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Background: Being overweight or obese is associated with poor outcomes and an increased risk of failure after rotator cuff (RC) surgery. However, the effect of obesity on enthesis healing has not been well characterized. Hypotheses: Diet-induced obesity (DIO) would result in inferior enthesis healing in a rat model of RC repair, and a dietary intervention in the perioperative period would improve enthesis healing. Study Design: Controlled laboratory study. Methods: Male Sprague-Dawley rats were divided into 3 weight-matched groups (n = 26 per group): control diet (CD), high-fat diet (HFD), or HFD until surgery and then CD thereafter (HF-CD). After 12 weeks, the left supraspinatus tendon was detached, followed by immediate repair. Animals were sacrificed, and RCs were harvested at 2 and 12 weeks after surgery for biomechanical and histological evaluations. Metabolic end points were assessed using dual-energy X-ray absorptiometry and plasma analyses. Results: DIO was established in the HFD and HF-CD groups before surgery and subsequently reversed in the HF-CD group after surgery. At 12 weeks after surgery, the body fat percentage ( P = .0021) and plasma leptin concentration ( P = .0025) were higher in the HFD group compared with the CD group. Histologically, the appearance of the repaired entheses was poorer in both the HFD and HF-CD groups compared with the CD group at 12 weeks after surgery, with semiquantitative scores of 6.20 ( P = .0078), 4.98 ( P = .0003), and 8.68 of 15, respectively. The repaired entheses in the HF-CD group had a significantly lower load to failure ( P = .0278) at 12 weeks after surgery compared with the CD group, while the load to failure in the HFD group was low but not significantly different ( P = .0960). There were no differences in the biomechanical and histological results between the groups at 2 weeks after surgery. Body mass at the time of surgery, plasma leptin concentration, and body fat percentage were negatively correlated with histology scores and plasma leptin concentration was correlated with load to failure at 12 weeks after surgery. Conclusion: DIO impaired enthesis healing in this rat RC repair model, with inferior biomechanical and histological outcomes. Restoring a normal weight with dietary changes after surgery did not improve healing outcomes. Clinical Relevance: Obesity is a potentially modifiable factor that impairs RC healing and increases the risk of failure after surgery. Exploring interventions that improve the metabolic state of obese patients and counseling patients appropriately about their modest expectations after repair should be considered.

Monocarboxylate transporter 4 (MCT4) is a high‑capacity lactate transporter in cells and the alteration in MCT4 expression harms cellular survival. The present study investigated whether hypothermia affects tumor necrosis factor‑α (TNF‑α) and MCT4 immunoreactivity in the subfield cornu ammonis 1 (CA1) following cerebral ischemia/reperfusion (IR) in gerbils. Hypothermia was induced for 30 min before and during ischemia. It was found that IR‑induced death of pyramidal neurons was markedly augmented and occurred faster under hyperthermia than under normothermia. TNF‑α immunoreactivity in the pyramidal cells started to increase at 3 h after IR and peaked at 1 day after IR under normothermia. However, in hyperthermic control and sham operated gerbils, TNF‑α immunoreactivity was significantly increased compared with the normothermic gerbils, and IR under hyperthermia caused a more rapid and significant increase in TNF‑α immunoreactivity in pyramidal neurons than under normothermia. In addition, in the normothermic gerbils, MCT4 immunoreactivity began to decrease in pyramidal neurons from 3 h after IR and markedly increased at 1 and 2 days after IR. On the other hand, MCT4 immunoreactivity in pyramidal neurons of the hyperthermic gerbils was significantly increased from 3 h after IR, maintained until 1 day after IR and markedly decreased at 2 days after IR. These results indicate that acceleration of IR‑induced neuronal death under hyperthermia might be closely associated with early alteration of TNF‑α and MCT4 protein expression in the gerbil hippocampus after IR.

Nuclear receptor related 1 protein (Nurr1), a member of the nuclear receptor 4 family of orphan nuclear receptors, has been reported to display anti‑inflammatory properties. The present study investigated the alteration of Nurr1 immunoreactivity in the gerbil hippocampus proper following 5 min of transient global cerebral ischemia. In sham operated gerbils, Nurr1 immunoreactivity was observed in pyramidal neurons in all cornu ammonis 1‑3 (CA1‑3) subfields of the hippocampus proper. In ischemia‑operated gerbils, Nurr1 immunoreactivity was altered in the CA1 subfield. Nurr1 immunoreactivity in CA1 pyramidal neurons gradually decreased until 2 days post‑ischemia, and, at 4 days post‑ischemia, Nurr1 immunoreactivity was concentrated in CA1 pyramidal neurons. Additionally, Nurr1 immunoreactivity was newly expressed in microglia in the CA1 subfield at 4 days post‑ischemia. Conversely, in the CA2/3 subfield, time‑dependent alteration of Nurr1 immunoreactivity was not identified at any time following ischemia. These results indicated that the alteration of Nurr1 expression in the CA1 subfield in the hippocampus may be associated with the death of CA1 pyramidal neurons.

Bisphosphonates are used in treating patients with breast cancer. In vitro studies have shown that bisphosphonates act directly on tumour cells, inhibiting cell proliferation and inducing apoptosis. In most such studies, drugs were added to culture media exposing cells to high bisphosphonate concentrations in solution. However, since bisphosphonates bind to bone hydroxyapatite with high affinity and remain bound for very long periods of time, these experimental systems are not an optimal model for the action of the drugs in vivo. The aim of this study was to determine whether bone-bound zoledronate has direct effects on adjacent breast cancer cells. Bone slices were pre-incubated with bisphosphonate solutions, washed, and seeded with cells of the breast cancer cell lines, MCF7 or MDA-MB-231. Proliferation was assessed by cell counts and thymidine incorporation for up to 72 h. Inhibition of the mevalonate pathway was tested by measuring the levels of unprenylated Rap1A, and apoptosis was examined by the presence of cleaved caspase-8 on western blots. The proliferation rate of breast cancer cells on zoledronate-treated bone was significantly lower compared to cells on control bone. Other bisphosphonates showed a similar inhibitory effect, with an order of potency similar to their clinical potencies. Unprenylated Rap1A accumulated in MCF7 cells on zoledronate-treated bone, suggesting zoledronate acted through the inhibition of the mevalonate pathway. Accumulation of cleaved caspase-8 in MDA-MB-231 cells on bisphosphonate-treated bone indicated increased apoptosis in the cells. In conclusion, bone-bound zoledronate inhibits breast cancer cell proliferation, an activity that may contribute to its clinical anti-tumour effects.

