Changes in the composition of molecular species of covalently bound and free ceramides [EOS], and their correlation with disease severity in atopic dermatitis.

Ceramides are major constituents of stratum corneum (SC) intercellular lipids involved in skin barrier function. The ratio of molecular species of ceramides and their correlation with disease severity was examined in patients with atopic dermatitis (AD). Thirty-eight patients with AD and 32 healthy controls (HCs) were assessed for transepidermal water loss, SC collection and clinical assessment. The ceramide content of different molecular species in the samples was quantified using high-performance liquid chromatography coupled with tandem mass spectrometry. Unsaturated acyl chains of both covalently bound and free ceramides [EOS] were higher in AD lesional skin than those in AD non-lesional or normal HC skin. The proportion of unsaturated acyl chains (C30:1, C32:1 and C34:1) was higher than other ceramide molecular species among covalently bound and free ceramides [EOS] in patients with AD. The proportion of unsaturated acyl chains in covalently bound ceramides was positively correlated with transepidermal water loss (r = 0.600) when considering the total number of non-lesional and lesional skin. Additionally, thymus and activation-regulated chemokine (TARC) showed a positive correlation with unsaturated acyl chains proportion in AD non-lesional (r = 0.676) and lesional (r = 0.503) skin. Our study is the first to show the increase in unsaturated acyl chains of both covalently bound and free ceramides [EOS] in lesional and non-lesional skin in AD for each molecular species. This increase is associated with dryness and impaired barrier function, which correlates with TARC levels, a marker for the degree of type 2 inflammation. We speculate that type 2 inflammation exacerbation leads to abnormal epidermal lipid metabolism in the skin of patients with AD.

Long-term outcomes of transtrochanteric rotational osteotomy for non-traumatic osteonecrosis of the femoral head.

AIMS: Transtrochanteric rotational osteotomy (TRO) is performed for young patients with non-traumatic osteonecrosis of the femoral head (ONFH) to preserve the hip. We aimed to investigate the long-term outcomes and the risk factors for failure 15 years after this procedure. PATIENTS AND METHODS: This study included 95 patients (111 hips) with a mean age of 40 years (21 to 64) who underwent TRO for ONFH. The mean follow-up was 18.2 years (3 to 26). Kaplan-Meier survivorship analyses were performed with conversion to total hip arthroplasty (THA) and radiological failure due to secondary collapse of the femoral head or osteoarthritic changes as the endpoint. Multivariate analyses were performed to assess risk factors for each outcome. RESULTS: Survival rates at 15 years with conversion to THA and radiological failure as the endpoint were 59% (95% confidence interval (CI) 49 to 67) and 30% (95% CI 22 to 39), respectively. Necrotic type C2 ONFH (lesions extending laterally to the acetabular edge) (hazards ratio (HR) 3.9) and age > 40 years (HR 2.5) were risk factors for conversion to THA. Stage > 3a ONFH (HR 2.0) and age > 40 years (HR 1.9) were risk factors for radiological failure. CONCLUSION: The 15 year outcomes after TRO for ONFH are unfavorable because osteoarthritic changes occur after five years post-operatively. Cite this article: Bone Joint J 2017;99-B:175-83.

A pre-operative predictive score for the outcome of eccentric rotational acetabular osteotomy in the treatment of acetabular dysplasia and early osteoarthritis of the hip in adults.

AIMS: The influence of identifiable pre-operative factors on the outcome of eccentric rotational acetabular osteotomy (ERAO) is unknown. We aimed to determine the factors that might influence the outcome, in order to develop a scoring system for predicting the prognosis for patients undergoing this procedure. PATIENTS AND METHODS: We reviewed 700 consecutive ERAOs in 54 men and 646 women with symptomatic acetabular dysplasia or early onset osteoarthritis (OA) of the hip, which were undertaken between September 1989 and March 2013. The patients' pre-operative background, clinical and radiological findings were examined retrospectively. Multivariate Cox regression analysis was performed using the time from the day of surgery to a conversion to total hip arthroplasty (THA) as an endpoint. A risk score was calculated to predict the prognosis for conversion to THA, and its predictive capacity was investigated. RESULTS: The congruity of the hip, age, the pre-operative minimum width of the joint space and range of abduction were identified as factors predicting conversion to THA. For three groups of patients (scoring 0 to 5, 6 to 7, and 8 to 9 points), the Kaplan-Meier event-free rates of survival at 15 years post-operatively for conversion to THA were 99.6%, 85.2% and 67.3%, respectively. CONCLUSION: These four pre-operative factors are easily measured and predict the prognosis for patients following ERAO. They may be used for decision making when offering surgical treatment to patients with acetabular dysplasia and early onset osteoarthritis. Cite this article: Bone Joint J 2016;98-B:1326-32.

