Bone infection: a clinical priority for clinicians, scientists and educators.

Bone infection has received increasing attention in recent years as one of the main outstanding clinical problems in orthopaedic-trauma surgery that has not been successfully addressed. In fact, infection may develop across a spectrum of patient types regardless of the level of perioperative management, including antibiotic prophylaxis. Some of the main unknown factors that may be involved, and the main targets for future intervention, include more accurate and less invasive diagnostic options, more thorough and accurate debridement protocols, and more potent and targeted antimicrobials. The underlying biology dominates the clinical management of bone infections, with features such as biofilm formation, osteolysis and vascularisation being particularly influential. Based on the persistence of this problem, an improved understanding of the basic biology is deemed necessary to enable innovation in the field. Furthermore, from the clinical side, better evidence, documentation and outreach will be required to translate these innovations to the patient. This review presents the findings and progress of the AO Trauma Clinical Priority Program on the topic of bone infection.

Juzentaihoto extract suppresses adipocyte hypertrophy and improves hyperglycemia in KKAy mice.

Juzentaihoto is a herbal medicine with reported anti-inflammatory effects, and it is predicted to improve inflammation and insulin sensitivity within obesity. In the present study, juzentaihoto hot water extract (JTT) was administered to obese type 2 diabetic model mice (KKAy) for 56 days. In addition, the effects of JTT on the adipose tissue, glucose metabolism, and blood lipids were evaluated for examining its impact on insulin sensitivity and obesity. As a result of JTT administration, KKAy mice exhibited suppressed adipocyte hypertrophy, decreased the mRNA levels of tumor necrosis factor α, and increased the mRNA levels of adiponectin in epididymal fat tissue. In addition, fasting blood glucose levels, blood triglyceride, and total cholesterol decreased. In summary, these data indicated that JTT administration suppressed the production of inflammatory cytokines and increased adiponectin levels in the adipose tissue. Therefore, with improved insulin sensitivity, blood glucose, and lipid decreased.

Functional analysis of a novel G87V TNFRSF1A mutation in patients with TNF receptor-associated periodic syndrome.

Tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) is an autoinflammatory disease that is caused by heterozygous mutations in the TNFRSF1A gene. Although more than 150 TNFRSF1A mutations have been reported to be associated with TRAPS phenotypes only a few, such as p.Thr79Met (T79M) and cysteine mutations, have been functionally analyzed. We identified two TRAPS patients in one family harboring a novel p.Gly87Val (G87V) mutation in addition to a p.Thr90Ile (T90I) mutation in TNFRSF1A. In this study, we examined the functional features of this novel G87V mutation. In-vitro analyses using mutant TNF receptor 1 (TNF-R1)-over-expressing cells demonstrated that this mutation alters the expression and function of TNF-R1 similar to that with the previously identified pathogenic T79M mutation. Specifically, cell surface expression of the mutant TNF-R1 in transfected cells was inhibited with both G87V and T79M mutations, whereas the T90I mutation did not affect this. Moreover, peripheral blood mononuclear cells (PBMCs) from TRAPS patients harboring the G87V and T90I mutations showed increased mitochondrial reactive oxygen species (ROS). Furthermore, the effect of various Toll-like receptor (TLR) ligands on inflammatory responses was explored, revealing that PBMCs from TRAPS patients are hyper-responsive to TLR-2 and TLR-4 ligands and that interleukin (IL)-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) are likely to be involved in the pathogenesis of TRAPS. These findings suggest that the newly identified G87V mutation is one of the causative mutations of TRAPS. Our findings based on unique TRAPS-associated mutations provide novel insight for clearer understanding of inflammatory responses, which would be basic findings of developing a new therapeutic and prophylactic approach to TRAPS.

Probiotic Lactobacillus gasseri SBT2055 improves insulin secretion in a diabetic rat model.