Spinal cord ischemia can result from cardiac arrest. It is an important cause of severe spinal cord injury that can lead to serious spinal cord disorders such as paraplegia. Hypothermia is widely acknowledged as an effective neuroprotective intervention following cardiac arrest injury. However, studies on effects of hypothermia on spinal cord injury following asphyxial cardiac arrest and cardiopulmonary resuscitation (CA/CPR) are insufficient. The objective of this study was to examine effects of hypothermia on motor deficit of hind limbs of rats and vulnerability of their spinal cords following asphyxial CA/CPR. Experimental groups included a sham group, a group subjected to CA/CPR, and a therapeutic hypothermia group. Severe motor deficit of hind limbs was observed in the control group at 1 day after asphyxial CA/CPR. In the hypothermia group, motor deficit of hind limbs was significantly attenuated compared to that in the control group. Damage/death of motor neurons in the lumbar spinal cord was detected in the ventral horn at 1 day after asphyxial CA/CPR. Neuronal damage was significantly attenuated in the hypothermia group compared to that in the control group. These results indicated that therapeutic hypothermia after asphyxial CA/CPR significantly reduced hind limb motor dysfunction and motoneuronal damage/death in the ventral horn of the lumbar spinal cord following asphyxial CA/CPR. Thus, hypothermia might be a therapeutic strategy to decrease motor dysfunction by attenuating damage/death of spinal motor neurons following asphyxial CA/CPR.

Erigeron annuus (L.) PERS. (EALP) and Clematis mandshurica RUPR. (CMR) have been used in traditional remedies due to their medicinal effects. Recently, we reported that pretreatment with 200 mg/kg of YES-10® (a combination of extracts from leaves of EALP and CMR) displayed neuroprotective effects against brain ischemia and reperfusion injury. The present study analyzed the major ingredients of YES-10® and investigated whether neuroprotection from YES-10® was dependent upon antioxidant effects in the cornu ammonis 1 (CA1) field in the gerbil hippocampus, after transient forebrain ischemia for 5 min. YES-10® was demonstrated to predominantly contain scutellarin and chlorogenic acid. Pretreatment with YES-10® significantly increased protein levels and the immunoreactivity of copper/zinc-superoxide dismutase (SOD1) and manganese-superoxide dismutase (SOD2) was in the pyramidal neurons of the hippocampal CA1 field when these were examined prior to transient ischemia induction. The increased SODs in CA1 pyramidal neurons following YES-10® treatment were maintained after ischemic injury. In this case, the CA1 pyramidal neurons were protected from ischemia-reperfusion injury. Oxidative stress was significantly attenuated in the CA1 pyramidal neurons, and this was determined by 4-hydroxy-2-nonenal immunohistochemistry and dihydroethidium histofluorescence staining. Taken together, the results indicated that YES-10® significantly attenuated transient ischemia-induced oxidative stress and may be utilized for developing a protective agent against ischemic insults.

Oxcarbazepine (OXC), a voltage-gated sodium channel blocker, is an antiepileptic medication and used for the bipolar disorders treatment. Some voltage-gated sodium channel blockers have been demonstrated to display strong neuroprotective properties in models of cerebral ischemia. However, neuroprotective effects and mechanisms of OXC have not yet been reported. Here, we investigated the protective effect of OXC and its mechanisms in the cornu ammonis 1 subfield (CA1) of gerbils subjected to 5 min of transient global cerebral ischemia (tGCI). tGCI led to death of most pyramidal neurons in CA1 at 5 days after ischemia. OXC (100 and 200 mg/kg) was intraperitoneally administered once at 30 min after tGCI. Treatment with 200 mg/kg, not 100 mg/kg OXC, significantly protected CA1 pyramidal neurons from tGCI-induced injury. OXC treatment significantly decreased superoxide anion production, 4-hydroxy-2-nonenal and 8-hydroxyguanine levels in ischemic CA1 pyramidal neurons. In addition, the treatment restored levels of superoxide dismutases, catalase, and glutathione peroxidase. Furthermore, the treatment distinctly inhibited tGCI-induced microglia activation and significantly reduced levels of pro-inflammatory cytokines (interleukin-1β and tumor necrosis factor-α). In particular, OXC treatment significantly enhanced expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream protein heme oxygenase-1 in ischemic CA1. The neuroprotective effects of OXC were abolished by brusatol (an inhibitor of Nrf2). Taken together, these results indicate that post-treatment of OXC can display neuroprotection against brain injuries following ischemic insults. This neuroprotection may be displayed by attenuation of oxidative stress and neuroinflammation, which can be mediated by activation of Nrf2 pathway.

Transient brain ischemia triggers selective neuronal death/loss, especially in vulnerable regions of the brain including the hippocampus. Laminarin, a polysaccharide originating from brown seaweed, has various pharmaceutical properties including an antioxidant function. To the best of our knowledge, few studies have been conducted on the protective effects of laminarin against ischemic injury induced by ischemic insults. In this study, we histopathologically investigated the neuroprotective effects of laminarin in the Cornu Ammonis 1 (CA1) field of the hippocampus, which is very vulnerable to ischemia-reperfusion injury, following transient forebrain ischemia (TFI) for five minutes in gerbils. The neuroprotective effect was examined by cresyl violet staining, Fluoro-Jade B histofluorescence staining and immunohistochemistry for neuronal-specific nuclear protein. Additionally, to study gliosis (glial changes), we performed immunohistochemistry for glial fibrillary acidic protein to examine astrocytes, and ionized calcium-binding adaptor molecule 1 to examine microglia. Furthermore, we examined alterations in pro-inflammatory M1 microglia by using double immunofluorescence. Pretreatment with 10 mg/kg laminarin failed to protect neurons in the hippocampal CA1 field and did not attenuate reactive gliosis in the field following TFI. In contrast, pretreatment with 50 or 100 mg/kg laminarin protected neurons, attenuated reactive gliosis and reduced pro-inflammatory M1 microglia in the CA1 field following TFI. Based on these results, we firmly propose that 50 mg/kg laminarin can be strategically applied to develop a preventative against injuries following cerebral ischemic insults.