Pharmacokinetic Evaluation of a Drug Transporter Cocktail Consisting of Digoxin, Furosemide, Metformin, and Rosuvastatin.

This article reports the clinical investigation of a probe drug cocktail containing substrates of key drug transporters. Single oral doses of 0.25 mg digoxin (P-gp), 5 mg furosemide (OAT1 and OAT3), 500 mg metformin (OCT2, MATE1, and MATE2-K), and 10 mg rosuvastatin (OATP1B1, OATP1B3, and BCRP) were administered separately or as a cocktail in a randomized six-period crossover trial in 24 healthy male volunteers. As a cocktail, relative bioavailabilities of digoxin and metformin and furosemide AUC0-tz were similar to separate dosing. However, when administered as a cocktail the Cmax of furosemide was 19.1% lower and the Cmax and AUC0-tz of rosuvastatin were 38.6% and 43.4% higher, respectively. In addition, the effects of increased doses of metformin or furosemide on the cocktail were investigated in 11 and 12 subjects, respectively. The cocktail explored in this trial has the potential to be used for the in vivo screening of transporter-mediated drug-drug interactions. © 2016 American Society for Clinical Pharmacology and Therapeutics.

Three-day regimen of oseltamivir for postexposure prophylaxis of influenza in wards.

Inpatients who had been in close contact with patients with influenza were given oseltamivir [75mg capsules once daily for adults or 2mg/kg (maximum of 75mg) once daily for children] for three days as postexposure prophylaxis (PEP). The index patients with influenza were prescribed a neuraminidase inhibitor and were discharged immediately or transferred to isolation rooms. The protective efficacy of oseltamivir for three days was 93% overall [95% confidence interval (CI) 53-99%; P=0.023] and 94% for influenza A (95% CI 61-99%; P=0.017), which is comparable to that of seven- to 10-day regimens of oseltamivir as PEP.

Long-term kinetics of AA amyloidosis and effects of inflammatory restimulation after disappearance of amyloid depositions in mice.

Amyloid A (AA) amyloidosis is characterized by extracellular pathogenic deposition of insoluble fibril protein in various body organs. Deposited amyloid generally remains in a variety of organs for long periods, but its disappearance has been reported after the precursor protein is diminished. The kinetics of AA deposition are not completely understood and, in particular, the roles of cells and cytokines in the deposition and clearance of amyloid remain unclear. In this study, we investigated the disappearance of amyloid depositions in mice over a 1-year period. AA amyloidosis was induced experimentally in mice by injecting amyloid-enhancing factor (AEF) and silver nitrate. Mice were killed at different time-points to examine the occurrence and disappearance of amyloid depositions. Maximum levels of amyloid depositions were observed at 20 days after inoculation. Clearance of amyloid depositions was observed from the 40th day onwards, with only minute traces of amyloid present by 240 days. A second inflammatory stimulus consisting of AEF and silver nitrate was given at 330 or 430 days, after amyloid depositions had disappeared almost completely. After that, serum amyloid A was overproduced and redeposition of amyloid was observed, indicating that all mice were primed for aggressive amyloid depositions. After administration of the inflammatory stimuli, the proinflammatory environment was found to have increased levels of interleukin (IL)-6, while anti-inflammatory conditions were established by IL-10 as regression of amyloid deposition occurred. These results suggest that the proinflammatory and anti-inflammatory status have key roles in both amyloid deposition and clearance.

Ten-year experience of recombinant activated factor VII use in surgical patients with congenital haemophilia with inhibitors or acquired haemophilia in Japan.