The probiotic Lactobacillus gasseri SBT2055 (LG2055) has a protective effect against metabolic syndrome in rats and humans. Metabolic syndrome increases the risk of type 2 diabetes mellitus. In this study, Goto-Kakizaki rats were used as a diabetic model and fed diets containing LG2055-fermented or nonfermented skim milk for 4 wk. Indices of diabetes such as blood glucose levels, serum glucagon levels, plasma levels of insulin, C-peptide, and glucagon-like peptide-1, tissue glycogen contents, and pancreatic mRNA levels were measured. The plasma C-peptide levels and pancreatic mRNA levels of insulin genes (Ins1 and Ins2) and Pdx1 (a transcriptional factor of insulin genes) were increased in LG2055 diet-fed rats. The increase in insulin secretion corresponded to an improvement in serum and pancreatic inflammatory status, associated with decreases in serum levels of serum amyloid P and pancreatic levels of granulocyte colony-stimulating factor. Insulin resistance in Goto-Kakizaki rats was ameliorated by increased glycogen storage in the liver and quadriceps femoris muscles and decreased serum free fatty acid levels. This improvement may be related to the increased cecal production of short-chain fatty acids. In conclusion, dietary LG2055 improved insulin secretion in diabetic rats by improving the inflammatory status in the pancreas and serum.

Quality control of hospital preparations: Establishment of a simple and rapid method for quantifying ulinastatin in vaginal suppositories.

Ulinastatin vaginal suppositories, used to prevent threatened premature delivery, are frequently used in hospitals. However, there is no established method for quantifying ulinastatin contained in suppositories. Therefore, we investigated a simple and efficient method for quantifying ulinastatin contained in suppositories. Our analytical method involved removal of the base; optimising the enzyme inhibition reaction time and enzyme reaction time; and measuring the absorbance. The modified method was reproducible, operation time was significantly shortened, and cost was reduced to approximately 1/17 of that of the previously reported method. This simple and rapid quantitative method could contribute to the improvement of quality control of ulinastatin vaginal suppositories as an extemporaneous hospital preparation.

FOLFIRI plus bevacizumab as second-line therapy in patients with metastatic colorectal cancer after first-line bevacizumab plus oxaliplatin-based therapy: the randomized phase III EAGLE study.

BACKGROUND: A targeted agent combined with chemotherapy is the standard treatment in patients with metastatic colorectal cancer (mCRC). The present phase III study was conducted to compare two doses of bevacizumab combined with irinotecan, 5-fluorouracil/leucovorin (FOLFIRI) in the second-line setting after first-line therapy with bevacizumab plus oxaliplatin-based therapy. PATIENTS AND METHODS: Patients were randomly assigned to receive FOLFIRI plus bevacizumab 5 or 10 mg/kg in 2-week cycles until disease progression. The primary end point was progression-free survival (PFS), and secondary end points included overall survival (OS), time to treatment failure (TTF), and safety. RESULTS: Three hundred and eighty-seven patients were randomized between September 2009 and January 2012 from 100 institutions in Japan. Baseline patient characteristics were well balanced between the two groups. Efficacy was evaluated in 369 patients (5 mg/kg, n = 181 and 10 mg/kg, n = 188). Safety was evaluated in 365 patients (5 mg/kg, n = 180 and 10 mg/kg, n = 185). The median PFS was 6.1 versus 6.4 months (hazard ratio, 0.95; 95% confidence interval [CI] 0.75-1.21; P = 0.676), and median TTF was 5.2 versus 5.2 months (hazard ratio, 1.01; 95% CI 0.81-1.25; P = 0.967), respectively, for the bevacizumab 5 and 10 mg/kg groups. Follow-up of OS is currently ongoing. Adverse events, including hypertension and hemorrhage, occurred at similar rates in both groups. CONCLUSION: Bevacizumab 10 mg/kg plus FOLFIRI as the second-line treatment did not prolong PFS compared with bevacizumab 5 mg/kg plus FOLFIRI in patients with mCRC. If bevacizumab is continued after first-line therapy in mCRC, a dose of 5 mg/kg is appropriate for use as second-line treatment. CLINICAL TRIAL IDENTIFIER: UMIN000002557.