Clematis mandshurica RUPR. (CMR) and Erigeron annuus (L.) PERS. (EALP) have pharmacological effects including anti-inflammatory activity and been used in traditional medicines in Asia. However, neuroprotective effects of CMR and/or EALP extracts against brain ischemic insults have never been addressed. Thus, the aim of this study was to examine neuroprotective effects of YES-10, a combination of extracts from CMR and EALP (combination ratio, 1:1), in the hippocampus following ischemia/reperfusion in gerbils. Protection of neurons was investigated by cresyl violet staining, fluoro-jade B histofluorescence staining and immunohistochemistry for neuronal nuclei. In addition, attenuation of gliosis was studied by immunohistochemistry for astrocytic and microglial markers. Treatments with 50 or 100 mg/kg YES-10 failed to protect neurons in the hippocampus after ischemia/reperfusion injury. However, administration of 200 mg/kg YES-10 protected neurons from ischemia/reperfusion injury and attenuated reactive gliosis. These findings strongly suggest that a combination of extracts from CMR and EALP can be used as a prevention approach/drug against brain ischemic damage.

BACKGROUND/AIMS The objective of this study was to determine the efficacy and safety of add-on therapy with certolizumab pegol (CZP) in active rheumatoid arthritis (RA) patients of a single ethnicity. METHODS In this 24-week, phase 3, randomized, double-blind, placebo-controlled trial, eligible patients (n = 127) were randomized 2:1 to subcutaneous CZP + methotrexate (MTX; 400 mg at week 0, 2, and 4 followed by 200 mg every 2 weeks) or placebo + MTX. RESULTS At week 24, the American College of Rheumatology criteria for 20% (ACR20) response rate was significantly greater with CZP + MTX than with placebo (66.7% vs. 27.5%, p < 0.001). Differences in ACR20 response rates for CZP vs. placebo were significant from week 1 (p < 0.05) and remained significant through week 24. The CZP group reported significant improvement in physical function and disability compared to the placebo group (p < 0.001) at week 24, as assessed by Korean Health Assessment Questionnaire-Disability Index (KHAQ-DI). Post hoc analysis indicated that the proportion of patients who had ACR70 responses, Disease Activity Score 28 (DAS28) low disease activity, and DAS28 remission at week 24 was greater in CZP + MTX-treated patients who achieved a decrease in DAS28 ≥ 1.2 (43.8%) at week 4 than in nonresponders. Among 18 (22.2%) and 14 patients (35.0%) in CZP and placebo groups who had latent tuberculosis (TB), none developed active TB. Most adverse events were mild or moderate. CONCLUSION CZP treatment combined with MTX in active RA patients with moderate to severe disease activity and an inadequate response to MTX resulted in rapid onset of efficacy, which is associated with better clinical outcome at week 24 and has an acceptable safety profile, especially in an intermediate TB-burden population.

INTRODUCTION Congenital myopathies are muscle diseases characterized by specific histopathologic features, generalized hypotonia from birth, and perinatal complications, although some cases develop during childhood or, rarely, in adulthood. We undertook this study to characterize congenital myopathies among patients registered at our institution. METHODS Clinical, histopathologic, and genetic features were evaluated in 34 patients recruited for this study. RESULTS The majority of patients experienced a childhood onset, and no disease-related mortality was recorded during follow-up. Functional outcomes were no better for those with late-onset disease, indicating later disease progression can be significant. Nemaline myopathy was the most frequent pathology, followed by central core disease and centronuclear myopathy. Among the 18 (54.5%) genetically confirmed patients, NEB and RYR1 mutations were the most common, followed by DNM2 mutations. DISCUSSION This study shows features not previously reported and suggests that congenital myopathy should be considered an important issue among adult patients. Muscle Nerve 58: 235-244, 2018.

Nemaline myopathy (NM), the most common of the congenital myopathies, is caused by various genetic mutations. In this study, we attempted to identify the causative mutations of NM and to reveal any specific genotype-phenotype relationship in Korean patients with this disease. We investigated the clinical features and genotypes in 15 pathologically diagnosed NM patients, using whole exome sequencing (WES) combined with targeted sequencing and array-based comparative genomic hybridization. This strategy revealed pathogenic causative mutations in seven patients (46.7%), among whom mutations in the nebulin gene (NEB) were the most frequent (5 patients, 33.3%). Copy number variation (CNV) abnormality in NEB was not observed in any of our patients. In those with NEB-associated NM, the clinical spectrum was highly variable regardless of the mutation type. However, the majority of patients showing anterior lower leg weakness were associated with mutations located between NEB exons 166 and 177. We concluded that the combination of WES and targeted Sanger sequencing is an effective strategy for analyzing genotypes in patients with NM, and that CNV in NEB may not be a frequent cause of this disease among Koreans.

Introduction Several clinical studies using tacrolimus revealed reasonable therapeutic mechanisms and efficacy in patients with myasthenia gravis (MG). However, long-period studies in a large number of patients with MG are limited; therefore, the aim of this study was to investigate the therapeutic efficacies and safety of tacrolimus in patients with MG during a 12-month follow-up period. Methods Tacrolimus was administered to 150 patients with MG who were recruited based on the inclusion criteria. Fifteen medical centers in Korea participated in this study. The efficacy of tacrolimus was assessed using MG composite scales (MGCS) and the prednisolone-sparing effect. And the adverse drug reactions (ADRs) of tacrolimus were monitored in each patient from the beginning of tacrolimus treatment to the end of the follow-up period. Results After starting tacrolimus, the 32 patients were affected by ADRs, and consequentially 134 patients of the enrolled patients were followed up for 12 months. They showed that the mean prednisolone dosage significantly decreased (6.1 ± 7.6 mg/day), compared to that in the baseline (11.3 ± 9.5 mg/day), and MGCS significantly improved after 12 months of tacrolimus treatment, compared to that at the baseline. Conclusions Our study showed that tacrolimus would be an effective immunosuppressant as an initial therapeutic agent in patients with MG; in addition, it showed tolerable safety profiles during the 12-month follow-up evaluation.

Cell-cell communication plays a crucial role in bone development and maintenance, in health and disease. Our knowledge of the signalling pathways that regulate the communication of osteoblasts, osteoclasts and osteocytes is improving with time, giving us a better understanding of the regulation of the bone remodelling process. Bone cell communication has wider significance than the local tissue environment, however, with bone derived factors playing a role in whole-body homeostasis, including energy metabolism and kidney function. Similarly, there are a range of systemic conditions which affect the maintenance of bone mass. Understanding these complex interactions, the autocrine, paracrine and endocrine signalling involved in the regulation of bone health is paramount to a better understanding of bone disease, and ultimately developing targets for pharmaceutical intervention.