Patients with congenital haemophilia with inhibitors or acquired haemophilia are at risk of bleeding complications during surgery. In these patients, replacement therapy for the missing coagulation factor is ineffective, and a bypassing agent such as recombinant activated factor VII (rFVIIa) is required to manage bleeding. To evaluate the safety and haemostatic efficacy of rFVIIa treatment in Japanese patients with congenital haemophilia with inhibitors to FVIII/FIX or acquired haemophilia undergoing surgery. Postmarketing surveillance data from May 2000 to March 2010 were analysed to assess the haemostatic efficacy of 38 procedures in 22 patients with congenital haemophilia A, 13 procedures in seven patients with congenital haemophilia B, and five procedures in five patients with acquired haemophilia. Postoperative bleeding control was judged to be effective (bleeding was stopped completely or reduced considerably) for 34/38 procedures (89%) in patients with congenital haemophilia A, 10/13 procedures (77%) in patients with congenital haemophilia B, and 4/5 procedures (80%) in patients with acquired haemophilia. Tranexamic acid was used concomitantly for 36/56 procedures (64%). Safety was analysed for 66 procedures in 37 patients. Adverse effects potentially related to rFVIIa treatment included mild superficial thrombophlebitis, mild decrease in platelet count, and mild elevation of the serum alanine transaminase level in one patient each. All adverse effects resolved without treatment. Administration of rFVIIa provided adequate haemostasis without serious adverse effects in the majority of cases. The efficacy and safety data in Japanese patients were similar to previously published data from other countries.

Factors associated with an unfavourable outcome after Salter innominate osteotomy in patients with unilateral developmental dysplasia of the hip: does occult dysplasia of the contralateral hip affect the outcome?

Salter innominate osteotomy is an effective reconstructive procedure for the treatment of developmental dysplasia of the hip (DDH), but some children have a poor outcome at skeletal maturity. In order to investigate factors associated with an unfavourable outcome, we assessed the development of the contralateral hip. We retrospectively reviewed 46 patients who underwent a unilateral Salter osteotomy at between five and seven years of age, with a mean follow-up of 10.3 years (7 to 20). The patients were divided into three groups according to the centre-edge angle (CEA) of the contralateral hip at skeletal maturity: normal (> 25°, 22 patients), borderline (20° to 25°, 17 patients) and dysplastic (< 20°, 7 patients). The CEA of the affected hip was measured pre-operatively, at eight to nine years of age, at 11 to 12 years of age and at skeletal maturity. The CEA of the affected hip was significantly smaller in the borderline and dysplastic groups at 11 and 12 years of age (p = 0.012) and at skeletal maturity (p = 0.017) than in the normal group. Severin group III was seen in two (11.8%) and four hips (57.1%) of the borderline and dysplastic groups, respectively (p < 0.001). Limited individual development of the acetabulum was associated with an unfavourable outcome following Salter osteotomy.

Early and late fracture following extensive limb lengthening in patients with achondroplasia and hypochondroplasia.

Two types of fracture, early and late, have been reported following limb lengthening in patients with achondroplasia (ACH) and hypochondroplasia (HCH). We reviewed 25 patients with these conditions who underwent 72 segmental limb lengthening procedures involving the femur and/or tibia, between 2003 and 2011. Gender, age at surgery, lengthened segment, body mass index, the shape of the callus, the amount and percentage of lengthening and the healing index were evaluated to determine predictive factors for the occurrence of early (within three weeks after removal of the fixation pins) and late fracture (> three weeks after removal of the pins). The Mann­Whitney U test and Pearson's chi-squared test for univariate analysis and stepwise regression model for multivariate analysis were used to identify the predictive factor for each fracture. Only one patient (two tibiae) was excluded from the analysis due to excessively slow formation of the regenerate, which required supplementary measures. A total of 24 patients with 70 limbs were included in the study. There were 11 early fractures in eight patients. The shape of the callus (lateral or central callus) was the only statistical variable related to the occurrence of early fracture in univariate and multivariate analyses. Late fracture was observed in six limbs and the mean time between removal of the fixation pins and fracture was 18.3 weeks (3.3 to 38.4). Lengthening of the tibia, larger healing index, and lateral or central callus were related to the occurrence of a late fracture in univariate analysis. A multivariate analysis demonstrated that the shape of the callus was the strongest predictor for late fracture (odds ratio: 19.3, 95% confidence interval: 2.91 to 128). Lateral or central callus had a significantly larger risk of fracture than fusiform, cylindrical, or concave callus. Radiological monitoring of the shape of the callus during distraction is important to prevent early and late fracture of lengthened limbs in patients with ACH or HCH. In patients with thin callus formation, some measures to stimulate bone formation should be considered as early as possible.

Biodistribution of locally or systemically transplantedosteoblast-like cells.

