Cell therapy for medication-related osteonecrosis of the jaw: update on treatment strategies.

Bioactive glasses (BAG) are used as bone-graft substitutes in orthopaedic surgery. A specific BAG scaffold was developed by sintering BAG-S53P4 granules. It is hypothesised that this scaffold can be used as a bone substitute to fill bone defects and induce a bioactive membrane (IM) around the defect site. Beyond providing the scaffold increased mechanical strength, that the initial inflammatory reaction and subsequent IM formation can be enhanced by coating the scaffolds with poly(DL-lactide-co-glycolide) (PLGA) is also hypothesised. To study the immunomodulatory effects, BAG-S53P4 (± PLGA) scaffolds were placed on monolayers of primary human macrophage cultures and the production of various pro- and anti-inflammatory cytokines was assessed using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and ELISA. To study the osteogenic effects, BAG-S53P4 (± PLGA) scaffolds were cultured with rabbit mesenchymal stem cells and osteogenic differentiation was evaluated by RT-qPCR and matrix mineralisation assays. The scaffold ion release was quantified and the BAG surface reactivity visualised. Furthermore, the pH of culture media was measured. BAG-S53P4 scaffolds had both anti-inflammatory and osteogenic properties that were likely attributable to alkalinisation of the media and ion release from the scaffold. pH change, ion release, and immunomodulatory properties of the scaffold could be modulated by the PLGA coating. Contrary to the hypothesis, the coating functioned by attenuating the BAG surface reactions and subsequent anti-inflammatory properties, rather than inducing an elevated inflammatory response compared to BAG-S53P4 alone. These results further validated the use of BAG-S53P4 (± PLGA) scaffolds as bone substitutes and indicate that scaffold properties can be tailored to a specific clinical need.

Mechanism of motor coordination of masseter and temporalis muscles for increased masticatory efficiency in mice.

The demand for the use of mice as animal models for elucidating the pathophysiologies and pathogeneses of oral motor disorders has been increasing in recent years, as more and more kinds of genetically modified mice that express functional disorders of the stomatognathic system become available. However, the fundamental characteristics of mouse jaw movements during mastication have yet to be fully elucidated. The purpose of this study was to investigate the roles of the masseter and temporalis muscles, and the mechanisms of motor coordination of these muscles for increasing masticatory efficiency in the closing phase in mice. Twenty-two male Jcl:ICR mice were divided into control (n = 8), masseter-hypofunction (n = 7) and temporalis-hypofunction groups (n = 7). Botulinum neurotoxin type A (BoNT/A) was used to induce muscle hypofunction. The masticatory movement path in the horizontal direction during the occlusal phase became unstable after BoNT/A injection into the masseter muscle. BoNT/A injection into the temporalis muscle decreased antero-posterior excursion of the late-closing phase corresponding to the power phase of the chewing cycle. These results suggest that the masseter plays an important role in stabilizing the grinding path, where the food bolus is ground by sliding the posterior teeth from back to front during the occlusal phase. The temporalis plays a major role in retracting the mandible more posteriorly in the early phase of closing, extending the grinding path. Masticatory efficiency is thus increased based on the coordination of activities by the masseter and temporalis muscles.

Gender differences of muscle and crural fascia origins in relation to the occurrence of medial tibial stress syndrome.

Although women reportedly have a higher prevalence of medial tibial stress syndrome (MTSS) than men, the possible role of gender-based anatomical differences has not been investigated. The aim of the present study was to investigate the presence of gender-based differences in the range of muscle attachments along the entire medial tibia, the proportion of muscle attachment at the middle and distal thirds of the medial margin of the tibia, the structure of the crural fascia, and chiasm position. The specimens were 100 legs of 55 Japanese cadavers. Statistical analysis was carried out using a chi-square test to compare anatomical features between the sexes. The flexor digitorum longus (FDL) had a higher proportion of attachment to the middle and distal thirds of the medial margin of the tibia than the soleus (SOL; P < 0.001). The proportion of the SOL attachment to the middle and distal thirds of the medial margin of the tibia was 33.3% in men and 72.5% in women (P < 0.001). The soleal aponeurosis was not observed in any specimen. In all specimens the FDL formed the top layer of both chiasms. These results suggest that the higher prevalence of MTSS reported among women may be the result of gender-based anatomical differences.