Oxcarbazepine, an antiepileptic drug, has been reported to modulate voltage-dependent sodium channels, and it is commonly used in epilepsy treatment. In this study, we investigated the neuroprotective effect of oxcarbazepine in the hippocampus after transient ischemia in gerbils. Gerbils randomly received oxcarbazepine 100 or 200 mg/kg before and after transient ischemia. We examined its neuroprotective effect in the cornu ammonis 1 subfield of the gerbil hippocampus at 5 days after transient ischemia by using cresyl violet staining, neuronal nuclei immunohistochemistry and Fluoro-Jade B histofluorescence staining for neuroprotection, and by using glial fibrillary protein and ionized calcium-binding adapter molecule 1 immunohistochemistry for reaction of astrocytes and microglia, respectively. Pre- and post-treatment with 200 mg/kg of oxcarbazepine, but not 100 mg/kg of oxcarbazepine, protected pyramidal neurons of the cornu ammonis 1 subfield from transient ischemic damage. In addition, pre- and post-treatment with oxcarbazepine (200 mg/kg) significantly ameliorated astrocytes and microglia activation in the ischemic cornu ammonis 1 subfield. In brief, our current results indicate that post-treatment as well as pre-treatment with 200 mg/kg of oxcarbazepine can protect neurons from ischemic insults via attenuation of the glia reaction.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels have been known to participate in the regulation of neuronal excitability, synaptic transmission and long-term potentiation in the hippocampus. The present study investigated transient ischemia-induced changes of HCN1 and HCN2 expressions in the Cornu Ammonis 1 (CA1) subfield of the hippocampus in gerbils subjected to 5 min transient global cerebral ischemia (tgCI). Neuronal death was exhibited in pyramidal neurons of the striatum pyramidale in the CA1 subfield 4 days after tgCI. HCN1 and HCN2 immunoreactivities were demonstrated in intact CA1 pyramidal neurons, and were transiently and markedly increased in the CA pyramidal neurons at 6 h after ischemia. Thereafter, they gradually decreased in a time-dependent manner. A total of 4 days after ischemia, HCN1 and HCN2 immunoreactivities were barely detected in the CA1 pyramidal neurons; however, HCN1 and HCN2 were began to be expressed in pericytes and astrocytes at 4 days after ischemia. The results indicated that HCN1 and HCN2 expression levels were apparently changed in the gerbil hippocampal CA1 subfield following tgCI and suggested that ischemia-induced alterations in HCN1 and HCN2 expression levels may be closely associated with the death of CA1 pyramidal neurons following 5 min of tgCI.

Histone-binding protein RbAp48 has been known to be involved in histone acetylation, and epigenetic alterations of histone modifications are closely associated with the pathogenesis of ischemic reperfusion injury. In the current study, we investigated chronological change of RbAp48 expression in the hippocampus following 5 min of transient ischemia in gerbils. RbAp48 expression was examined 1, 2, 5, and 10 days after transient ischemia using immunohistochemistry. In sham operated gerbils, RbAp48 immunoreactivity was strong in pyramidal and non-pyramidal cells in the hippocampus. After transient ischemia, RbAp48 immunoreactivity was changed in the cornu ammonis 1 subfield (CA1), not in CA2/3. RbAp48 immunoreactivity in CA1 pyramidal neurons was gradually decreased and not detected at 5 and 10 days after ischemia. RbAp48 immunoreactivity in non-pyramidal cells was maintained until 2 days post-ischemia and significantly increased from 5 days post-ischemia. Double immunohistofluorescence staining revealed that RbAp48 immunoreactive non-pyramidal cells were astrocytes. At 5 days post-ischemia, death of pyramidal neurons occurred only in the CA1. These results showed that RbAp48 immunoreactivity was distinctively altered in pyramidal neurons and astrocytes in the hippocampal CA1 following 5 mins of transient ischemia. Ischemia-induced change in RbAp48 expression may be closely associated with neuronal death and astrocyte activation following 5 min of transient ischemia.

Neuronal death and reactive gliosis are major features of brain tissue damage following transient global cerebral ischemia (tgCI). This study investigated long-term changes in neuronal death and astrogliosis in the gerbil hippocampus for 180 days after 5 min of tgCI. A massive loss of pyramidal neurons was found in the hippocampal CA1 area (CA1) area between 5 and 30 days after tgCI by Fluoro-Jade B (FJB, a marker for neuronal degeneration) histofluorescence staining, but pyramidal neurons in the CA2/3 area did not die. The reaction of astrocytes (astrogliosis) was examined by glial fibrillary acidic protein (GFAP) immunohistochemistry. Morphological change or degeneration (death) of the astrocytes was found in the CA1 area after tgCI, but, in the CA2/3 area, astrogliosis was hardly shown. GFAP immunoreactive astrocytes in the CA1 area was significantly increased in number with time and peaked at 30 days after tgCI, and they began to be degenerated or dead from 40 days after tgCI. The effect was examined by double immunofluorescence staining for FJB and GFAP. The number of FJB/GFAP+ cells (degenerating astrocytes) was gradually increased with time after tgCI. At 180 days after tgCI, FJB/GFAP+ cells were significantly decreased, but FJB+ cells (dead astrocytes) were significantly increased. In brief, 5 min of tgCI induced a progressive degeneration of CA1 pyramidal neurons from 5 until 30 days with an increase of reactive astrocytes, and, thereafter, astrocytes were degenerated with time and dead at later times. This phenomenon might be shown due to the death of neurons.

