receptor antibodies (TRAbs) have been known to be involved in Graves' disease and primary hypothyroidism. We previously isolated and reconstituted immunoglobulin (Ig) genes of Epstein-Barr virus-transformed B cell clones producing monoclonal TRAbs obtained from Graves' patients. In the present study, we performed a similar experiment using a B cell clone, 32A-5, derived from a patient with primary hypothyroidism. The variable region genes of Ig heavy (H) and light (L) chains were isolated and sequenced from the 32A-5 clone. A significant number of somatic mutations were found in variable regions of H and L chain gene segments. Each pair of H and L chain cDNAs was ligated into an expression vector for IgG1 production and stably introduced into myeloma cells. The transfectants were injected ip into BALB/c mice to yield ample volume of the antibody for following applications. Interactions of recombinant 32A-5 with Graves' sera with varying thyroid-stimulating antibody (TSAb) activities were studied. The recombinant antibody tended to suppress TSAb activities in 10 of 15 Graves' sera, in which four were significantly inhibited. In summary, this is the first study to analyze human monoclonal TSH-stimulation blocking antibodies (TSBAb) at the molecular level. Use of human recombinant monoclonal TSBAb may be an analytical tool for molecular-basis etiology and an alternative therapeutic path for Graves' disease.