Complications after intraoral vertical ramus osteotomy: relationship to the shape of the osteotomy line.

Intraoral vertical ramus osteotomy (IVRO) is used widely to correct mandibular prognathism. However, several disadvantages of this procedure have been reported, such as condylar luxation and bony interference at the osteotomy site. The aim of this study was to survey the incidence of complications (condylar luxation and bony interference) based on the shape of the osteotomy line. One hundred and eighty-five rami in 118 patients with jaw deformities, which were treated with IVRO, were examined retrospectively. The shape of the osteotomy line and the postoperative complications were examined on panoramic radiographs. Osteotomy lines were classified into three types: vertical, C-shaped, and oblique. Of the 185 osteotomy sites, 98 were vertical, 37 C-shaped, and 50 oblique. Condylar luxation was found in six rami (3.2%); four had undergone vertical osteotomy and two had undergone C-shaped osteotomy. Bony interference occurred in seven rami (3.8%), all with vertical type osteotomy lines. Most complications occurred in the vertical type cases and no complications were found in oblique type cases. Condylar luxation was found mainly in unilateral IVRO cases and bony interference was found in bilateral IVRO cases. These results suggest that the oblique type of osteotomy line has the advantage of avoiding complications.

Impact of maternal bifidobacteria and the mode of delivery on Bifidobacterium microbiota in infants.

The aim of this study is to examine the influence of maternal intestinal and vaginal bifidobacteria on the colonisation of bifidobacteria in the gut of infants. Faecal samples from 120 healthy pregnant mothers within 1 month of delivery and from their infants at 1 month of age and 98 vaginal swabs from the mothers at the time of delivery were collected at a maternity hospital in Chiang Mai, Thailand. The faecal and vaginal samples were assayed by real-time PCR assays to detect Bifidobacterium species and to estimate the bifidobacterial copy numbers. After adjusting for the numbers of each Bifidobacterium species, delivery mode, and antibiotic use in infants by the age of 1 month, total counts of bifidobacteria in the mothers' faeces were associated with increased copy numbers of bifidobacteria in the faeces of breastfed infants. A caesarean section was also significantly associated with a decrease in the copy numbers of bifidobacteria in the faeces of infants. No significant correlation was found between the bifidobacterial copies of the vaginal swabs and those of the infants' faeces. The intestinal bifidobacterial status of exclusively breastfed infants was significantly positive affected by vaginal delivery and high bifidobacterial copy numbers in their mothers' gut.

Effects of feeding a soft diet and subsequent rehabilitation on the development of the masticatory function.

It has been suggested that feeding a soft diet could possibly inhibit normal development of the masticatory function. However, the consequences of such changes in the alimentary habits have yet to be fully clarified. Therefore, the aim of this study was to determine whether a soft diet prevents the development of masticatory function and whether a critical period for programming the masticatory system exists. To examine these hypotheses, we used a three-dimensional jaw-movement tracking device and jaw muscle electromyography (EMG) to analyse masticatory function changes in mice. Jcl:ICR mice were divided into three groups, with the normal group fed a hard diet, the hypofunctional group fed a soft diet, and the rehabilitation group first fed a soft diet that was then changed to a hard diet. Our results showed that the excursion and duration of late-closing phase (occlusal phase) of the chewing cycle and EMG activity in the masseter muscle were not only reduced in the hypofunctional but also in the rehabilitation group as compared with the normal group. These results suggest that optimisation of the chewing pattern and acquisition of appropriate masticatory function are impeded by feeding a soft diet during the animal's growth period and that no catch-up effect of the masticatory function is observed when there is a prolonged period of time prior to changing the diet from soft to hard. In conclusion, masticatory function can only be fully developed through a learning process such as exposure to chewing various kinds of foods with different food textures.

Autophagy in superficial spinal dorsal horn accelerates the cathepsin B-dependent morphine antinociceptive tolerance.