Ischaemic brain damage induces autoimmune responses, including the production of autoantibodies with potential neuroprotective effects. Platelets share unexplained similarities with neurons, and the formation of anti-platelet antibodies has been documented in neurological disorders. The aim of this study was to investigate the presence of anti-platelet antibodies in the peripheral blood of patients after ischaemic stroke and determine any clinical correlations. Using a flow cytometry-based platelet immunofluorescence method, we detected platelet-reactive antibodies in 15 of 48 (31%) stroke patients and two of 50 (4%) controls (p &lt, 0.001). Western blotting revealed heterogeneous reactivities with platelet proteins, some of which overlapped with brain proteins. Stroke patients who carried anti-platelet antibodies presented with larger infarcts and more severe neurological dysfunction, which manifested as higher scores on the National Institutes of Health Stroke Scale (NIHSS, p = 0.009), but they had a greater recovery in the NIHSS by the time of hospital discharge (day 7 &plusmn, 2) compared with antibody-negative patients (p = 0.043). Antibodies from stroke sera reacted more strongly with activated platelets (p = 0.031) and inhibited platelet aggregation by up to 30.1 &plusmn, 2.8% (p &lt, 0.001), suggesting the potential to interfere with thrombus formation. In conclusion, platelet-reactive antibodies can be found in patients soon after ischaemic stroke and correlate with better short-term outcomes, suggesting a potential novel mechanism limiting thrombosis.

To date, hypothermia has focused on improving rates of resuscitation to increase survival in patients sustaining cardiac arrest (CA). Towards this end, the role of body temperature in neuronal damage or death during CA needs to be determined. However, few studies have investigated the effect of regional temperature variation on survival rate and neurological outcomes. In this study, adult male rats (12 week-old) were used under the following four conditions: (i) whole-body normothermia (37 ± 0.5 °C) plus (+) no asphyxial CA, (ii) whole-body normothermia + CA, (iii) whole-body hypothermia (33 ± 0.5 °C)+CA, (iv) body hypothermia/brain normothermia + CA, and (v) brain hypothermia/body normothermia + CA. The survival rate after resuscitation was significantly elevated in groups exposed to whole-body hypothermia plus CA and body hypothermia/brain normothermia plus CA, but not in groups exposed to whole-body normothermia combined with CA and brain hypothermia/body normothermia plus CA. However, the group exposed to hypothermia/brain normothermia combined with CA exhibited higher neuroprotective effects against asphyxial CA injury, i.e. improved neurological deficit and neuronal death in the hippocampus compared with those involving whole-body normothermia combined with CA. In addition, neurological deficit and neuronal death in the group of rat exposed to brain hypothermia/body normothermia and CA were similar to those in the rats subjected to whole-body normothermia and CA. In brief, only brain hypothermia during CA was not associated with effective survival rate, neurological function or neuronal protection compared with those under body (but not brain) hypothermia during CA. Our present study suggests that regional temperature in patients during CA significantly affects the outcomes associated with survival rate and neurological recovery.

FHL1-related myopathies are clinically heterogeneous, involving skeletal and cardiac muscles. Overlapping clinical features include joint contractures, rigid spine, scapuloperoneal weakness and cardiac diseases. Histopathologically, reducing bodies are the most characteristic finding, but not present in all FHL1-related cases. Non-specific dystrophic pathology without reducing body is usual in the forms of X-linked myopathy with postural muscle atrophy, Emery-Dreifuss muscular dystrophy and isolated hypertrophic cardiomyopathy. Here, we describe a patient with mild weakness with ankle contracture. We finally concluded he has a FHL1-related myopathy at an extreme end of phenotypic spectrum of FHL1 myopathy, which one might miss to recognize as a form of myopathy. The genetic variant was detected by whole exome sequencing, and its pathogenicity was clearly confirmed with pathological and biochemical studies. This is the first FHL1 case with a mildest phenotype backed by biochemical/genetic evidence. This report will help clinicians hesitating to further evaluate mild cases to better correlate the genotype to the phenotype.

OBJECTIVES: This study aims to examine the effects of tocilizumab therapy on serum levels of interleukin (IL)-33 and IL-6 in rheumatoid arthritis (RA) patients. PATIENTS AND METHODS: Interleukin-33 and IL-6 levels in serum samples from 83 RA patients (10 males, 73 females; mean age 51.9 years; range, 26 to 77 years) and 12 healthy controls (2 males, 10 females; mean age 52.2 years; range, 39 to 62 years) were compared by cross-sectional, case control analysis. Of the RA patients, 40 received 24 weeks of tocilizumab therapy and were assigned to the prospective cohort. These 40 patients were then divided into two subgroups as responders and non-responders according to the American College of Rheumatology (ACR) 20. The remaining 43 RA patients did not receive tocilizumab therapy. Serum cytokine levels were analyzed at baseline and after 24 weeks of tocilizumab therapy in these patients. RESULTS: Interleukin-33 and IL-6 concentrations were significantly higher in RA patients than in healthy controls (p

Background:The treatment of symptomatic acromioclavicular joint (ACJ) injury in the rotator cuff (RC) tear has not been well clarified.Purpose:To compare the clinical results between patients who had distal clavicle resection (DCR) and those who did not during RC repair.Study Design:Randomized controlled trial; Level of evidence, 1.Methods:From August 2008 to December 2009, a total of 56 consecutive patients (58 shoulders) were included. All patients had either a full-thickness or high-grade (>50%) RC tear, ACJ tenderness, arthritic change visible on plain radiographs, and a positive ACJ lidocaine injection test the day before surgery. Patients were randomized into 2 groups: DCR and RC repair (DCR+RCR group) and RC repair only (isolated RCR). Evaluation was performed preoperatively, at 6 months postoperatively, and at a final follow-up a minimum of 24 months postoperatively using the American Shoulder and Elbow Surgeons (ASES) score, the Constant shoulder score, range of motion examination, and pain visual analog scale (VAS).Results:After simple randomization, 26 shoulders were allocated in the DCR+RCR group, and 32 were placed in the isolated RCR group. Five shoulders in the DCR+RCR group and 6 in the isolated RCR group were excluded from analysis due to loss of follow-up. Therefore, the evaluation was performed for 21 shoulders in the DCR+RCR group and 26 shoulders in the isolated RCR group. The mean follow-up period was 44.2 months in the DCR+RCR group and 44.0 months in the isolated RCR group. There were no differences in age, sex, symptom duration, RC tear size, or preoperative ASES, Constant, and VAS scores between the 2 groups ( P > .05). At final follow-up, the ASES, Constant, and VAS scores were significantly improved in both groups ( P < .001). There were no differences in ASES, Constant, and VAS scores between the 2 groups at final follow-up ( P > .05), and there was no difference in residual ACJ tenderness (7 in the DCR+RCR group and 5 in the isolated RCR group) between the 2 groups ( P = .270).Conclusion:There was no difference in the clinical evaluations between the combined arthroscopic DCR and RCR group and the isolated RCR group at a minimum 24-month follow-up. Arthroscopic DCR should be carefully considered in patients who have symptomatic ACJ arthritis with RC tears.