OBJECTIVES: In order to ensure safety of the cell-based therapy for bone regeneration, we examined in vivo biodistribution of locally or systemically transplanted osteoblast-like cells generated from bone marrow (BM) derived mononuclear cells. METHODS: BM cells obtained from a total of 13 Sprague-Dawley (SD) green fluorescent protein transgenic (GFP-Tg) rats were culture-expanded in an osteogenic differentiation medium for three weeks. Osteoblast-like cells were then locally transplanted with collagen scaffolds to the rat model of segmental bone defect. Donor cells were also intravenously infused to the normal Sprague-Dawley (SD) rats for systemic biodistribution. The flow cytometric and histological analyses were performed for cellular tracking after transplantation. RESULTS: Locally transplanted donor cells remained within the vicinity of the transplantation site without migrating to other organs. Systemically administered large amounts of osteoblast-like cells were cleared from various organ tissues within three days of transplantation and did not show any adverse effects in the transplanted rats. CONCLUSIONS: We demonstrated a precise assessment of donor cell biodistribution that further augments prospective utility of regenerative cell therapy. Cite this article: Bone Joint Res 2014;3:76-81.

Transmission of systemic AA amyloidosis in animals.

Amyloidoses are a group of protein-misfolding disorders that are characterized by the deposition of amyloid fibrils in organs and/or tissues. In reactive amyloid A (AA) amyloidosis, serum AA (SAA) protein forms deposits in mice, domestic and wild animals, and humans that experience chronic inflammation. AA amyloid fibrils are abnormal ß-sheet-rich forms of the serum precursor SAA, with conformational changes that promote fibril formation. Extracellular deposition of amyloid fibrils causes disease in affected animals. Recent findings suggest that AA amyloidosis could be transmissible. Similar to the pathogenesis of transmissible prion diseases, amyloid fibrils induce a seeding-nucleation process that may lead to development of AA amyloidosis. We review studies of possible transmission in bovine, avian, mouse, and cheetah AA amyloidosis.

Keratan sulfate expression in microglia is diminished in the spinal cord in experimental autoimmune neuritis.

Experimental autoimmune neuritis (EAN) is an animal model of Guillain-Barré syndrome, an inflammatory demyelination disease of the peripheral nervous system. Although this disease has been extensively studied on peripheral nerves, the pathology of the central nervous system has not been fully understood. Previous studies demonstrate that expression of keratan sulfate (KS), the sugar chain of proteoglycan, is associated with activated microglia/macrophages accumulated after neuronal injuries. Unexpectedly, we found here that KS is rather diminished in rat EAN. KS was restrictively expressed in microglia in the spinal cord of normal rats. KS was positive in 50% microglia in the ventral horn and 20% in the dorsal horn. In EAN, microglia increased in number and expressed the activation marker CD68, but KS expression was abolished. Concomitantly, pro-inflammatory cytokines, i.e., interferon (IFN)-γ, interleukin (IL)-1ß, and tumor necrosis factor (TNF)-α, were increased in the spinal cord of EAN rats, whereas anti-inflammatory cytokines, such as IL-4 and IL-10, were decreased. In addition, silencing of KSGal6ST attenuated KS expression on the primary cultured microglia and upregulated expression of some activation markers (TNF-α, IL-1ß, and iNOS) under the stimulation with lipopolysaccharide and IFN-γ. This study demonstrates for the first time a close association of EAN and disappearance of KS on microglia. KS expression could be a useful marker to evaluate the status of polyneuropathy.

Total spondylectomy following carbon ion radiotherapy to treat chordoma of the mobile spine.

The main form of treatment of a chordoma of the mobile spine is total en bloc spondylectomy (TES), but the clinical results are not satisfactory. Stand-alone carbon ion radiotherapy (CIRT) for bone and soft-tissue sarcomas has recently been reported to have a high rate of local control with a low rate of local recurrence. We report two patients who underwent TES after CIRT for treating a chordoma in the lumbar spine with good medium-term outcomes. At operation, there remained histological evidence of viable tumour cells in both cases. After the combination use of TES following CIRT, neither patient showed signs of recurrence at the follow-up examination. These two cases suggest that CIRT should be combined with total spondylectomy in the treatment of chordoma of the mobile spine.

AA amyloidosis in vaccinated growing chickens.