Opioids are the most widely used analgesics in the treatment of severe acute and chronic pain. However, opioids have many adverse side effects, including the development of antinociceptive tolerance after long-term use. The antinociceptive tolerance of opioids has limited their clinical use. A recent study has reported that autophagy is responsible for morphine-induced neuronal injury. However, little is known about the role of autophagy in morphine antinociceptive tolerance. In the present study, chronic morphine administration was found to induce the expression of autophagy-related proteins, including Beclin1 and microtubule-associated protein light chain 3 (LC3)-II, in GABAergic interneurons in the superficial layer (lamina I-II) of the spinal cord. A single intrathecal administration of autophagy inhibitors, 3-methyladenine (3MA) or wortmannin, inhibited the development of antinociceptive tolerance in a dose-dependent manner. Autophagy in the lamina I-II neurons was associated with increased level of cathepsin B (CatB), a lysosomal cysteine protease. The pharmacological blockade or gene deletion of CatB markedly prevented the development of morphine antinociceptive tolerance. Furthermore, the intrathecal administration of 3MA suppressed the upregulation of CatB 5 days after morphine administration. Finally, CatB deficiency inhibited the increased release probability of glutamate in the lamina I neurons after chronic morphine treatment. These observations suggest that the dysfunction of the spinal GABAergic system induced by CatB-dependent excessive autophagy is partly responsible for morphine antinociceptive tolerance following chronic treatment.

Calcium-independent opening of lid1 of a family I.3 lipase by a single Asp to Arg mutation at the calcium-binding site.

A family I.3 lipase from Pseudomonas sp. MIS38 (PML) has two lids, lid1 and lid2, which are open when it exhibits activity. A single calcium ion is required to anchor lid1 in the open conformation by coordination with two acidic residues (Asp153 and Asp157) in lid1 and three other residues. Lid1 adopts a long α-helix in the open conformation, whereas it is sharply bent within this helix, such that Asp153 and Asp157 are distantly located to each other, in the closed conformation. To examine whether the mutation of Asp153 or Asp157 to a positively charged residue allows two residues at Positions 153 and 157 to come close with each other and thereby stabilizes the open conformation of lid1 even in the absence of calcium ions, five single mutant proteins (D153K-, D153R-, D153A-, D157K- and D157R-PMLs) and two double mutant proteins (D153A/D157A- and D153R/D157N-PMLs) were constructed. Of these mutant proteins, only D153R-PML exhibited activity in the absence of calcium ions. Its lipase and esterase activities were 7-fold lower and 4-fold higher than those of PML, respectively. These activities were lost by the mutation of Asp157 to Asn. These results suggest that lid1 of D153R-PML opens even in the absence of calcium ions due to electrostatic attraction between Arg153 and Asp157.

Commensal microbiota modulate murine behaviors in a strictly contamination-free environment confirmed by culture-based methods.

BACKGROUND: There is increasing evidence suggesting the existence of an interaction between commensal microbiota, the gut and the brain. The aim of this study was to examine the influence of commensal microbiota on the host behaviors in a contamination-free environment, which was verified by culture-based methods. METHODS: Open-field and marble-burying tests were used to analyze anxiety-like behaviors and locomotor activity in gnotobiotic BALB/c mice with a common genetic background in a sterile isolator. The monoamine levels in several regions of the brain were measured in germfree (GF) mice and commensal fecal microbiota-associated mice (EX-GF). KEY RESULTS: A 24-h exposure to the environment outside the sterile isolators rendered GF mice less anxious than those not contaminated, while there was no change in the locomotion. EX-GF mice, the gnotobiotic mice with normal specific pathogen-free microbiota, were less anxious and active than GF mice using open-field and marble-burying tests. The norepinephrine, dopamine, and serotonin turnover rates were higher in the EX-GF mice than in the GF mice in most regions of the brain, suggesting that monoaminergic neurotransmission might increase in the EX-GF mice comparing the GF mice. Monoassociation with Brautia coccoides reduced the anxiety level, but it did not affect the locomotor activity. In contrast, colonization with Bifidobacterium infantis decreased the locomotor activity, while having little effect on the anxiety level. CONCLUSIONS & INFERENCES: These results strongly support the current view that gut microorganisms modulate brain development and behavior.