Functional variants in the thromboxane A2 receptor gene are associated with lung function in childhood-onset asthma.

BACKGROUND: The thromboxane A2 receptor (TBXA2R) gene is associated with asthma, but no functional genetic variations are known to associate with the disease or its related phenotypes. OBJECTIVE: To investigate the association of TBXA2R polymorphisms with asthma susceptibility and related phenotypes and to identify functionally relevant polymorphisms. METHODS: We performed comprehensive sequencing of the TBXA2R gene in 48 Japanese control subjects and found a set of variants (SNP1 G>T rs2238634, SNP2 T>G rs2238633, SNP3 C>T rs2238632 and SNP4 G>A rs2238631) in intron 1 in linkage disequilibrium with c.795 T>C rs1131882, which was previously reported to be associated with asthma and related phenotypes. To investigate the effect of four common haplotypes (H1, H2, H3 and H4) on transcriptional activity, we performed a luciferase assay in primary bronchial smooth muscle cells (BSMCs) and human airway epithelial cells (BEAS-2B). We also studied the haplotype association with lung function, TBXA2R mRNA levels, and eosinophil fraction/count in peripheral blood in childhood-onset asthma patients and/or controls. RESULTS: H2 and H4, containing minor alleles of SNP2 and SNP3, had significantly higher transcriptional activities than H1 consisting of major alleles (P < 0.001 in BSMCs and BEAS-2B). Homozygotes for redefined haplotype h2 corresponding to minor alleles of SNP2 and SNP3 were associated with lower lung function in childhood-onset asthma patients compared to other zygotes (baseline Forced expiratory volume in one second (FEV1)/ Forced vital capacity (FVC) and Forced expiratory flow between 25% and 75% of the FVC (%FEF(25-75%)): P = 0.00201 and 0.0128, respectively, and post-bronchodilator FEV1/FVC and %FEF(25-75%): P = 0.00224 and 0.0393 respectively). Haplotype h2 was also associated with higher mRNA levels in control peripheral blood cells and higher blood eosinophil fractions and counts in female controls. CONCLUSIONS AND CLINICAL RELEVANCE: Genetic variants were identified in the TBXA2R gene that influenced transcriptional activity and were associated with asthma-related phenotypes. Thromboxane pathways may therefore play important roles in airway inflammation and remodelling in asthma patients.

Evaluation of automated 2D-3D image overlay system utilizing subtraction of bone marrow image for EVAR: feasibility study.

OBJECTIVE: To evaluate the automated 2D-3D image overlay system ("3D Roadmap") for use during endovascular aneurysm repair (EVAR) in the hybrid operating theater. METHODS: Datasets of preoperative CT images were modified to subtract dense bone marrow to improve the visualization of vasculature on the overlaid image, and allow for accurate navigation of the endovascular devices. The 3D-CT overlay image was registered on the 2D fluoroscopy image to mark the iliac crest and lumbar vertebrae on both images as landmarks. RESULTS: Arteriography was performed only twice to confirm the precision of the position of renal artery and the final evaluation. Twenty patients underwent EVAR with Medtronic Endurant, Gore Excluder, or COOK Zenith using "3D Roadmap". The origin of the renal artery and iliac bifurcation were registered with complete accuracy in 10 patients (50%). The lower renal artery deviated toward the cranial side less than 3 mm in six patients. In all cases, EVAR was successful, and completed with the volume of contrast material limited to 43.8 ± 3.1 mL. CONCLUSION: "3D Roadmap" was confirmed to be valuable for visualization of vessel origin in a fused image and for reduction of contrast material during EVAR.

A rare case of Lemierre`s syndrome caused by Porphyromonas asaccharolytica.