Purpose The purpose of this study was to compare morphologic features of the acromion after 2 different repair methods (single-row [SR] repair with a minimum of 4 knots and suture-bridge [SB] repair with minimal knots) in medium to large rotator cuff tears. Methods From May 2005 to July 2012, 1,693 rotator cuff repairs were performed, among them medium to large tears requiring more than 2 anchors for repair; those who had 6-month postoperative magnetic resonance imaging (MRI) scans were included (221 shoulders). They were divided into 2 groups; group A (SR repair) and group B (SB repair). Acromial morphologic characteristics were evaluated using MRI 6 months postoperatively. An acromial defect was defined as an irregular defect or erosion on the flat acromion. Clinical measurements were performed using the American Shoulder and Elbow Surgeons (ASES) score, Constant score, visual analogue scale (VAS) pain score, and range of motion (ROM). Results Erosion in the acromion was observed in 2 of 118 patients (1.7%) in group A and in 1 of 103 (1%) patients in group B. There was no statistically significant difference between the 2 groups ( P = .796). A statistically significant improvement was observed in the clinical scores measured ( P = .0043). ROM was not fully recovered to the preoperative level at 6 months postoperatively. Acromioplasty was performed in 2 of 3 patients with acromial erosion. There was acromial erosion in one patient in group A without performing subacromial decompression. Conclusions Our study showed that there was no difference in acromial erosion in high-profile knots made by an SR compared with double-row (DR) SB low-profile repairs. Level of Evidence Level III, retrospective comparative study.

The present study aimed to investigate the vitamin D status in patients with early inflammatory arthritis (EIA). We conducted a retrospective study among patients who presented with EIA at the outpatient rheumatology clinic of a tertiary referral center between March 2012 and February 2013. In total, 101 subjects with EIA (≥1 swollen joint and symptom duration of ≤6 months, not explained by another disease) and 101 healthy controls matched for age, sex, and the month of serum vitamin D measurements were enrolled. Serum 25-hydroxy vitamin D (25-OHD) concentrations were assessed by radioimmunoassay. Vitamin D “deficiency” and “severe deficiency” were defined as serum 25-OHD levels

Anticipation is a phenomenon characterized by decreasing age at onset and increasing severity of symptoms of a disease in successive generations within a pedigree. Anticipation mostly occurs in neurodegenerative diseases with expansion of unstable trinucleotide repeats. However, it has not been previously pointed out in episodic ataxia type 2 (EA2). Clinical and genetic analyses were performed in nine members from three consecutive generations of a Korean family with EA2. We performed a polymerase chain reaction (PCR)-based direct sequence analysis of all coding regions of CACNA1A using genomic DNA. The clinically affected family members showed recurrent vertigo, interictal nystagmus, and childhood epilepsy. There is a decrease in the age onset (possible genetic anticipation) in three succeeding generations of the family. Genetic analysis identified a splice site mutation (p.Val1465Glyfs13X) and normal trinucleotide repeats in CACNA1A in all clinically affected and one unaffected members. Recognizing anticipation would aid in genetic counseling in EA2.

We applied a comprehensive set of clinical and radiological criteria for the diagnosis of hypochondroplasia (HCH) in 160 patients with short stature 58 of whom were diagnosed to have HCH. Taking into account the genotypic and phenotypic variations in HCH, we conducted a study with these 58 patients and tested them for mutations in the fibroblast growth factor receptor 3 (FGFR3) and the short stature homeobox (SHOX) gene. We characterized the phenotypes by clinical and radiologic findings. In the patients with HCH, 19 were included in Group I (FGFR3 mutations-mutations of definite significance), and 39 were in Group II (6 SHOX mutations and 33 negative for disease-causing FGFR3 mutations). The clinical findings were similar in two groups regardless of the presence or absence of mutations. More than 95% of the patients had mesomelic proportions. In Group I, the radiological findings of mesomelia of upper and lower limbs and, L1/L4 ratio in anterior-posterior and lateral view were more typical than in Group II. This study proposes comprehensive clinico-radiological criteria for the diagnosis of HCH, which would help in detecting the true incidence of this underdiagnosed condition. The presence of SHOX mutations suggest genotypic-phenotypic overlap between HCH and Leri-Weill dyschondrosteosis, though further investigation is needed to effectively elucidate the importance of these mutations. Also, the 56.9% of HCH patients with negative mutations for FGFR3 suggests that there are other undiscovered gene mutations associated with this phenotypic entity.

Interleukin (IL) 17 plays an important role in synovial inflammation and bone destruction in rheumatoid arthritis (RA), while IL-27 exerts a regulatory role in T cell-mediated immune responses. Our aim was to study the influence of IL-17 on IL-27 production in RA. Following injection of IL-17 in the knee joint of CIA mice, synovium was examined for IL-17 and IL-27 expression by western blot, real-time PCR, and immunohistochemistry. IL-17 and IL-27 levels were measured by ELISA in mouse bone marrow-derived dendritic cells (BM-DCs) and in synovial fluid (SF) macrophages from RA patients. IL-17 exacerbated disease progression in CIA mice. Histological analysis showed increased pannus formation associated with cartilage and bone erosion following injection with IL-17. The expression of IL-27 was increased in CIA mice. The expression of IL-17 and IL-27 was increased more in IL-17-injected CIA mice than in control mice. The majority of cells expressing IL-27 were co-localized with synovial macrophages. Increased expression of IL-27 by application of recombinant IL-17 was confirmed in CIA BM-DCs and in SF macrophages from RA patients. IL-17 enhanced expression of IL-27 in synovial macrophages from RA patients and CIA mice, indicating an interaction between IL-17 and IL-27 as an autoregulatory mechanism.

Objective To compare the frequency of osteoporosis and bone mineral density (BMD) below the expected range for age between female patients with rheumatoid arthritis (RA) and healthy subjects and to determine risk factors for bone loss in female patients with RA. Method Two hundred and ninety-nine patients with RA and 246 age-matched healthy subjects were included in this study. BMD in the lumbar spine, femoral neck and total hip were measured with dual-energy X-ray absorptiometry. A T-score of −2.5 or lower in postmenopausal women was defined as osteoporosis, and a Z-score −2.0 or lower in females prior to menopause was defined as below the expected range for age. Result The frequency of osteoporosis in the RA patients (22.1%) was significantly higher than in healthy subjects (11.4%) at either the spine or hip (P = 0.014). The occurrence of BMD below the expected range for age in RA patients (7.8%) was also significantly higher than in healthy subjects (1.0%, P = 0.015). In 299 female patients with RA, higher age, lower body mass index and postmenopausal status were significantly associated with the lumbar spine and hip BMD reduction. Of disease-related variables, glucocorticoid use was independently associated with reduction of hip BMD. Conclusion The prevalence of osteoporosis in the RA patients was 1.9 times higher than in healthy subjects. Glucocorticoid use was a risk factor for generalized bone loss in female RA patients.