Intracellular glutathione redox status in human dendritic cells regulates IL-27 production and T-cell polarization.

BACKGROUND: Glutathione redox status, changes in intracellular reduced (GSH) or oxidized (GSSG) glutathione, plays a significant role in various aspects of cellular function. In this study, we examined whether intracellular glutathione redox status in human dendritic cells (DCs) regulates the polarization of Th1/Th2 balance. METHODS: Human monocyte-derived DCs (MD-DCs) treated with glutathione reduced form ethyl ester (GSH-OEt) or L-buthionine-(S,R)-sulfoximine (BSO) were stimulated by lipopolysaccharide (LPS), and the levels of polarization cytokines were measured. Next, DCs matured by LPS or thymic stromal lymphopoietin (TSLP) were cocultured with allogeneic CD4(+) naive T cells and Th1/Th2 balance was evaluated by cytokine production from the primed T cells. RESULTS: Monocyte-derived DCs exposed to GSH-OEt and BSO had increased and decreased intracellular GSH contents, respectively. Lipopolysaccharide-induced interleukin (IL)-27 production was enhanced by GSH-OEt and suppressed by BSO, but neither GSH-OEt nor BSO affected the expression of HLA-DR, CD80, CD83, or CD86. Mature GSH-OEt-treated MD-DCs enhanced interferon (IFN)-γ production from CD4(+) T cells compared with nontreated MD-DCs, and small interfering RNA (siRNA) against IL-27 suppressed the effect of GSH-OEt on IFN-γ production. Additionally, although human myeloid DCs activated by TSLP (TSLP-DCs) prime naïve CD4(+) T cells to differentiate into Th2 cells, treatment of TSLP-DCs with GSH-OEt reduced IL-13 production and enhanced IFN-γ production by CD4(+) T cells. Interleukin-27 siRNA attenuated the inhibitory effect of GSH-OEt on Th2 polarization. CONCLUSION: Our results reveal that Th1 and Th2 responses are controlled by intracellular glutathione redox status in DCs through IL-27 production.

Importance of an extreme C-terminal motif of a family I.3 lipase for stability.

A five-residue sequence motif (VTLVG) located at positions 15-19 from the C-terminus of family I.3 lipase from Pseudomonas sp. MIS38 (PML) and an extreme C-terminal motif (DGIVIA) located at the C-terminus of PML are relatively well conserved in the passenger proteins of type 1 secretion system (T1SS). To analyze the role of these motifs, four mutant proteins of PML (PMLΔ5, PMLΔ10, 3A-PML and 2A-PML) were constructed. PMLΔ5 and PMLΔ10 lack the C-terminal 5 and 10 residues of PML, respectively. 3A-PML has triple mutations within an extreme C-terminal motif and 2A-PML has double mutations within a five-residue sequence motif. Secretion of these proteins was analyzed using Escherichia coli DH5 cells carrying Lip system (T1SS for family I.3 lipase). The secretion level of 2A-PML was dramatically reduced when compared with that of PML, whereas the secretion level of 3A-PML was comparable to that of PML, indicating that a five-residue sequence motif, instead of an extreme C-terminal motif, is required for secretion of PML. None of the mutations and truncations seriously affects the enzymatic activity of PML. However, 3A-PML, PMLΔ5 and PMLΔ10 were less stable than PML by 2.1, 7.6 and 7.6°C in T(1/2), respectively, and by 5.0, 21.3 and 17.9 kJ/mol in ΔG(H(2)O), respectively. These results indicate that an extreme C-terminal motif of PML is important for stability.

Cloning of the RNase H genes from a metagenomic DNA library: identification of a new type 1 RNase H without a typical active-site motif.