Lemierre's syndrome is only very rarely caused by Porphyromonas asaccharolytica. Here, we report the case of a 35-year-old man who developed a left peritonsillar abscess, thrombophlebitis of the left internal jugular vein, and septic embolization of both lungs. Anaerobic P. asaccharolytica was isolated in the blood cultures, and we subsequently confirmed the diagnosis as Lemierre's syndrome. Our case indicates that although P. asaccharolytica is not commonly found in oral cavities, this organism may still cause Lemierre's syndrome. Consequently, when it is detected in blood cultures, the treating physician should perform the medical examination while keeping in mind the possibility that the patient could have Lemierre's syndrome.

Protein-Ligand Interaction Analyses with Nuclear Magnetic Resonance Spectroscopy Enhanced by Dissolution Triplet Dynamic Nuclear Polarization.

Solution-state nuclear magnetic resonance spectroscopy (NMR) is a powerful method for the analysis of intermolecular interactions within a biomolecular system. However, low sensitivity is one of the major obstacles of NMR. We improved the sensitivity of solution-state 13C NMR for the observation of intermolecular interactions between protein and ligand using hyperpolarized solution samples at room temperature. Eutectic crystals composed of 13C-salicylic acid and benzoic acid doped with pentacene were hyperpolarized by dynamic nuclear polarization using photoexcited triplet electrons, and a 13C nuclear polarization of 0.72 ± 0.07% was achieved after dissolution. The binding of human serum albumin and 13C-salicylate was observed with several hundred times sensitivity enhancement under mild conditions. The established 13C NMR was applied for pharmaceutical NMR experiments by observation of the partial return of the 13C chemical shift of salicylate by competitive binding with other non-isotope-labeled drugs.

Potential role of autoantibody in severe neutropenia of a patient with Kawasaki Syndrome.

Neutropenia associated with Kawasaki Syndrome (KS) has been rarely reported, and the detailed mechanisms responsible for this state are not yet elucidated. The aim of this study was to clarify the mechanisms of neutropenia in KS. We examined antibodies to known neutrophil antigens (HNA1a, HNA1b, HNA null, HNA2, HNA3, HNA4 and non-HLA antigen 9a) in a KS patient with neutropenia. We also performed the granulocyte immunofluorescence test (GIFT) using patient or control neutrophils incubated with the patient's serum at serial time points over the patient's clinical course. No specific antibody to known neutrophil antigens was detected. Flow cytometric analysis showed that autoantibodies bound to immature CD13-positive myeloid cells, which resulted in myeloid lineage maturation arrest in the bone marrow. GIFT showed that neutrophil-specific autoantibodies were produced by the patient, and the amount of autoantibody inversely correlated with the patient's neutrophil counts. The presence of an autoantibody to a novel antigen on immature myeloid cells or neutrophils is the likely the cause of severe neutropenia in this patient with KS.

Is there any circadian variation consequence on acute case fatality of stroke? Takashima Stroke Registry, Japan (1990-2003).

BACKGROUND: Circadian periodicity in the onset of stroke has been reported. However, it is unclear whether this variation affects the acute stroke case fatality. Time of the day variation in stroke case fatality was examined using population-based stroke registration data. METHODS: Stroke event data were acquired from the Takashima Stroke Registry, which covers a stable population of approximately 55,000 in Takashima County in central Japan. During the period of 1990-2003, there were 1080 (549 men and 531 women) cases with classifiable stroke onset time. Stroke incidence was categorized as occurring at night (midnight-6 a.m.), morning (6 a.m.-noon), afternoon (noon-6 p.m.), and evening (6 p.m.-midnight). The 28-day case fatality rates and 95% confidence intervals (95% CI) were calculated by gender, age, and stroke subtype across the time blocks. After adjusting for gender, age at onset, and stroke severity at onset, the hazard ratios for fatal strokes in evening, night, and morning were calculated, with afternoon serving as the reference. RESULTS: For all strokes, the 28-day case fatality rate was 23.3% (95% CI:19.4-27.6) for morning onset, 16.9% (95% CI:13.1-21.6) for afternoon onset, 18.3% (95% CI:13.6-24.1) for evening onset, and 21.0% (95% CI:15.0-28.5) for the night onset stroke. The case fatality for strokes during the morning was higher than the case fatality for strokes during afternoon. This fatality risk excess for morning strokes persisted even after adjusting for age, gender, and stroke severity on onset in multivariate analysis. CONCLUSION: In the examination of circadian variation of stroke case fatality, 28-day case fatality rate tended to be higher for the morning strokes.