IL-17 plays important roles in synovial inflammation and bone destruction in the mouse model of autoimmune arthritis and in rheumatoid arthritis (RA). Cadherin-11 determines the behavior of synovial cells in their proinflammatory and destructive tissue response in inflammatory arthritis, and promotes the invasive behavior of fibroblast-like synoviocytes (FLS). The purpose of this study was to examine the effect of IL-17 on the expression of cadherin-11 in autoimmune experimental arthritis and in RA synovium. The severity of synovial inflammation and bone destruction were examined in IL-17-injected knee joints of mice with collagen-induced arthritis (CIA). Cadherin-11 expression was examined in the synovium of mice with CIA, of IL-1 receptor antagonist (IL-1Ra)-deficient mice and of patients with RA and osteoarthritis (OA). Cadherin-11 expression was also examined in the synovium of IL-17 injected knee joints from CIA mice and in IL-17-stimulated FLS of CIA mice and RA patients. IL-17 aggravated synovial inflammation and bone destruction in CIA. By immunohistochemistry, cadherin-11 expression was increased in the synovium of mice with CIA and IL-1Ra-deficient mice and in patients with RA. Synovial cadherin-11 expression in IL-17-injected knee joints, measured by real-time RT-PCR, Western blot and immunohistochemistry, was increased in CIA. Cadherin-11 expression was significantly increased by IL-17 in cultured FLS of CIA mice and RA patients, and these increases were blocked by NF-κB inhibitors. IL-17 increased the expression of cadherin-11 in vivo and in vitro, which implies that an IL-17-induced increase of cadherin-11 is involved in IL-17-induced aggravation of joint destruction and inflammation.

The aim of this study was to investigate the expressions of Toll-like receptor (TLR) 2, TLR4, TLR9, and their correlations with the expression of cytokines that are associated with activation of CD4(+) T cells and inflammation including interferon gamma (IFNgamma), interleukin 4 (IL4), interleukin 17 (IL17), and tumor necrosis factor alpha (TNFalpha) in muscle tissues of patients with dermatomyositis (DM) and polymyositis (PM). The expressions of TLR2, TLR4, TLR9, IFNgamma, IL4, IL17, and TNFalpha were measured by real-time reverse transcription-polymerase chain reaction in muscle tissues from 14 patients with DM and PM (nine patients with DM, five patients with PM) and three controls. The expressions of TLR2, TLR4, and TLR9 were also localized with immunohistochemistry. The expression levels of TLR2, TLR4, TLR9, IFNgamma, IL4, IL17, and TNFalpha were significantly high in patients with DM and PM compared with those in the controls, and the expression levels of TLR4 and TLR9 had significant positive correlations with the expressions of IFNgamma, IL4, IL17, and TNFalpha. Immunohistochemistry showed that TLR2, TLR4, and TLR9 were expressed by infiltrating cells of perimysium in DM, whereas they were expressed by infiltrating cells of endomysium in PM. These results suggest that the involvement of TLR4 and TLR9 in immunopathogenesis of DM and PM might be connected with activation of CD4(+) T cells.

Diabetic motor neuropathy is expressed as the loss of function and the contracture of the intrinsic muscles of the foot, leading to the classic claw toe deformity. This deformity predisposes the foot to ulcerations on the dorsum or tip of the toes or an interdigital ulcer over a condyle between the toes. We present our results of a modified resection arthroplasty for the treatment of this difficult problem.In this study, 72 toes (57 feet) with a deformity in the second to fifth toe accompanied by chronically infected ulcers were involved. All patients underwent modified resection arthroplasty of the PIP or DIP joint depending on the ulcer location. The second toe was involved in 27 cases (38%), the third toe in 11 cases (15%), the fourth toe in 19 cases (26%), and the fifth toe in 15 cases (21%). With the exception of 4 patients, all had a positive culture, including 7 cases of MRSA. The mean followup was 28.7 +/- 8.1 months.The mean wound healing time was 25.6 +/- 6.2 days. Three cases eventually required toe amputation but there was no proximal spread of infection. No recurrence of a claw toe or ulcer occurred in the remaining toes.We believe that modified resection arthroplasty for toe deformities with chronic infected ulcers in diabetic patients is a good treatment alternative to toe amputation.

T helper 17-related cytokines have been implicated in rheumatoid arthritis (RA) pathogenesis. The study aimed to identify cytokines associated with the treatment response of RA patients to tocilizumab (TCZ), a humanized monoclonal antibody against the interleukin- (IL-) 6 receptor. As an independent substudy of the 24-week, randomized, double-blinded CWP-TCZ301 trial of TCZ in RA patients with an inadequate response to disease-modifying antirheumatic drugs, serum levels of cytokines including tumor necrosis factor-alpha, IL-17A, IL-21, IL-23, IL-6, and soluble IL-6 receptor were measured. Baseline IL-17A levels were significantly lower in RA patients who achieved disease activity score 28 (DAS28) remission at 12 weeks of TCZ treatment, compared to patients not in remission. Patients were stratified into IL-17A low group and IL-17A high group. Significantly more patients in the IL-17A low group achieved remission as compared to the IL-17A high group (47.6 versus 17.4%,P=0.032). DAS28 improvement was significantly better in the IL-17A low group than in the IL-17A high group at 12 weeks(P=0.045)and 24 weeks(P=0.046)after adjustment. Other baseline cytokines were not associated with treatment response to TCZ. The data demonstrate that low baseline IL-17A levels are associated with better clinical response to TCZ treatment in RA patients.