AIMS: The study aimed to combine a metagenomics approach with complementary genetics to identify novel bacterial genes with orthologous functions, with the identification of novel RNase H genes as a test case. METHODS AND RESULTS: A metagenomic DNA library was prepared from leaf-and-branch compost and used to screen for the RNase H genes by their abilities to complement the temperature-sensitive growth phenotype of the rnhA mutant Escherichia coli strain MIC3001. Determination of the nucleotide sequences of the cloned DNA fragments allowed us to identify 12 different genes encoding type 1 RNases H. Eleven of them encode novel RNases H, which show 40-72% amino acid sequence identities to those available from database. One of them lacks a typical DEDD/E active-site motif, which is almost fully conserved in various RNases H. CONCLUSIONS: Functional screening of environmental DNA without cultivation of microbes is a useful procedure to isolate novel RNase H genes. SIGNIFICANCE AND IMPACT OF THE STUDY: One of the identified RNase H genes had no sequence similarity to a previously assumed conserved motif, suggesting multiple catalytic mechanisms exist. This test case illustrates that metagenomics combined with complementary genetics can identify novel genes that are orthologous without sequence similarity to those from cultivated bacteria.

Subtilisin-like serine protease from hyperthermophilic archaeon Thermococcus kodakaraensis with N- and C-terminal propeptides.

The genome of the hyperthermophilic archaeon Thermococcus kodakaraensis contains three genes encoding subtilisin-like serine proteases, Tk-1689, Tk-0076 and Tk-subtilisin. Of them, the structure and function of Tk-subtilisin have been extensively studied. To examine whether Tk-1689 is matured to an active form and functions as a hyperthermostable protease as is Tk-subtilisin, the gene encoding the Tk-1689 derivative without a putative N-terminal signal sequence, termed Pro-Tk-SP, was overexpressed in Escherichia coli. Pro-Tk-SP is composed of 640 amino acid residues and its molecular mass is 68.6 kDa. The recombinant protein was purified, however, as an active 44 kDa protease, termed Tk-SP, which lacks the N-terminal 113 and C-terminal 101 amino acid residues. This result suggests that Pro-Tk-SP consists of an N-terminal propeptide (Ala1-Ala113), a mature domain (Tk-SP, Val114-Val539) and a C-terminal propeptide (Asp540-Gly640). Like Tk-subtilisin, Tk-SP showed a broad substrate specificity and was highly thermostable. Its optimum temperature for activity was approximately 100 degrees C and its half-life at 100 degrees C was 100 min. It was fully resistant to treatment with 5% SDS, 8 M urea or 10% Triton X-100. However, unlike Tk-subtilisin and bacterial subtilisins, Tk-SP requires neither Ca2+ nor propeptide for folding. As a result, Tk-SP was fully active even in the presence of 10 mM EDTA. Thus, Tk-SP has a great advantage over other proteases in high resistance to heat, denaturants, detergents and chelating agents and therefore has great potential for application in biotechnology fields.

Clinical effects of kestose, a prebiotic oligosaccharide, on the treatment of atopic dermatitis in infants.

BACKGROUND: Oligosaccharides may have beneficial properties of the prevention of atopic dermatitis (AD). Kestose, a fructo-oligosaccharide, stimulates the activity of bifidobacteria. OBJECTIVE: To assess the clinical effect of kestose on the treatment of AD in infants. METHODS: A randomized, double-blind, placebo-controlled trial was carried out using 15 and 14 infants with AD in the kestose group and placebo groups, respectively. One to 2 g kestose and maltose were administered to the subjects in the kestose and placebo groups, respectively, everyday for 12 weeks. Clinical evaluations of AD using Severity Scoring of Atopic Dermatitis (SCORAD) and the enumeration of bifidobacteria in the feces using real-time PCR were performed at Weeks 0, 6, and 12. RESULTS: The medians of the SCORAD score were significantly lower in the kestose group than in the placebo group on both Week 6 (25.3 vs. 36.4; P=0.004) and Week 12 (19.5 vs. 37.5; P<0.001). No significant correlation was found between the improvement of the SCORAD score and the count of bifidobacteria. CONCLUSION: Kestose was found to exert a beneficial effect on the clinical symptoms in infants with AD. The mechanism how does kestose improve the symptoms of AD remains to be elucidated.

Importance of the Ca2+-binding sites in the N-catalytic domain of a family I.3 lipase for activity and stability.