Prevalence of Parkinson's disease and atypical parkinsonian syndromes in a rural Japanese district.

OBJECTIVES: To investigate the prevalence of Parkinson's disease (PD) and atypical parkinsonian syndromes (APS) in a rural Japanese district. METHOD: Collaboration with the medical institutions, the long-term care insurance system facilities, and the public health office. RESULTS: The crude prevalence rates were 175 per 100,000 (95% CI: 143-206) for PD, 18 (8-28) for progressive supranuclear palsy, 17 (7-26) for multiple system atrophy (MSA), and 9 (2-16) for corticobasal degeneration. The age-adjusted prevalence rates were 109 per 100,000 (88-134), 10 (2-17), 13 (4-21), and 6 (0-12), for each condition. There was a preponderance of women with PD and of men with APS. Nine of the 116 PD patients and 7 of the 29 APS patients were newly diagnosed in this study. CONCLUSIONS: There are high prevalence rates for PD and APS and suboptimal recognition of APS. This is the first epidemiological prevalence study of MSA from Japan.

Oocyte apoptosis is suppressed by disruption of the acid sphingomyelinase gene or by sphingosine-1-phosphate therapy.

The time at which ovarian failure (menopause) occurs in females is determined by the size of the oocyte reserve provided at birth, as well as by the rate at which this endowment is depleted throughout post-natal life. Here we show that disruption of the gene for acid sphingomyelinase in female mice suppressed the normal apoptotic deletion of fetal oocytes, leading to neonatal ovarian hyperplasia. Ex vivo, oocytes lacking the gene for acid sphingomyelinase or wild-type oocytes treated with sphingosine-1-phosphate resisted developmental apoptosis and apoptosis induced by anti-cancer therapy, confirming cell autonomy of the death defect. Moreover, radiation-induced oocyte loss in adult wild-type female mice, the event that drives premature ovarian failure and infertility in female cancer patients, was completely prevented by in vivo therapy with sphingosine-1-phosphate. Thus, the sphingomyelin pathway regulates developmental death of oocytes, and sphingosine-1-phosphate provides a new approach to preserve ovarian function in vivo.

Purification and identification of lactoperoxidase in milk basic proteins as an inhibitor of osteoclastogenesis.

A milk protein fraction with alkaline isoelectric points (milk basic protein, MBP) inhibits both bone resorption and osteoclastogenesis for in vitro models. We previously identified bovine angiogenin as a component of MBP that inhibits bone resorption. However, purified angiogenin had no effect on osteoclastogenesis, suggesting that MBP contains unidentified component(s) that inhibit osteoclast formation. In this study, we purified lactoperoxidase (LPO) as the predominant inhibitor of osteoclastogenesis in MBP. The LPO treatment downregulated levels of reactive oxygen species in osteoclasts. Signaling by receptor activator of NF-kappa-B ligand/receptor activator of NF-kappa-B (RANKL/RANK) was downregulated in LPO-treated cells, and, in particular, the ubiquitination of tumor necrosis factor receptor associate factor 6 (TRAF6) and activation of downstream signaling cascades (JNK, p38, ERK, and NFκB) were suppressed. Ultimately, LPO treatment led to decreased expression of c-Fos and NFAT2. These results suggest that MBP contains at least 2 components that independently suppress bone resorption through a unique mechanism: angiogenin inhibits bone resorption and LPO inhibits RANKL-induced osteoclast differentiation. These data explain many of the positive aspects of milk consumption on bone health.

Formation of an antral follicle-like structure of bovine cumulus-oocyte complexes embedded individually or in groups in collagen gels.