A twenty-year old male presented with diffuse limb muscle weakness and exertional dyspnea since childhood. The diagnosis of nemaline myopathy was given based on the muscle pathology findings that revealed nemaline rods on light and electron microscopy and discovery of a novel mutation, E239K, in ACTA1. Incidentally, the patient had hypertrophic cardiomyopathy (HCM) as shown by echocardiography. In nemaline myopathy, a few cases of HCM have been reported, albeit rarely and always fatal, but only one patient had ACTA1 mutation. This present report describes an infantile onset of nemaline myopathy with a milder clinical course and non-fatal HCM as compared with previous cases, showing clinical diversity in skeletal and cardiac manifestations of conditions associated with ACTA1 mutations.

Dysferlinopathy comprises a group of autosomal recessive muscular dystrophies caused by mutations in the DYSF gene. Due to the large size of the gene and its lack of mutational hot spots, analysis of the DYSF gene is time-consuming and laborious using conventional sequencing methods. By next-generation sequencing (NGS), DYSF gene analysis has previously been validated through its incorporation in multi-gene panels or exome analyses. However, individual validation of NGS approaches for DYSF gene has not been performed. Here, we established and validated a hybridization capture-based target-enrichment followed by next-generation sequencing to detect mutations in patients with dysferlinopathy. With this approach, mean depth of coverage was approximately 450 fold and almost all (99.3%) of the targeted region had sequence coverage greater than 20 fold. When this approach was tested on samples from patients with known DYSF mutations, all known mutations were correctly retrieved. Using this method on 32 consecutive patient samples with dysferlinopathy, at least two pathogenic variants were detected in 28 (87.5%) samples and at least one pathogenic variant was identified in all samples. Our results suggested that the NGS-based screening method could facilitate efficient and accurate genetic diagnosis of dysferlinopathy.

Background: While animal studies have shown better healing with a longer duration of protection without motion exercise after rotator cuff repair, supporting clinical studies are rare. The purpose of this study was to assess the effect of immobilization following rotator cuff repair and to determine whether there was any difference in clinical outcome related to the duration of immobilization. Methods: One hundred patients who underwent arthroscopic single-row repair of a posterosuperior rotator cuff tear (mean, 2.3 cm in the coronal-oblique plane and 2.0 cm in the sagittal-oblique plane) were prospectively randomized to be treated with immobilization forfouroreightweeks.During the immobilizationperiod, nopassiveoractive range-of-motion exercise,including pendulum exercise, was allowed. According to the intention-to-treat protocol and full analysis set, eighty-eight patients were evaluated clinically and with magnetic resonance imaging postoperatively, after exclusion of twelve patients without postoperative clinical evaluation. Ranges of motion, clinical scores, and retear rates were compared between the four and eight-week groups. Ninety-eight patients were contacted by telephone at a mean of thirty-five months to investigate the clinical outcomes. Results: The mean duration of immobilization was 4.1 weeks in the four-week group and 7.3 weeks in the eight-week group. There were nine full-thickness retears (10%), and 89% of the patients rated their result as excellent or good. There were five full-thickness retears in the four-week group and four in the eight-week group (p = 0.726). At the time of final follow-up, the two groups showed no differences in range of motion or clinical scores. However, the proportion showing stiffness was higher in the eight-week group (38% compared with 18%, p = 0.038). Conclusions: Eight weeks of immobilization did not yield a higher rate of healing of medium-sized rotator cuff tears compared with four weeks of immobilization. Level of Evidence: Therapeutic Level I. See Instructions for Authors for a complete description of levels of evidence.

Background:One of the goals of rotator cuff repair is to restore the torn tendon to its original insertion anatomically. However, it is sometimes difficult to restore the entire footprint.Purpose:This study was undertaken to evaluate the variables affecting this repair coverage and to discern the differences in retear rate and clinical results between complete and incomplete footprint coverage in rotator cuff surgery.Study Design:Case series; Level of evidence, 4.Methods:From 2007 to 2009, a total of 85 consecutive repairs for medium-to-large rotator cuff tears were identified as having complete or incomplete coverage of their original footprints. We defined the complete footprint coverage (CC) group as patients who had >50% of their footprint covered during repair and the incomplete (IC) group as Results:Fifty-seven repairs were defined in the CC group and 28 repairs in the IC group. Preoperatively, age, tear size in coronal oblique and sagittal oblique planes, Goutallier fatty infiltration, and atrophy of the supraspinatus affected the amount of footprint coverage in univariate analysis. In multivariable analysis, however, tear size in the coronal plane was the only independent factor affecting footprint coverage in rotator cuff repair. On postoperative MRI, 45.6% of the CC group had an intact tendon, 45.6% had a delaminated partial retear, and 8.8% had a full-thickness retear; in the IC group, 17.9% had an intact tendon, 60.7% had a delaminated partial retear, and 21.4% had a full-thickness retear. There was a statistically significant difference in the proportion of tendon integrity between groups ( P = .028). Clinical scores and range of motion at final follow-up showed no difference between the 2 groups.Conclusion:Tear size in the coronal plane was the only independent factor affecting the amount of footprint coverage. Repair quality based on retear classification was different between the 2 groups. However, both complete and incomplete footprint coverage in rotator cuff repair showed no differences in clinical scores and range of motion at short-term follow-up.

The goal of this study was to prospectively compare the early clinical results of intra-articular injection of hyaluronate or corticosteroid in patients with idiopathic adhesive capsulitis. The authors’ hypothesis was that there would be no difference between groups. Sixty-eight patients with idiopathic adhesive capsulitis were equally randomized to receive either corticosteroid or hyaluronate injection. All patients underwent standard physical examination and magnetic resonance imaging. Intra-articular injection was performed through an anterior approach by the same orthopedic surgeon without image guidance. Patients were followed up 2 and 12 weeks after completion of the injection. The primary outcome was the Constant score at week 12. Secondary outcomes included the visual analog scale (VAS) pain score, the American Shoulder and Elbow Surgeons (ASES) score, and range of motion at each time point. No significant differences were noted in preoperative demographic features or baseline shoulder function between groups. After treatment, no significant differences were noted in early clinical outcomes (at weeks 2 and 12) with VAS, ASES, and Constant scores between groups (all P >.05). Evaluation of range of motion showed no difference in forward elevation or external rotation at each time point. Internal rotation was significantly lower at week 2 in the hyaluronate group compared with the corticosteroid group ( P =.015). Internal rotation improved at week 12, with no significant difference between groups. Patients treated with intra-articular injection of hyaluronate and corticosteroid for idiopathic adhesive capsulitis showed significant improvement in early clinical scores and range of motion without significant differences between groups.