A family I.3 lipase from Pseudomonas sp. MIS38 (PML) contains three Ca(2+)-binding sites (Ca1-Ca3) in the N-catalytic domain. Of them, the Ca1 site is formed only in an open conformation. To analyze the role of these Ca(2+)-binding sites, three mutant proteins D157A-PML, D275A-PML and D337A-PML, which are designed to remove the Ca1, Ca2 and Ca3 sites, respectively, were constructed. Of them, the crystal structures of D157A-PML and D337A-PML in a closed conformation were determined. Both structures are nearly identical to that of the wild-type protein, except that the Ca3 site is missing in the D337A-PML structure. D157A-PML was as stable as the wild-type protein. Nevertheless, it exhibited little lipase and very weak esterase activities. D275A-PML was less stable than the wild-type protein by approximately 5 degrees C in T(1/2). It exhibited weak but significant lipase and esterase activities when compared with the wild-type protein. D337A-PML was also less stable than the wild-type protein by approximately 5 degrees C in T(1/2) but was fully active. These results suggest that the Ca1 site is required to make the active site fully open by anchoring lid 1. The Ca2 and Ca3 sites contribute to the stabilization of PML. The Ca2 site is also required to make PML fully active.

[Pulmonary atresia with intact ventricular septum showing Fontan-like circulation after biventricular repair].

A 6-year-old girl was referred to our institute for cardiac evaluation. She had been diagnosed as pulmonary atresia with intact ventricular septum (PAIVS) at 16 days after birth and she had underwent balloon atrial septostomy and bilateral Blalock-Taussig shunts. A cardiac catheterization at 5 months showed that her right ventricular end diastolic volume was 58% of normal, the Z value (standard deviation units) of the diameter of the tricuspid valve was -3.3, and a biventricular repair was performed. After the operation, she suffered from severe congestive heart failure for 10 months. A cardiac catheterization at the age of 6 years demonstrated that the pulmonary blood flow was generated during the diastolic phase like Fontan circulation. Although biventricular repair had been performed at 5 months, the circulation may be less advantageous for long term survival than if the patient had undergone the staged Fontan procedure. Careful and continuous hemodynamic assessment is essential for surgical therapy of PAIVS.

Effect of intestinal microbiota on the induction of regulatory CD25+ CD4+ T cells.

When oral tolerance was induced in either specific pathogen-free (SPF) or germ-free (GF) mice, ovalbumin (OVA) feeding before immunization induced oral tolerance successfully in SPF mice. On the other hand, OVA-specific immunoglobulin G1 (IgG1) and IgE titres in OVA-fed GF mice were comparable to those in phosphate-buffered saline-fed GF mice, thus demonstrating that oral tolerance could not be induced in GF mice. The frequencies of CD25(+) CD4(+)/CD4(+) cells in the mesenteric lymph node (MLN) and the absolute number of CD25(+) CD4(+) cells in the Peyer's patches and MLN of naive GF mice were significantly lower than those in naive SPF mice. In an in vitro assay, the CD25(+) CD4(+) cells from the naive SPF mice suppressed more effectively the proliferation of responder cells in a dose-dependent manner than those from the GF mice. In addition, the CD25(+) CD4(+) regulatory T (T(reg)) cells from the naive SPF mice produced higher amounts of interleukin (IL)-10 and transforming growth factor (TGF)-beta than those from the GF mice. When anti-TGF-beta neutralizing antibody, but not anti-IL-10 neutralizing antibody, was added to the in vitro proliferation assay, the suppressive effect of the CD25(+) CD4(+) T(reg) cells from the SPF mice was attenuated to the same level as that of the CD25(+) CD4(+) cells from the GF mice. In conclusion, the TGF-beta-producing CD25(+) CD4(+) T(reg) cells from the MLN of SPF mice played a major role in oral tolerance induction. In addition, as the regulatory function of the CD25(+) CD4(+) cells from the naive GF mice was much lower than that of the CD25(+) CD4(+) T(reg) cells from the SPF mice, indigenous microbiota are thus considered to contribute to the induction and maintenance of CD25(+) CD4(+) T(reg) cells.