Culture techniques of antral follicle-like structure (AFLS) derived from cumulus-oocyte complexes (COCs) might provide important insights into follicular development and oocyte maturation. This study was undertaken to investigate the effects of embedding bovine COCs individually (one COC) or in groups (4-5 COCs) in collagen gels on the formation of AFLS and the meiotic status of oocytes. The observations of AFLS formation were performed every second day for 14 days. The AFLS was formed at Day 2 or 4 after the start of culture (Day=0), irrespective of the culture methods. The mean diameters of AFLS during Days 4-14 using the individual culture method were significantly higher (p<0.05) than those using the group culture method. However, the AFLS formation rate in the individual culture method was significantly lower compared to that in the group culture method (26.1% vs 62.7%, p<0.01). Almost all oocytes had undergone the germinal vesicle breakdown stage, irrespective of the culture method or AFLS formation. In conclusion, comparison with the individual culture method revealed that the mean diameters of AFLS in the group culture method were smaller, but more COCs formed AFLS. The group culture method might be useful for evaluating the various hypotheses of follicular formation and interfollicular communication. However, improvement of the group culture system is necessary to prevent the meiotic resumption of oocytes, because the AFLS formation is dependent on the cumulus/granulosa cells surrounding oocytes.

Neurokinin 3 receptor-selective agonist, senktide, decreases core temperature in Japanese Black cattle.

Heat stress disrupts reproductive function in cattle. In summer, high ambient temperature and humidity elevate core body temperature, which is considered to be detrimental to reproductive abilities in cattle. Neurokinin B (NKB) is a factor that generates pulsatile GnRH and subsequent LH secretion in mammals. Recent studies have reported that NKB-neurokinin 3 receptor (NK3R) signaling is associated with heat-defense responses in rodents. The present study aimed to clarify the role of NKB-NK3R signaling in thermoregulation in cattle. We examined the effects of an NK3R-selective agonist, senktide, on vaginal temperature as an indicator of core body temperature in winter and summer. In both seasons, continuous infusion of senktide for 4 h immediately decreased vaginal temperature, and the mean temperature change in the senktide-treated group was significantly lower than that of both vehicle- and GnRH-treated groups. Administration of GnRH induced LH elevation, but there was no significant difference in vaginal temperature change between GnRH- and vehicle-treated groups. Moreover, we investigated the effects of senktide on ovarian temperature. Senktide treatment seemed to suppress the increase in ovarian temperature from 2 h after the beginning of administration, although the difference between groups was not statistically significant. Taken together, these results suggest that senktide infusion caused a decline in the vaginal temperature of cattle, in both winter and summer seasons, and this effect was not due to the gonadotropin-releasing action of senktide. These findings provide new therapeutic options for senktide to support both heat-defense responses and GnRH/LH pulse generation.

Age-associated characteristics of murine hematopoietic stem cells.

Little is known of age-associated functional changes in hematopoietic stem cells (HSCs). We studied aging HSCs at the clonal level by isolating CD34(-/low)c-Kit(+)Sca-1(+) lineage marker-negative (CD34(-)KSL) cells from the bone marrow of C57BL/6 mice. A population of CD34(-)KSL cells gradually expanded as age increased. Regardless of age, these cells formed in vitro colonies with stem cell factor and interleukin (IL)-3 but not with IL-3 alone. They did not form day 12 colony-forming unit (CFU)-S, indicating that they are primitive cells with myeloid differentiation potential. An in vivo limiting dilution assay revealed that numbers of multilineage repopulating cells increased twofold from 2 to 18 mo of age within a population of CD34(-)KSL cells as well as among unseparated bone marrow cells. In addition, we detected another compartment of repopulating cells, which differed from HSCs, among CD34(-)KSL cells of 18-mo-old mice. These repopulating cells showed less differentiation potential toward lymphoid cells but retained self-renewal potential, as suggested by secondary transplantation. We propose that HSCs gradually accumulate with age, accompanied by cells with less lymphoid differentiation potential, as a result of repeated self-renewal of HSCs.