Systemic amyloid-A (AA) amyloidosis in birds occurs most frequently in waterfowl such as Pekin ducks. In chickens, AA amyloidosis is observed as amyloid arthropathy. Outbreaks of systemic amyloidosis in flocks of layers are known to be induced by repeated inflammatory stimulation, such as those resulting from multiple vaccinations with oil-emulsified bacterins. Outbreaks of fatal AA amyloidosis were observed in growing chickens in a large scale poultry farm within 3 weeks of vaccination with multiple co-administered vaccines. This study documents the histopathological changes in tissues from these birds. Amyloid deposits were also observed at a high rate in the tissues of apparently healthy chickens. Vaccination should therefore be considered as a potential risk factor for the development of AA amyloidosis in poultry.

Minocycline selectively inhibits M1 polarization of microglia.

Minocycline is commonly used to inhibit microglial activation. It is widely accepted that activated microglia exert dual functions, that is, pro-inflammatory (M1) and anti-inflammatory (M2) functions. The in vivo status of activated microglia is probably on a continuum between these two extreme states. However, the mechanisms regulating microglial polarity remain elusive. Here, we addressed this question focusing on minocycline. We used SOD1(G93A) mice as a model, which exhibit the motor neuron-specific neurodegenerative disease, amyotrophic lateral sclerosis. Administration of minocycline attenuated the induction of the expression of M1 microglia markers during the progressive phase, whereas it did not affect the transient enhancement of expression of M2 microglia markers during the early pathogenesis phase. This selective inhibitory effect was confirmed using primary cultured microglia stimulated by lipopolysaccharide (LPS) or interleukin (IL)-4, which induced M1 or M2 polarization, respectively. Furthermore, minocycline inhibited the upregulation of NF-κB in the LPS-stimulated primary cultured microglia and in the spinal cord of SOD1(G93A) mice. On the other hand, IL-4 did not induce upregulation of NF-κB. This study indicates that minocycline selectively inhibits the microglia polarization to a proinflammatory state, and provides a basis for understanding pathogeneses of many diseases accompanied by microglial activation.

Relationship between pre-radiographic cartilage damage following anterior cruciate ligament injury and biomarkers of cartilage turnover in clinical practice: a cross-sectional observational study.

OBJECTIVE: To determine whether differences in synovial fluid (SF) biomarkers of collagen and proteoglycan turnover are associated with pre-radiographic damage to articular cartilage and menisci following anterior cruciate ligament (ACL) injury and are of clinical value. METHOD: SF samples from ACL injured knees of 108 patients were obtained when damage to cartilages and menisci was evaluated arthroscopically. Concentrations of SF collagenase-generated cleavage neoepitope of type II collagen (C2C) were determined using ELISA and aggrecan-derived disaccharides of chondroitin-4-sulfate (Δdi-C4S), chondroitin-6-sulfate (Δdi-C6S), and keratan sulfate (KS), were measured in SF by High performance liquid chromatography (HPLC). RESULTS: Radiographic examination failed to detect any intra-articular degenerative changes. The number of high-grade cartilage lesions was positively associated with age, duration after injury and the level of C2C, and negatively with the level of KS. There was no association between the number of high-grade cartilage and meniscal lesions. Multivariable logistic regression revealed significant associations of increased C2C (adjusted Odds ratio (OR) of the upper quartile to remainder of 2.49, 95% Confidence interval (CI) = 0.85-7.27) and decreased KS (adjusted OR of the lower quartile to the remainder of 3.32, 95% CI = 1.19-9.24) with the presence of three or more high-grade cartilage lesions, independent of age and duration after injury. The combined impact of increased C2C and decreased KS was 22.8 (95% CI = 1.95-265.9), far exceeding the impact of each independent biomarker. CONCLUSION: Combinations of the C2C and KS as described here may offer greater ability to identify patients with early pre-radiographic high-grade cartilage damage compared to single clinical or biomarker parameters.

Novel Compound Heterozygous Mutations in the Cathepsin K Gene in Japanese Female Siblings with Pyknodysostosis.

We report on female siblings with pyknodysostosis who showed common clinical and radiographic features including disproportionate short stature, dental abnormalities, increased bone density, open fontanelle, and acroosteolysis. Sequence analysis of the cathepsin K (CTSK) gene demonstrated compound heterozygous mutations (935 C>T, A277V and 489 G>C, R122P) in the affected siblings and a heterozygous mutation in their parents. The former missense mutation has previously been reported in 6 unrelated patients, and the latter seemed to be a novel mutation. Atomic model assessment of the CTSK gene revealed that the R122P mutant could disrupt hydrogen bonds binding with chondroitin 4-sulfate leading to a decrease in the collagen-degrading activity of cathepsin K.